水稻同源四倍体三系法杂种优势利用研究

水稻是重要的粮食作物，其产量的增加和品质的改良都是关系国计民生的大事。就我国现阶段的国情而言，水稻产量在现有水平上稳步提升仍是未来十几年甚至几十年农业生产最重要的目标之一。尽管根据“超级杂交水稻育种”的战略设想和水稻育种实践，通过不断地改进育种技术可望在更高的产量水平上进行水稻杂种优势利用，在稻属植物内还具有很大的产量潜力可以挖掘。然而，仅仅从现有的种质基础出发，要更大幅度提高水稻单产，实现“超级杂交稻”的目标也存在一些困难：现有的推广品种是二倍体，尽管种类众多，但是其基因组的来源相对单一；同时，水稻基因组DNA含量也是作物中最少的，基因组内寻求开发潜力有一定困难；水稻作为C3植物，光合利用效率不高也是制约水稻产量提高的因素之一。因此，寻求常规手段以外的技术突破或者方法创新，是实现“超级杂交稻”的目标的迫切需求。本研究利用秋水仙素能抑制细胞分裂中纺锤丝的收缩、使细胞染色体加倍的作用，对水稻幼穗诱导的愈伤组织细胞进行加倍，并分化出再生植株；创制出水稻同源四倍体新的种质材料，在此基础上选育水稻同源四倍体雄性不育三系材料，并实现水稻同源四倍体的三系配套，开展水稻同源四倍体杂种优势利用和四倍体杂交水稻选育研究，建立水稻同源四倍体杂种优势利用的新技术体系。这不仅有助于倍性水平杂种优势的开拓和利用，同时也将为我国新世纪“超级稻”育种研究开辟一条新的技术途径。 水稻幼穗诱导愈伤组织并分化成苗是一项成熟、简单的组织培养技术。本研究以普通二倍体水稻亲本为材料，用秋水仙素进行水稻的多倍体化诱导，创制同源四倍体水稻三系亲本材料并对其进行鉴定。多倍体化以秋水仙素诱导的愈伤组织培养为基础，研究不同秋水仙素浓度梯度和愈伤组织诱导培养基组合对诱导四倍体植株的影响。结果表明在MS+2,4 D 1.0mg/L+ KT0.2mg/L+ IAA0.2mg/L 和500mg/L的秋水仙素处理下，水稻愈伤组织染色体加倍（有最高的效率）效果较好，平均加倍频率可达25.26%，其中，材料CDR22和IR26诱导较易成功，加倍频率分别达到75%和26.5%；相对材料94109 1.3%加倍频率和冈46B 10.8%加倍频率，诱导率差异极显著。 对水稻四倍体材料进行了形态学鉴定结果表明，与二倍体水稻对照相比其株高、穗长、花粉育性等主要农艺性状，确定四倍体材料在穗长和千粒重两方面极显著提高，种子的长度和宽度也显著增长。对花粉育性鉴定，确认水稻四倍体不育系材料仍为不育，保持系材料自交和杂交可育，恢复系材料自交和杂交可育。对四倍体材料进行细胞形态、染色体数目等方面进行细胞学鉴定，经核型分析表明水稻四倍体材料具有48条染色体，是二倍体水稻的两倍。水稻四倍体材料根尖分生组织细胞与二倍体的根尖分生组织细胞相比，细胞体积、细胞核和核仁显著增大。四倍体三系材料在细胞有丝分裂中期均可规则排列在赤道板，并能均等地移向两极；后期观察中没有发现染色体分离滞后现象，分裂末期细胞能够形成大小相对均一的子细胞。水稻同源四倍体三系材料细胞分裂未见异常，植株生长发育正常。 从1996年至2006年，针对结实率、有效分蘖、着粒数和穗长等主要农艺性状，通过系谱选育的方法，对培育的同源四倍体水稻亲本材料进行了连续选择和改良，取得较好成效。表现为结实率的改良效果极佳，所有改良材料的平均结实率均呈上升趋势，如D237（29.70％→72.70％）、DTB（19.55％→53.21％）等。有效分蘖总体呈现上升趋势，但在不同的年份，如1998和2002存在较大的负向波动。部分材料改良效果明显，如D19B（5.87→13.50）、D什香 (7.00→12.00)等；同时一些材料如DTB和D明恢63虽然总体略有提高，但在不同的年份波动很大，因此存在较大改良阻力，原因还有待进一步研究。着粒数的改良上升趋势比较显著，除保持系的DTB之外，其余材料的平均着粒数有显著提高。穗长的改良阻力较大，虽然不同材料总体上有所提高，但效果并不显著，并且不同年份有较大负向波动（2001）。此外还对株高、剑叶长等性状也进行了选择，但效果不显著，原因有待进一步提高。同源四倍体材料产量相关性状遗传改良幅度不一致，保持系和恢复系间的遗传改良效果也存在差异。这为同源四倍体水稻的进一步利用打下了良好的基础。 籼稻和粳稻亚种间杂交及杂种优势利用的主要障碍就是其低的结实率。而同源四倍体杂交水稻的研究为提高杂交水稻的杂种优势利用创造了新的途径。本研究通过随机区组设计方案，挑选性状优良的二倍体水稻材料，包括雄性不育系，保持系和恢复系进行秋水仙素诱导加倍，从而获得同源四倍体水稻对应的三系材料。利用选育的优良水稻同源四倍体三系材料，配制7个杂交组合，杂交F1代与其恢复系亲本进行比较，用于计算超亲优势（HB）；而杂交F1代与生产上大面积推广的二倍体杂交品种汕优63进行比较，用于计算杂种优势。结果显示，同源四倍体杂交水稻的超亲优势表现为：每株有效穗变化幅度为1.4%至105.9%，总粒数为0.5%至74.3%，每穗实粒数为17.6%至255.7%，结实率为9.6%至130.4%。这些农艺性状的改良使得这7个杂种F1的理论产量的超亲优势高达64.8%至672.7%。小区试验中四倍体杂交水稻组合T461A/T4002和T461A/T4193分别比二倍体对照汕优63提高46.3%和38.3%以上，除一个品种以外所有品种产量均接近或高于汕优63的产量。同源四倍体水稻强大的杂种优势表明，亚种间杂交育性低的问题可通过四倍体化及强化选择来解决。此外，同源四倍体杂交水稻器官的巨大性也是其产量提高的有利因素，水稻同源四倍体三系杂种优势利用研究具有一定的理论价值和商业生产潜力。 Rice is one of the major food crops, the improvement of the production and quality of it is an important thing related to the people's livelihood. On China's current national conditions, steadily increase of the rice yield based on the current level is still one of the most important goals in the next decade or even decades of agricultural production. According to the "super hybrid rice breeding" the strategic and rice breeding practice, improvement of the use of hybrid rice heterosis through continuous improvements in breeding technology is expected to get a higher level of rice yield, there are also a great yield potential can be exploited. However, there are also some difficulties to increase rice yield obviously and implement the goal of "super hybrid rice" based on the existing germplasm: Rice varieties in promotion are diploid, although there are many varieties, but their genome are from a comparatively single source; Meanwhile, the rice genome DNA are the least among the crops, it is difficult to exploit the development potential within the genome; Rice as C3 plants, photosynthetic efficiency is not high, it is one of the factors constraint rice yield. Therefore, seeking technological breakthroughs or innovative methods different from conventional means is the urgent needs to reach the target of "super hybrid rice". Using colchicine inhibit spindle contraction during cell division, double the cell chromosome, we induced callus cells from rice panicle to be doubled, and differentiated regeneration; we created a new autotetraploid rice germplasm material, and on that basis we bred male sterility three line autotetraploid rice materials, and the achieved the three line rice autotetraploid matchmaking, researched in autotetraploid rice heterosis usage and tetraploid hybrid rice breeding, constituted a new technology system of autotetraploid hybrid rice heterosis utilization. This not only helps the tetraploid rice heterosis exploration and use, but also inaugurates a new technical means for China in the new century "super rice" breeding research. We chose ordinary diploid rice as materials, using colchicine to induce the polyploidization, created the autotetraploid rice three-line materials and identified them. The polyploidization was based on the colchicine-induced callus tissue culture, and we experimented different colchicine concentrations and culture mediums to induce tetraploid plants, confirmed that the optimal concentration for inducement was 500 mg/L, the average induce rate was 25.26 %. Among all the materials, CDR22 and IR26 had higher induced rate; in contrary, 94109 and GANG46B had lower induced rate, the difference was significant. Autotetraploid materials was identified of both morphological and cytological, compared plant height, length of pollen sterility, and other major agronomic traits with a diploid rice as the control plant, identified that the autotetraploid materials had very significant advantages in ear length and thousand-grain weight, as well as the size of the seeds. Cytology identification included observation of the cell morphology, the number of chromosomes, and karyotype analysis on the autotetraploid materials confirmed that their chromosome number was 48, twice of the diploid rice. Mitoses in the three lines were common: chromosomes arrayed normally in metaphase and separated balanced into the two poles, chromosome moved without lagging in anaphase and daughter cells normally formed in telophase except one. It has been proved that tetraploid rice has normal meiosis as their diploid relatives, which usually including series of sub-phases as interphase, prophase I (five sub-phases), prophase II, metaphase I, II, anaphase I, II and telophase I, II. However, abnormal phenomena, such as formation of tetravalent, trivalent and univalent, chromosome lagging and so on, which would finally block meiosis. Configurations of chromosome in metaphaseⅠwere versatile in structure and form accept the bivalent. That condition varied in different strain, suggesting more complex paring configurations and more versatile genetic characters in tetraploid rice. All these abnormalities in meiosis contributed to low fertility of gamete and might consequently resulted in low seed setting. Successive selection and improvement on seed set, productive tiller per plant, total grains per panicle, panicle length and so on had been carried out from 1996 to 2006. The raise of seed sets was significant in both restorers and maintainers. Seed sets of some strains were improved more significantly than others, for example D237（29.70％→72.70％）、DTB（19.55％→53.21％）and et al.. Productive tiller per plant was improved to some extant. The tendency of improvement was rising on the whole but changed in some years such as 1998 and 2002. Part of the stains increased greatly, such as D19B（5.87→13.50）、Dshixiang (7.00→12.00) and so on, but some strains including DTB and Dminghui63 only increased little and decreased in some years by unknown reason. Total grains per panicle increased significantly and all strains except DTB increased. Improvement of panicle length termed to be hard. Different strains showed different capacities for improvement and floating existed in different years for example 2001. It has been proved that other agronomical traits including plant length, flag leaf length and so on could be improved but not significantly by selection also. In a word, agronomical traits could be raised by successive selection that is prerequisite for further utility of autotetraploid rice. Poor fertility is the main barrier for utilizing heterosis between the two rice (Oryza stiva L.) sub-species, indica and japonica. Recently, the development of autotetraploid hybrids (2n=4x=48) has been suggested as a new method for increasing heterosis in hybrid rice. Using standard experimental protocols, the elite diploid rice male sterile, maintainer, and restorer lines were colchine-doubled and autotetraploid counterparts were obtained. Seven resulting hybrids were analyzed for heterobeltiosis (HB), where the F1 was compared to the male parent, and the degree of heterosis, where the F1 was compared to the diploid commercial hybrid, Shanyou 63. The HB among the autotetraploid hybrids ranged from 1.4 to 105.9% for the productive panicles per plant, 0.5 to 74.3% for total kernels per panicle, 17.6 to 255.7% for filled kernels per panicle, and 9.6 to 130.4% for seed set. Improvements in these yield components resulted in the HB for kernel yield ranging from 64.8 to 672.7% among the seven hybrids. Hybrids T461A/T4002 and T461A/T4193 yielded 46.3 and 38.3% more, respectively than Shanyou 63, and all other hybrids but one yielded the same or more than Shanyou 63. The high heterosis for yield suggests that hybrid sterility between two rice sub-species may be overcome by using tetraploid lines followed by intensive selection. Also, the gigantic features of the autotetraploid hybrids may establish a plant structure able to support the higher yield.

不同小麦品质类型与生态环境关系的研究

小麦（Triticum aestivum L.）是世界上种植面积最大，总产量最高，食物加工种类最丰富的粮食作物，占世界人口35 ％－40 ％的人们以此为主要食物。因此小麦产量的高低和品质的优劣直接影响人们对食物需求的安全和满意程度，也影响着人类的营养平衡以及面粉和食品加工业的发展。随着生活水平的提高，人们对于小麦的品质越来越重视。培育优质专用小麦新品种，制定优质专用小麦品种品质生态区划，从而在不同程度上实现小麦的区域化种植和产业化经营具有重要的意义。 影响小麦品质的因素主要是遗传因素和环境因素，其中环境因素又包括各种自然生态因素和人为因素。研究表明，小麦品质的环境间的差异大于品种间的差异，气候条件是影响小麦品质的最重要的因子，小麦品质的地域间的差异反映出了小麦的品质区域分布规律。为了满足市场对不同品质小麦的需求，对小麦进行区域化研究具有重要的理论和现实意义。本研究结合四川的地理、气候特点，研究不同品质类型与生态环境的关系，为在复杂的生态环境内进行品质区划提供依据。 本研究首先根据四川省小麦种植区域的生态特点，在四川省多个典型生态区：川南丘陵的荣县、川西南高原的西昌、川西平原的双流布点种植，采用的小麦试验材料为不同品质类型：中筋小麦川育12、川育14、川育16由本所提供；弱筋小麦川麦32和强筋小麦川麦36由四川省农科院作物所提供。通过研究品质性状与品种及各个生态因子包括地点、土壤土质差异等的关系，明确不同生态环境中适宜种植的小麦品种类型，强筋小麦、中筋小麦更适合于在荣县、双流地区种植，弱筋小麦更适合于在西昌地区种植，为品种品质区划奠定基础。 其次，选择了本课题组育成的稳定中间品系，对其品质性状SDS沉降值进行了多年测定。分析了品质性状SDS沉降值与多种气候因子的相关性，结果表明SDS沉降值与日均温、日照时数成正相关，与降水量成负相关，为品质育种提供了理论依据。 此外，以中筋小麦新品种小麦川育14为材料，应用三元二次正交旋转回归模型设计试验，研究主要栽培因子播期、密度和施肥量对产量的影响，并建立函数模型。经计算机模拟寻优，筛选出了高产高效栽培组合措施，并确定了置信域。结合四川省不同的地理情况，在平原和丘陵地区分别进行实验，并各自建立了高产高效栽培组合措施，为川育14品种的推广提供了理论指导。 Wheat is one of the most important crops in the world. About 35%-40% people all over the world, take the wheat as their most important food. So the quality, as well as the quantity of the wheat makes a direct effect on people’s demands of food and their satisfaction. It also effects on human’s healthy, and the development of the Food processing industry. With the development of the living standard, people pay more attention to the quality of wheat. So, we set a special ecology zoning for wheat. It is significant to carry out planting the wheat in special zoning in varying degrees. The main factors affecting wheat quality are heredity and environment including many ecological factors and the factors in cultivation. As to the quality，the difference between ecology and cultivation is more important than the difference between special wheat. In so many factors, climate is the most important one. From the difference in quality between different zones，we can conclude the rule of distribution abort quality of wheat. Finding out the intersection of numerous wheat not only can meet the demand of food production，but also has important signification in theory and realism。In our research, according to the complex geography in Sichuan province, we study the relationship between numerous kinds of quality characters in wheat and the ecology. So, we can set a foundation for more research. In this research, firstly, we plant wheat in some typical ecological regions of SICHUAN province: RONGXIAN（south of SC）、XICHANG（south of SC）, SHUANGLIU(west of SC). The materials of the experiments: ChuanYu12, ChuanYu14, ChuanYu16(from our institute), Chuanmai32, Chuanmai36 (from the Chinese academy of agriculture sciences of Sichuan. Through the research on the relationship between the quality of wheat and those ecology factors, we can make a definition that which area is perfect matched with which kind of wheat. And it can satisfy the demand of people. Secondly, select many sorts of wheat from our research group. All of them are selected and bred more than 3 years(2003-2005). And we make every-year determination as well. We’ve gotten SDS value from those 9, and various data on factors of climate. We also got to know the relation ship between those numbers. Thirdly, use Chuanyu14 as material, the mathematical model of the relation between the production of wheat and main agricultural measures such as date, density and fertilizer. The model was established by association of three elements two return, rotate and regression. We set a suitable model and get a suitable method which can make high harvest. Based on various kinds of geographical regions in Sichuan province, we set different models which can be used in plain and hill. So, we can plant Chuan Yu 14 in Sichuan province under the result in research.

中药穿龙薯蓣、黄山药和盾叶薯蓣的分子鉴别研究

为建立对中药穿龙薯蓣、黄山药和盾叶薯蓣分子鉴别的方法，我们首先研究薯蓣叶绿体psbA-trnH片段遗传多样性，探讨该片段用于中药穿龙薯蓣、黄山药和盾叶薯蓣种间分子鉴别和系统学研究中的意义。在对不同类群薯蓣种的叶绿体psbA-trnH基因间区进行ＰＣＲ扩增并测序，获得了该区间的完整序列的基础上，将所得序列用软件MEGA3.0进行相关分析。穿龙薯蓣的psbA-trnH片段全长274bp，黄山药全长279bp，盾叶薯蓣植物个体内的叶绿体DNA则有两种psbA-trnH片段，长度分别为241bp和503bp。MEGA3.0软件分析，三种薯蓣种间psbA-trnH片段序列的遗传距离（p-distance）为0.00350-0.04545，各个薯蓣种内的不同类群该序列无差异。用UPGMA法根据psbA-trnH序列的遗传距离建立系统发生树，每个种不同产地的薯蓣很明确地聚在一起，和形态分类一致。所得结果显示psbA-trnH片段序列在所研究的三种薯蓣种内保守，在种间具有明显的较大差异，而三种薯蓣及薯蓣属的系统发生关系尚须进一步研究。 以穿龙薯蓣、黄山药和盾叶薯蓣的psbA-trnH片段序列分析结果为基础，我们根据三种薯蓣在该片段上的特征序列位点设计了用于识别三种薯蓣的寡核苷酸片段并作为PCR反应的引物，将三种薯蓣的特征标记引物与psbA-trnH片段引物配合使用建立了相互识别三种薯蓣的显性和共显性两种检测方式，辅以NaOH碱法快速提取薯蓣干燥根茎总DNA，为三种薯蓣药材相互间快速准确鉴别创造了条件。 To establish molecular method for identifying Chinese medicine Dioscorea nipponica/panthaica/zingiberensis, we firstly study the Genetic diversity of psbA-trnH of Dioscorea and discuss the value of the fragment to molecular identity and systematology to three Dioscorea species (Dioscorea nipponica/panthaica/zingiberensis). The psbA-trnH fragments of different species of Dioscorea are amplified and sequenced，analyzing by software MEGA3.0. The length of Dioscorea nipponica and panthaica are 274bp and 279bp; two psbA-trnH fragments exist in Dioscorea zingiberensis individual, length of the shorter fragment is 241bp and the longer is 503bp. The interspecific Genetic distance (p-distance) of the three species is 0.00350-0.04545, and no intraspecific diversity exists. According to the Genetic distance, phylogenetic tree is built by UPGMA Method, each species of different place gather obviously, which is same to the shape classification. Results show that the psbA-trnH fragment is highly conservative at intraspecific level and obvious diversity at interspecific level, however, phylogenetic study is necessary for the Dioscorea in the future. Based on analysis to psbA-trnH fragment of Dioscorea panthaica, nipponica and zingiberensis, according to the special point of the three Yam species, primers as marker are designed for distinguishing the three species. Cooperating with primer pair psbAf-trnHr, dominant and codominant way is established for discriminating them. Moreover, assisted with NaOH method to extract total DNA of Dry stenophora of Yam, rapid and correct identity to the three species can be done.

糯小麦杂交后代的基因型鉴定及Wx基因的效应研究

糯小麦在食品加工业、淀粉加工业及其它工业上有着重要用途，是近年来许多国家小麦研究的重要课题。国外糯小麦选育尚未突破高产与糯性相结合的难点，国内目前还没有培育出高蛋白强筋型的糯小麦品种，这在一定程度上与缺乏合适的育种方法和高效、实用的糯小麦分子标记辅助育种技术有关。国内外对Wx基因效应的研究主要利用缺体－四体系、重组自交系或近等基因系，还未见有利用遗传背景相同的BC5F2代回交改良群体的报道。 糯性位点近等基因系是小麦淀粉品质育种的重要材料，而我国目前还没有一套中国栽培小麦遗传背景的糯性位点近等基因系。为了选育部分糯小麦、全糯小麦和中国栽培小麦遗传背景的糯性位点近等基因系，我们利用Wx蛋白电泳和高效实用的分子标记技术体系来鉴定糯小麦杂交后代的基因型，结果证明该体系能有效地用于糯小麦的分子标记辅助育种。以中国春糯性位点全套近等基因系为研究材料，对小麦Wx基因的6个STS标记和1个CAPS标记进行了筛选，改良PCR扩增条件以及产物检测方式后，从这些标记中筛选出3个标记，包括鉴定Wx-A1、Wx-D1位点的2个共显性STS标记和Wx-B1位点的1个显性STS标记。利用上述3个分子标记从BC5F2 代回交改良群体中筛选出了8种Wx基因型，经卡方检验，其分离比符合3对基因的分离比例，其中基因型为aabbdd的植株有2株，直链淀粉含量分别为1.81%和0.82%，为全糯小麦；基因型为AAbbdd，aabbDD的部分糯性植株各有1株，直链淀粉含量分别为15.24%和17.57%。以上4株植株的农艺性状和品质性状接近回交亲本“川育12”，并明显优于全糯材料“98Y1441”，表明采用回交法与Wx基因分子标记辅助选择相结合，有助于培育高产、优质的全糯和部分糯小麦。同时，本研究中建立的分子标记技术体系，也为选育具有中国栽培小麦遗传背景的糯性位点近等基因系奠定了基础。 在基因型鉴定的基础上，利用糯小麦杂交后代BC5F2代回交改良群体研究了各基因缺失降低直链淀粉含量的效果和各基因合成直链淀粉的能力，以及直链淀粉含量与农艺性状、品质性状、淀粉糊化特性等的相关性。缺失不同Wx基因的8种基因型，直链淀粉含量差异显著。研究单缺失基因型发现，减少效应最大的是Wx-B1b基因，Wx-B1b和Wx-D1b基因没有显著差异，减少效应最小的是Wx-A1b基因。研究双缺失基因型发现，Wx基因合成直链淀粉的能力，Wx-B1a基因最高，Wx-A1a基因最低，而Wx-B1a和Wx-D1a基因差异很小；直链淀粉含量与株高、穗长、小穗数、穗粒数、千粒重等农艺性状相关不显著，表明淀粉品质育种可以与高产育种实现有机结合；直链淀粉含量与SDS-沉降值呈显著负相关(r=-0.726)，说明直链淀粉含量降低在一定程度上有利于提高小麦营养与加工品质，这一结果至今未见有文献报道；全糯类型的淀粉糊化特性与其他类型显著不同，具有最高的峰值粘度和稀懈值，最低的低谷粘度、最终粘度、反弹值、峰值时间、糊化温度、起始糊化温度，表明糯小麦淀粉在食品和工业上具有特殊用途；稀懈值与直链淀粉含量呈极显著负相关(r=-0.969)，其他粘度参数与直链淀粉含量呈显著正相关(最终粘度r=0.797，低谷粘度r=0.910、反弹值r=0.954、峰值时间r=0.970、糊化温度r=0.962、起始糊化温度r=0.932)。以上结论可为糯小麦品种选育和淀粉品质改良提供理论依据。 Waxy wheat is very important in food processing industry, starch processing industry and the other industries, so it is a focus of wheat research in many countries these years. Foreign wheat breeders have not conquered the difficulty of high yield combined with waxy character, and there is no waxy wheat variety with high protein and strong gluten in China at present，all of which were caused by lacking proper breeding methods and effective, applied molecular markers-assisted selection technique at a certain extent. Until now, research about the effect of waxy genes mainly depended on nullisomic-tetrasomic lines, recombinant lines or near-isogenic lines, and it is lacking the reports of using improved BC5F2 backcross progenies under the common genetic background. Near-isogenic lines at the Wx loci are important materials for wheat starch quality breeding. However, there are no such lines under the Chinese cultivated wheat genetic background. To develop partial waxy wheats, waxy wheats and near-isogenic lines at the Wx loci under the Chinese cultivated wheat genetic background, we use SDS-PAGE of waxy proteins and effective, applied molecular marker-assisted selection technical system to identify the genotype of waxy wheat’s progenies. The results indicated that such a system is applicable in waxy wheat’s molecular marker-assisted selection effectively. A series of Chinese Spring Wx loci near-isogenic lines were used to identify the specific bands of six STS markers and one CAPS marker of Wx genes. After optimizing PCR amplification and separating of PCR products, three co-dominant and dominant STS-markers were identified at the Wx-A1, Wx-D1 and Wx-B1 loci, respectively, which were used to identify the genotype of waxy wheat’s progenies. Eight Wx genotypes were developed from the improved BC5F2 backcross progenies, which follows Mendelian segregation. Among them, there were two aabbdd waxy plants whose amylose content in the BC5F3 seeds were 1.81% and 0.82%, respectively. In addition, there were partial waxy plants (AAbbdd and aabbDD) whose amylose content in the BC5F3 seeds were 15.24 % and 17.57%, respectively. Most agronomic and quality traits of these four plants resembled those of the recurrent parent “Chuanyu 12”, and superior to waxy wheat parent “98Y1441”. This shows that backcross approach in combination with molecular marker-assisted selection of waxy genes is helpful to develop partial and full waxy wheat with good traits in the waxy wheat breeding program. At the same time, molecular marker-assisted selection technical system in this paper, also establish the base for breeding the near-isogenic lines at the Wx loci under the Chinese cultivated wheat genetic background. According to the results of genotype identification, we use waxy wheat’s improved BC5F2 backcross progenies to verify the effects of null alleles on reducing amylose content and determine the amylose synthesis capacity of each Wx gene independently, and investigate the relativity among amylose content with agonomic traits, quality traits, starch pasting properties respectively. There was significant difference in amylose content of the eight genotypes carrying different null Wx alleles. The reducing effect of the single null alleles was the most significant in Wx-B1b, followed by Wx-D1b and Wx-A1b, and there was no significant difference between Wx-B1b and Wx-D1b. The results of the double null lines further demonstrated that for the capacity of amylose synthesis, Wx-B1a was the highest, followed by Wx-D1a and Wx-A1a, and there was no significant difference between Wx-B1a and Wx-D1a. Amylose contents of the eight genotypes were not significantly correlated with plant height, spike length, spikelets per spike, grains per spike, 1000-grain weight, which showed that starch quality breeding could integrate with high yield breeding. Amylose contents of the eight genotypes were negatively significantly correlated with SDS-sedimentation value(r=-0.726), which suggested that reduction in amylose content is propitious to improve quality at a certain extent. Starch pasting properties of the full waxy type was significantly different from the other seven types, with the highest peak viscosity and breakdown, and lowest valley viscosity, final viscosity, setback, peak time, pasting temperature and starting pasting temperature. It indicated that waxy wheat starch has special use in food and industry. Breakdown was negatively significantly correlated with amylose contents (r=-0.969), and the other parameters were positively significantly correlated with amylose contents (r=0.797 for final viscosity, r=0.910 for trough viscosity, r =0.954 for setback, r =0.970 for peak time, r=0.962 for pasting temperature and r=0.932 for starting pasting temperature ). The results of the study are very useful for waxy wheat variety breeding and starch quality improvement.

来自易变山羊草抗禾谷孢囊线虫基因的克隆与序列分析

禾谷孢囊线虫（Heterodera avenae）是严重危害禾谷类作物的病原线虫之一,它广泛分布于澳大利亚、欧洲、北美、印度和中国等世界主要小麦产区,使作物严重减产,造成巨大的经济损失。目前最有效的防治措施之一是将外源抗性基因导入栽培小麦(Triticum aestivum L.)，培育抗禾谷孢囊线虫的新品种。但迄今为止抗禾谷孢囊线虫基因克隆研究的相关报道却很少。 本实验根据此前从抗禾谷孢囊线虫材料E-10扩增得到的与来自节节麦（Aegilops tauschii）的抗禾谷孢囊线虫基因Cre3高度同源的序列Rccn4,设计出三条嵌套引物,采用SON-PCR(single oligonucleotide nested PCR)方法,从E-10基因组DNA中得到一个长为1264 bp的扩增产物(命名为Rccn-L),测序比对结果显示,这一序列将Rccn4的3’端延伸了1209 bp,与抗禾谷孢囊线虫Cre3基因核苷酸同源性为86﹪，核苷酸编码区长1026 bp，含一个不完整的开放阅读框,一个终止密码子，没有起始密码子和内含子结构,编码一个342个氨基酸残基的蛋白质。该蛋白质等电点为5.19，分子量为38112.6Da。从序列的第113位开始到第332位是NBS-LRR类抗病性基因LRR区,呈现XXLXXLXXL重复。LRR编码区内亮氨酸残基的含量达17﹪，与抗禾谷孢囊线虫Cre3基因LRR编码区的核苷酸和氨基酸同源性分别为89﹪和78﹪。本实验首次将SON-PCR成功地运用于植物基因克隆，为植物基因克隆提供了又一有效方法。 此外,还根据Cre3基因及其他的NBS-LRR类植物抗性基因的NBS和LRR区保守序列设计了两对特异性引物，从禾谷孢囊线虫抗性材料易变山羊草基因组DNA中扩增到两个相应的目标条带。测序分析结果表明，它们的长度分别为532bp和1175bp，构成了一个有32bp的共同序列的NBS-LRR编码区。其序列总长为1675bp（命名为RCCN），含有一个不完整的开放阅读框，没有起始密码子、终止密码子和内含子结构。其中编码序列为1673bp，可编码一个557个氨基酸的蛋白质，等电点（pI）为5.39，分子量为63537.5Da。与Cre3的核苷酸和氨基酸同源性分别为87.8﹪和77﹪。RCCN氨基酸序列中含有已知抗病基因NBS区域的几个保守模体：kinase2区的ILDD、kinase3的（ⅰ）ESKILVTTRSK，（ⅱ）KGSPLAARTVGG，（ⅲ）RRCFAYCS及EGF。RCCN NBS区与Cre3 NBS区的核苷酸和氨基酸的同源性分别为96.4﹪和94﹪。从氨基酸序列的274位到548位为LRR保守区，呈现不规则的aXXLXXLXXL（其中a代表I，V，L，F或M）重复，其中亮氨酸的含量为15.6﹪。该区域与Cre3的LRR区的核苷酸和氨基酸同源性分别为80.8﹪和74﹪。推测该序列可能为一个抗禾谷孢囊线虫的新基因。 本文对抗禾谷孢囊线虫基因的克隆研究，为进一步克隆基因全序列，探索其结构与功能，和研究该基因表达与调控提供了关键信息。同时也为通过基因工程途径将抗性基因向优良小麦品种高效、定向转移，最终培育出小麦抗禾谷孢囊线虫新品种奠定了基础。 Cereal cyst nematode (CCN) is a damaging pathogen of broad acre cereal crops in Australia, Europe, North America, India and China. It affects wheat, barley, oat and triticale and causes yield loss of up to 80%. At present, Transferring resistance genes against CCN into wheat cultivars and breeding varieties are considered one of the most effective methods for controlling the CCN. However, there are very limited reports concerning the cloning studies of resistance genes against the cereal cyst nematode. According to the sequence of Rccn4 which had high similarity to the nucleotide binding site (NBS) coding region of cereal cyst nematode resistance gene, Cre3， We designed three 3’ nested primers. Using single oligonucleotide nested PCR (SON-PCR) we successfully amplified one band, Rccn-L, of 1264bp from E-10 which is the wheat-Ae.variabilis translocation line containing the cereal cyst nematode resistance gene of Ae.variabilis. We found that this band of interesting is the 3’ flanking sequence of 1209bp in size of Rccn4. The coding region was 1026bp, which contained an incomplete open reading frame and a terminator codon, without initiation codon and intron, encoding a peptide of 342 amino acid residues, and shared 86﹪nucleotide sequence identity with Cre3. This peptide had a conserved LRR domain, containing the imperfect repeats,XXLXXLXXL, which contains 17﹪ leucine residues and shares, respectively, 89﹪ nucleotide sequence and 78﹪ amino acid sequence identity with the LRR sequence of Cre3 locus. This research firstly used SON-PCR in the research of plant genome successfully, which indicated that SON-PCR is another method of cloning plant gene. At the same time, According to the conversed motif of NBS and LRR region of cereal cyst nematode resistance gene Cre3 from wild wheat (Triticum tauschlii L.) and the known NBS-LRR group resistance genes, we designed two pairs of specific primers for NBS and LRR region respectively. One band of approximately 530bp was amplified using the specific primers for conversed NBS region and one band of approximately 1200bp was amplified with the specific primers for conversed LRR region. After sequencing, we found that these two sequences included 32bp common nucleotide sequence and have 1675 bp in total, which was registered as RCCN in the Genbank. RCCN contained a NBS-LRR domain and an incomplete open reading frame without initiation codon, terminator codon and inxon. Its exon encodes a peptide of 557 amino acid residues. The molecular weight of the protein from the amino acid was 63.537 KDa. The amino acid sequence of RCCN contained conserved motif: ILDD, ESKILVTTRSK, KGSPLAARTVGG, RRCFAYCS, EGF，LRR. RCCN shares 87.8﹪ nucleotide sequence and 77﹪ amino acid sequence identity with cereal cyst nematode gene Cre3. It might be a novel cereal cyst nematode resistance gene. These research results of cloning the resistance genes against cereal cyst nematode bring a great promise for transferring resistance genes into wheat cultivars and breeding new wheat varieties against cereal cyst nematode by gene engineering. And these results also lay the hard foundation for the expressing researches of these genes.

小麦胚乳蛋白组分和Glu-B3位点基因核心编码区差异与小麦品质关系的研究

小麦加工品质改良已成为我国小麦育种的主要目标之一。特别是我国加入WTO以后，对小麦产品的质量提出了更高的要求，小麦品质改良的任务将更加艰巨和重要，小麦胚乳蛋白是影响小麦加工品质性状的重要因素。因此，深入了解小麦胚乳蛋白对加工品质性状的影响及其分子基础，为品质改良提供理论依据和科学指导，对加速我国小麦品质育种和优质小麦生产具有重要意义。本研究选用在麦谷蛋白5个基因位点(Glu-A1、Glu-B1、Glu-D1、Glu-B3和Glu-D3)上均含不同等位基因的小麦品种99G45和京771及Pm97034和京771杂交F9代共164个麦谷蛋白纯合系，及228个中国推广普通小麦品种和高代育成品系为试材，研究了麦谷蛋白Glu-1和Glu-3位点基因等位变异对籽粒蛋白、湿面筋含量、Zeleny沉降值和SDS沉降值间的关系；本研究还利用小麦A、B和D基因组中低分子量麦谷蛋白亚基（LMW-GS）基因特异引物，通过PCR方法克隆了1个Glu-A3位点和3个Glu-B3位点LMW-GS基因片段，在此基础上分析了不同等位基因对品质造成差异的分子基础；另外，本研究对中国近年推广的部分品种和育成的高代品系资源的多样性进行了分析。现将主要研究结果简述如下： 1. 对来自三个麦区的148份材料的醇溶蛋白组成进行了分析，结果表明，各麦区醇溶蛋白模式具有较大差异。在ω区,A7、B、E、F、G、J、P、Q、S和U仅存在于西南秋播麦区；A3、M、N、R、W和X仅存在于黄淮特种麦区；K仅存在于北方冬麦区；A6是北方冬麦区出现频率最高的带型模式，而西南秋播麦区中D出现的频率最高。ω-区的E、H和M几种模式是以前国内外未曾报道的。且初步确定，这些模式对品质性状具有正效应。至于γ区,A、B、D、E和F在各区均有出现，其中B和E在各区出现的频率都很高，在26.1-39.6％之间。相反，H 仅出现在黄淮特种麦区，J仅限于西南秋播麦区。对于β-区醇溶蛋白，B型模式在所有区中都相当高，而模式A仅存在于第三区.对于α-区，模式A在Ⅲ区而模式D在Ⅱ区出现的频率很高。1BL.1RS易位系在中国小麦品种中出现频率高达41.2％，在I, II和Ⅲ麦区的出现频率分别为 45.5、43.5和35.2%。各生态区模式的差异可能是品种适应不同生态条件和人为选择的结果，但这有待进一步证明。由于醇溶蛋白位点（Gli-1）与LMW-GS位点（Glu-3）紧密连锁，本结果可为下面确定普通小麦LMW-GS等位基因变异所用。 2. 利用Gli-1与Glu-3的紧密连锁，以228个小麦品种/系为材料，首次对中国小麦品种麦谷蛋白亚基的6个位点进行综合分析，研究小麦籽粒蛋白与品质性状间的关系，结果表明6个高分子量（HMW）和低分子量（LMW）麦谷蛋白位点对蛋白质含量的效应大小为，Glu-D1>Glu-B3>Glu-A1=Glu-B1> Glu-A3=Glu-D3；对GMP含量的效应大小为, Glu-A3>Glu-B3>Glu-D1> Glu-B1>Glu-A1>Glu-D3；对湿面筋含量的效应大小为, Glu-B1>Glu-B3= Glu-D3>Glu-A3>Glu-A1>Glu-D1；对Zeleny沉降值的效应大小为, Glu-A1> Glu-B3>Glu-D3>Glu-D1>Glu-B1>Glu-A3；对SDS沉降值的效应大小为, Glu-B3>Glu-A1=Glu-D1=Glu-A3>Glu-D3>Glu-B1。对蛋白含量而言，各位点的最佳组合方式为1、17+18、5+10、Glu-A3e、Glu-B3g、Glu-D3b；对湿面筋含量而言，各位点的最佳组合方式为1、6+8、5+10、Glu-A3d、Glu-B3c、Glu-D3b；对Zeleny沉降值而言，各位点的最佳组合方式为N、17+18、5+10、Glu-A3d、Glu-B3d、Glu-D3b；对SDS沉降值而言，各位点的最佳组合方式为1、7+8、2.2+12、Glu-A3b、Glu-B3g、Glu-D3b。另外，分析了稀有亚基对5+12与2.2+12与品质性状的关系，认为5+12对品质有负效应，2.2+12对品质有正效应。在品质育种时，应对优异组合或优异亚基加以利用。 3. 首次利用重组自交系（RILs）为材料，研究麦谷蛋白亚基表达量与品质性状的关系，通过对重组自交系中各HMW-GS表达量的分析，认为，就单个亚基的表达量而言，7亚基最高；其次为2亚基、5亚基、12亚基和10亚基；亚基9和1的表达量最小；N亚基不表达。对成对出现的亚基对而言，x型和y型亚基的总表达量2+12>5+10>7+9>17+18。就单个亚基与品质性状的关系而言，仅有10亚基的表达量与蛋白含量的相关性达5%的显著水平，2亚基的表达量与湿面筋含量呈负相关，显著水平也达5%，其余单个亚基对品质性状均无显著影响；就x型/y型亚基的比例来看，2/12和5/10对湿面筋含量都有显著的负效应；对某一位点等位基因控制的亚基表达总量来看，2+12对SDS沉降值有显著负效应。另外，本研究得出：2＋12的亚基对的负效应主要体现在2亚基上，且在同一位点上，x型亚基的表达量大于y型。所以推导稀有亚基组合2+10很可能也是劣质亚基。 4. 以 Glu-A1、Glu-B1、Glu-D1、Glu-B3和Glu-D3作为5个因素对99G45/京771和Pm97034/京771杂交后代的蛋白质含量和SDS沉降值进行多因素方差分析。结果表明，Glu-A1和Glu-D3对蛋白含量的加性效应达5％显著水平；Glu-D1 * Glu-D3对蛋白质含量的互作效应也达5%显著水平；其余位点的加性和互作效应对蛋白质含量的影响均不显著。对SDS 沉降值而言，Glu-D1的加性效应最大,贡献率为4.2 % ,达1 %显著水平,其次是Glu-B1位点,贡献率为3.3% ,达5%显著水平。其余位点对SDS 沉降值的加性和互作效应均未达5%显著水平。总体而言, 各位点对蛋白含量的效应大小为Glu-D3 > Glu-A1 > Glu-D1>Glu-B1>Glu-B3；对SDS沉降值的效应大小为Glu-D1>Glu-B1> Glu-D3>Glu-A1> Glu-B3。Glu-D1和Glu-D3位点上等位基因变异对蛋白含量有显著或极显著影响，含Glu-D1d和Glu-D3 GD、Glu-D3 JD基因的株系分别比含Glu-D1a和Glu-D3 PD基因的株系有较高的蛋白含量；在该遗传背景下，麦谷蛋白各基因位点对蛋白含量的效应大小依次排列为：Glu-A1位点1>N；Glu-B1位点7+9>17+18>14+15；Glu-D1位点5+10>2+12；Glu-B3位点GB>JB>PB；Glu-D3位点GB>JB>PB。对SDS沉降值的效应大小依次排列为：Glu-A1位点1>N；Glu-B1位点7+9=17+18>14+15；Glu-D1位点5+10>2+12；Glu-B3位点GB>JB>PB；Glu-D3位点GB>JB>PB。所以，对蛋白含量和SDS沉降值均较好的组合为1，7+9，5+10，GB，GD。 5. 因为GB和PB对品质的效应有显著差异，选取LMW-GS位点特异扩增引物对京771、99G45和Pm97034的Glu-B3位点进行扩增，结果得到三个不一样的扩增片段（Genebank号为DQ539657－DQ539659），得到的基因片段与Genebank中已报道的同类序列高度同源。通过克隆片段组成的分析，发现对Pm97034的序列较京771和99G45段少一个7氨基酸的重复单元，这可能是它较另外两个片段对面筋强度影响小的主要原因；另外，在99G45的序列中，124位处出现L（亮氨酸）代替P（脯氨酸），158位处出现了T（苏氨酸）代换M(蛋氨酸)，这可能是99G45Glu-B3位点序列对SDS沉降值的效应显著优于Pm97034的原因。 6．通过对RILs各位点同普通小麦品种（系）各位点与品质关系的比较，发现对SDS沉降值的效应，各位点在不同研究材料中是不同的，普通小麦中：Glu-B3>Glu-A1=Glu-D1=Glu-A3>Glu-D3>Glu-B1，RILs中：Glu-D1>Glu-B1> Glu-D3>Glu-A1> Glu-B3。利用重组自交系材料（完全排除了1BL/1RS易位干扰）所得到的结果与Gupta and MacRitchie (1994)所得结论一致。进一步证实了1BL/1RS易位对小麦品质的重要影响。对蛋白含量而言，普通小麦品种（系）中，Glu-D1>Glu-B3>Glu-A1=Glu-B1> Glu-A3=Glu-D3，RILs中，Glu-D3 > Glu-A1 > Glu-D1>Glu-B1>Glu-B3，和对SDS沉降值的效应一样，推断在非1BL/1RS易位的情况下，各位点对其效应应为Glu-D3 > Glu-A1 > Glu-D1>Glu-B1>Glu-B3。 对同一位点的等位基因而言，普通小麦和重组自交系中Glu-A1和Glu-D1上的等位基因对品质性状的贡献是一致的，但Glu-B1上的等位基因对SDS沉降值的贡献发生了变化，普通小麦中17+18>7+9，RILs中7+9>17+18，这可能也是1BL/1RS造成的。 Baking quality improved is one of the main object of wheat bread in China. The overall objective of the present studies was to increase the understanding about protein quality in wheat, i.e. to make it possible to improve the production of wheat with desired quality for different end-uses. With the analysis of gluten protein in RILs, 99G45/Jing 771 and Pm97034/Jing, and 228 wheat cultivars or lines in China, the correlations between glutenin compositions and protein content, glutenin macropolymer(GMP), wet gluten content, Zeleny sedimentation value and SDS sedimentation value contentand breadmaking quality were studied. Also a rapid and efficient detection method of geneticpolymorphism at Glu-B3 loci in wheat was established using polymerase chain reaction(PCR).The results obtained were as follows: 1. Cultivated Chinese wheat germplasm has been a valuable genetic resource in international plant breeding. Patterns of gliadin among cultivated Chinese accessions are unknown, despite the proven value and potential novelty. The objective of this work was to analyse the diversity within improved Chinese wheat germplasm. The electrophoretic banding patterns of gliadin in common wheat cultivars and advanced lines were determined by acid-polyacrylamide gel electrophoresis. For 148 leading commercial cultivars and promising advanced lines used in our study, 48 patterns were identified, 29 corresponding to ω-gliadin, 9 to γ-gliadin, 5 to β-gliadin and 5 to α-gliadin. The most frequent patterns were A6 in ω; B in γ; B in β and A in the region of α. 116 band types appeared in the148 samples: 94 accessions had unique gliadin types, and 22 gliadin types while not unique were found in 54 accessions. The gliadin patterns of Chinese wheat cultivars and lines greatly differed from the patterns of wheat lines from other countries. Three patterns, E, J, H, M, N and O in the ω-zone had not previously been reported. Three wheat zones，the Northern Winter Wheat Region, the Yellow and Huai Valley River valleys Winter Wheat Region and the Southwestern Winter Wheat Region，in China showed different frequencies in their gliadin patterns. This information can be used to monitor genetic diversity with Chinese wheat germplasm. 2. To analyse the relationship between the loci and characteristics quality, we utilized the 228 cultivars/lines. The results showed that : For protein content, Glu-D1 >Glu-B3>Glu-A1=Glu-B1>Glu-A3=Glu-D3. For GMP content, Glu-A3>Glu-B3 >Glu-D1>Glu-B1>Glu-A1>Glu-D3. For wet gluten content, Glu-B1>Glu-B3= Glu-D3>Glu-A3>Glu-A1>Glu-D1. For Zeleny sedimentation value, Glu-A1>Glu-B3> Glu-D3>Glu-D1>Glu-B1>Glu-A3, For SDS sedimentation value, Glu-B3>Glu-A1= Glu-D1= lu-A3>Glu-D3>Glu-B1。For protein content, the best combination of 6 loci is (1,17+18,5+10,Glu-A3e, Glu-B3g,Glu-D3b). For wet gluten content, the best combination of 6 loci is (1,6+8,5+10,Glu-A3d,Glu-B3c,Glu-D3b). For Zeleny sedimentation value, the best combination of 6 loci is (N,17+18,5+10,Glu-A3d, Glu-B3d, Glu-D3b). For SDS sedimentation value, the best combination of 6 loci is(7+8,2.2+12,Glu-A3b, Glu-B3g,Glu-D3b)。Additional, we analysed the relationship between the subunits 5+12 and 2.2+12, think that 5+12 was negative for quality, 2.2+12 is postive for quality. It should be effective utilized. 3. It’s the first time to utilize RILs to study the relationship between subunits expression quantity and characteristics quality. The results showed that: For single subunit, the expression quantity of 7 is the highest. Then the 2, 5, 12 and 10. The expression of subunit 9 and 1 is the lowest. Subunit N is not expressed. For subunits, the expression quantity of x type and y type are 2+12>5+10>7+9>17+18. The significant relation of 5% only showed between the expression quantity of subunit 10 and protein content. The relationship between expression quantity of others and characteristic quality was not significant. For x type/ytype, 2/12 and 5/10 is negative relation insignificant level. For the subunit(s) in a loci, Only 2+12 effect SDS sedimentation value negative in significant level. 4. With RILs 99G45/Jing 771 and Pm97034/Jing 771, we found that: The effective of Glu-A1, Glu-D3 and Glu-D1 * Glu-D3 for protein content is significant at 5% level. The effect of other loci for protein wre not significant. For SDS sedimentation value, the effect of Glu-D1is the highest, which contribution is 4.2 % .Then the Glu-B1, contribution is 3.3%. The effect of other loci for SDS sedimentationvalue were not significant. In total, for protein content: Glu-D3 > Glu-A1 > Glu-D1>Glu-B1>Glu-B3; for SDS sedimentationvalue: Glu-D1>Glu-B1> Glu-D3>Glu-A1>Glu-B3. The effect of alleles in Glu-D1 and Glu-D3 loci are significant at 1% or 5%. In Glu-A1, 1>N; Glu-B1, 7+9>17+18>14+15; Glu-D, 5+10>2+12; Glu-B3, GB>JB>PB; Glu-D3, GB>JB>PB. For SDS sedimentation, Glu-A1, 1>N; Glu-B1, 7+9=17+18>14+15; Glu-D1, 5+10>2+12; Glu-B3, GB>JB>PB; Glu-D3, GB>JB>PB. The best combinations for SDS sedimentation value is 1，7+9，5+10，GB，GD. 5. Because of the difference of GB and PB for SDS sedimentation value, we selected the specific primer for LMW-GS loci to amplified the Glu-B3 of Jing771, 99G45and Pm97034. We got 3 amplify fragment (Gene Bank accession number are DQ539657－DQ539659）. We found that the fragment of Pm97034 were deleted a repetitive 7 amino acid domain, which is perhaps the reason effect the gluten strength. Furthermore, in the position 124 of sequence 99G45, L has been replaced with P. Position 158, T replaced M, which may be the reason why the Glu-B3 locus of 99G45 is prefer to Pm97034 when refer to SDS sedimentation value. 6. Comparing the results of RILs and common wheat, we found that perhaps just the1BL/1RS made the difference of loci in different accession.

青藏高原栽培青稞遗传多样性研究

青稞，是我国藏区居民对裸大麦的称谓，它不仅是藏民的主要食粮、燃料和牲畜饲料，而且也是啤酒、医药和保健品生产的原料；青稞不仅为藏区人民的健康和经济发展做出了很大的贡献，而且对人类健康和社会经济的可持续发展都有重要的意义。青藏高原是我国及世界上青稞分布和种植面积最大的地区，资源极其丰富。虽然从经典遗传直到分子标记对我国大麦遗传多样性都有研究，但研究手段、数量仍然不够深入，对我国大麦资源遗传多样性研究的信息非常有限，不能很好地满足大麦遗传研究和育种应用的需要，尤其是对西藏栽培大麦的遗传多样性的研究还只是刚刚开始，关于栽培青稞多态性的研究报道很少。本研究采用SSR标记和蛋白质电泳两类技术，从SSR标记位点、单体醇溶蛋白、B组醇溶蛋白和淀粉粒结合蛋白（SGP）等四个方面对我国青藏高原栽培青稞的遗传多样性进行了综合评价。 SSR标记具有基因组分布广泛、数量丰富、多态性高、容易检测、共显性、结果稳定可靠、实验重现性好、操作简单、经济、易于高通量分析等许多优点，被认为是用于遗传多样性、品种鉴定、物种的系统发育、亲缘关系及起源等研究的非常有效的分子标记。本研究采用SSR标记分析了64份青藏高原栽培青稞的遗传多样性，同时评估SSR标记在我国大麦育种和品种鉴定中的应用潜力。选择了30个已知作图位点SSR标记，其中25个标记与重要性状的控制位点连锁紧密。选择的30个SSR标记，5个未得到很好的扩增产物，3个无多态性。22个多态性SSR标记位点中，每位点检测出等位基因2~15个，共检测出等位基因132个，平均每位点6.0 个。各多态位点检测出基因型为2~11种，位点HVM33的基因型最多。各多态位点的多态信息指数为0.16~0.91, 平均为0.65。根据PIC值选择了13个SSR标记用于我国青藏高原栽培青稞基因型鉴定，这些标记的PIC值为0.6以上。结合PIC值和基因型差异，选择了8个多态信息含量高的SSR标记，构建了高效指纹图谱，此图谱能把64份材料完全区分。 贮藏蛋白电泳分析是研究相关编码蛋白基因多态性的非常有效的方法。大麦单体蛋白与小麦醇溶蛋白相对应，具有丰富的多态性，可用于大麦遗传多样性、品种鉴定和群体进化等研究。本研究通过A-PAGE电泳技术研究了84份青藏高原栽培青稞的单体醇溶蛋白多态性。大麦单体醇溶蛋白图谱与小麦醇溶蛋白电泳图谱类似，所分离的蛋白清晰地分为ω－，γ－，β－和α－四个部分。青藏高原栽培青稞单体醇溶蛋白具有丰富的多态性，84份青稞材料中存在43条不同的蛋白带，75种组合带谱；其中67种为单一材料所独有，另8种则分别包含了2-3份材料。每份材料中拥有醇溶蛋白带为6－16条，含有6-10条单体醇溶蛋白带材料较多。西藏和四川材料群体单体醇溶蛋白多态性不同，具有区域特异性。西藏材料中发现了40条不同蛋白带，3条特异带，46 种蛋白组合；四川材料中出现了40种不同蛋白带，26种条带组合, 3条特异带。基于单体蛋白多态性的聚类与材料的来源有一定的相关性。A-PAGE单体蛋白具有丰富的多态性，可作为遗传研究和品种鉴定的标记。 大麦醇溶蛋白（hordein）是大麦籽粒的主要贮藏蛋白，与大麦的营养品质和加工品质密切相关，而且具有丰富的多态性，广泛用于品种鉴定、种质筛选、遗传多样性和亲缘关系研究。B组醇溶蛋白是主要的醇溶蛋白组份，约占总醇溶蛋白的80%，而且具有丰富的多态性。本研究采用SDS-PAGE分析了72份青藏高原栽培青稞B组醇溶蛋白的遗传多样性。青藏高原栽培青稞B组醇溶蛋白具有丰富的多态性，72份青稞材料中存在15种蛋白带，30种组合带谱，其中15种为单一材料所独有，另15种则分别包含了2-10份材料。每份材料中B组醇溶蛋白条带数为4-8条，含5、6条的材料较常见。不同来源的群体材料间B组醇溶蛋白组成存在差异，西藏青稞含有26种蛋白组合带谱，其中有19种特异带谱；四川群体中共发现11种蛋白组合带型，其中有4种特有带谱。两群体中都存在稀有条带。聚类分析将材料分成三组，材料聚类与材料来源地没有明显的相关性。 淀粉粒蛋白（Starch granule proteins, SGPs）是一类与淀粉粒结合的微量蛋白，一些淀粉粒蛋白具有淀粉生化合成中主要的酶蛋白功能，其变异会影响淀粉含量和特性，从而影响淀粉的应用。关于我国大麦淀粉粒组成研究还未见报道。本实验首次开创了我国大麦淀粉粒结合蛋白的研究工作。采用SDS-PAGE电泳技术研究了青藏高原栽培青稞的SGP组成，并分析了不同SGP组合间淀粉含量的差异，初步探索了所分离的SGP蛋白与淀粉合成的关系。66份青稞材料中分离了10种主要的SGP，其表观分子量为40-100KD，低于60KD的SGP带有7条，共有16种组合带谱；各SGP蛋白和组合带谱出现的频率存在差异，青藏高原青稞的SGP组成存在多态性。西藏青稞和四川青稞的SGP组成有很大差异，SGP组成具有地域差异性，西藏青稞含有12种蛋白组合带谱，其中有9种特异带谱；四川群体中共发现7种蛋白组合带型，其中有4种特有带谱；两群体中仅有3种共同的蛋白组合带谱。SGP蛋白特性将66份青稞分为三组, 即Ⅰ、Ⅱ、Ⅲ，材料聚类与材料来源具有一定的相关性。不同组合带谱材料间淀粉含量差异显著性检验结果显示，不同带谱间材料的总淀粉含量、直链淀粉含量和支链淀粉含量有差异，带谱2（SGP1+3+7+9+10）和8（SGP1+2+4+6+8）的总淀粉含量及支链淀粉含量显著大于组合带谱3（SGP1+3+7+10）的总淀粉含量。组合带谱7（SGP1+2+6+8）的直链淀粉含量显著低于带谱11（SGP1+5+8）的直链淀粉。带谱SGP2、3、4、5、6、7、8、9、10可能参与淀粉合成，SGP9可能与高支链淀粉的合成相关。 SSR标记位点、单体醇溶蛋白、B组醇溶蛋白、淀粉结合蛋白等四个方面的研究结果表明青藏高原SSR标记多态性、单体醇溶蛋白多态性、B组醇溶蛋白多态性和SGP多态性都非常丰富，与青藏高原是栽培青稞的多样性分布中心的观点一致。 青藏高原栽培青稞的SSR标记、单体醇溶蛋白、B组醇溶蛋白和SGP多态性表现出很大差异。SSR标记覆盖了整个基因组，多态性非常高。单体蛋白、B组醇溶蛋白、SGP蛋白是育种中非常关注的性状，他们只是代表基因组中的某一区域或位点，多态性相对较低。但单体蛋白多态性很高，84份材料中检测出43条不同蛋白带，75种不同的组合带谱。SSR标记技术和单体蛋白技术都是遗传多样性研究的有力工具，但单体蛋白技术不仅多态性高，而且经济、操作简便，是种质鉴定的理想方法。 对不同标记的多态性材料数据进行聚类，聚类图能为我们提供各材料间的遗传相似信息，为材料选择提供参考。但材料聚类与材料来源的地理区域的相关性表现不一致。SSR聚类和B组醇溶蛋白聚类与材料的来源地无相关性，而单体醇溶蛋白和SGP聚类与材料来源地有一定相关性，即西藏群体和四川群体分别有集中类群，这可能是人为选择的附加效应。 不同来源的群体材料的遗传多样性不同，具有区域特异稀有基因，加强不同地区间资源的交换和配合使用，有利于增加群体遗传多样性和新品种培育。 青藏高原栽培青稞的麦芽浸提性状、淀粉性状、病虫及裸粒等重要农艺性状控制位点存在丰富的变异，遗传基础宽广，可能蕴藏着多种不同的等位基因，是研究重要性状遗传特性、基因资源挖掘和遗传育种的宝贵资源库。 Hulless barley, due to its favorable attributes such as high feed value, good human nutrition，rich dietary fiber and ease processing, attracts people,s attention . Hulless barley plays a very important role in Tibetan life, used as essential food crop, main animal feed and important fuel. In addition to tsampa (roasted barley flour), a main food for Tibetan, hulless barley is also made into cake, soup, porridge, recent naked barley liquor and cornmeal. Qinghai-Tibet Plateau is one of a few areas which plant naked barley widely in the world and also has a long growing history. Genetic diversity of the cultivated hulless barley in this region , however, has not been documented. The study of genetic diversity existing within this population is of particular interest in germplasm identification, preservation, and new cultivar development. This study analyzed the genetic diversity of the cultivated naked barley from Qinghai-Tibet plateau through the study of SSR marker loci and monomeric prolamins, B-horden and starch granule proteins. SSRs are present abundantly in genomes of higher organisms and have become a popular marker system in plant studies. SSRs offer a number of advantages, such as the high level of polymorphisms, locus specificity, co-dominance, reproducibility, ease of use through PCRand random distribution throughout the genome. In barley, several hundred SSRs have been developed and genetically mapped and can therefore be selected from specific genomic regions. The genetic diversity of 64 cultivated naked barley from Tibet and Sichuan was studied with 30 SSRs of known map location.Among the selected SSR markers, PCR products of 5 SSR markers were not obtained and 3 SSR marker loci were monomeric. A total of 132 alleles were identified at 22 polyomeric SSR loci. The number of alleles per locus ranged from 2 to 15, with an average of 6.0. The polymorphism information content values for the SSRs ranged from 0.08 to 0.94, with an average of 0.65. 13 SSR markers with the PIC value >0.6 have been selected for discrimination of Qinghai-Tibet naked barley genotypews. A finger Print map was developed through 7 SSR markers with the high PIC value. It could be used as an efficient tool for gene discovery and identification of gernplasm. Hordeins, the main storage proteins of the barley seed, are composed of momomeric and polymeric prolamins and divided into -A, B, C and D groups in order of decreasing electrophoretic mobility. Hordeins show high inter-genotypic variation and have been extensively used as markers for cultivar identification and analyzing the genetic diversity. This study analyzed the genetic diversity of B-hordein in 72 naked barley from Qinqhai-Tibet Plateau. Extensive diversity was observed. A total of 15 different bands and 30 distinct patterns were found. Jaccard's coefficient of similarity was calculated, and the accessions were divided into three　main groups by cluster analysis using UPGMA. Differentiation among the populations from different collecting regions based on the polymorphism of B-hordein was investigated. Monomeric prolamins show high inter-genotypic variation and have been used as molecular markers for cultivar identification, analyzing the genetic diversity in collections and investigating the evolution processes and structure of populations However, the cultivated hulless accessions from Qinghai-Tibet Pateau in China have never been examined with respect to monomeric prolamins. This study analyzed the genetic diversity of monomeric prolamins (protein fraction corresponding to wheat gliadins) using the Acid -PAGE technique in eighty-four cultivated hulless barley from Qinqhai-Tibet Plateau in China. Extensive diversity was observed. A total of 43 different bands were found, of which 21 different bands were in the region of ω group, 8 in the region of γ, 8 in the region of β, and 6 in the region of α group. Among the 86 accessions, 75 distinct patterns were identified. The number of bands ranged from 6 to 16, depending on the variety. Jaccard’s coefficient of similarity was calculated, and the lines were grouped by cluster analysis using UPGMA. A dendrogram was obtained from the analysis of the groups and five main clusters were identified. No relationship between the distribution in the dendrogram and growth habits and origins of the cultivars could be detected. Starch is the major constituent of the cereal endosperm, comprising approximately 65% of the dry weight of the mature wheat grain. The starch formed in all organs of plants is packaged into starch granules, which vary widely between species and cultivars in size and shape. Wheat endosperm starch granules contain about corresponding to the main biosynthase of starch. This report firstly dealed with intraspecific variation of the major SGPs in cultivated naked barley from Qinghai-Tibet plateau. A total of 10 major SGPs were observed in the range of 40KD-100KD and 16 types of patterns were found. Based on the variation of SGPs, accessions studied were classified into 3 groups. A geographical cline of electrophoregram was observed. In addition, significance test of the difference of starch content among groups and types of patterns were done, and the results indicated those SGPs could be related to the content of starch. Diagram obtained through cluster analysis exhibited a structuration of diversity and genetic relationship among cultivated hulless accessions. In breeding program, parents with genetically distant relationship for hybridization will increase genetic diversity of progenies. In conclusion, cultivated naked barley from Qinghai-Tibet Plateau in China presents a high variability with respect to monomeric prolamins，SSR markers , B- hordeins and SGPs. The result of this study supports Qinghai-Tibet Plateau is the center of cultivated hulless barley and the cultivated naked barley is considered to be a gene pool with large diversity and could be applied to breeding for cereal.

同源四倍体与二倍体水稻的遗传差异及产量、品质性状研究

本文以中国科学院成都生物研究所培育的同源四倍体水稻和二倍体水稻为材料，进行遗传差异及产量、品质性状的研究：1、以二倍体水稻为对照，研究了同源四倍体水稻在2004 年和2005 年的结实情况。结果同源四倍体的花粉育性、结实率均不同程度下降，尤其低代材料更是大幅度下降。F 检验表明，同源四倍体不同个体间的各产量性状均差异显著，说明其具有很大的遗传改良潜力。从1996 年到2005 年对部分同源四倍体水稻进行了连续选择改良，T 检验表明经过9 年的选择改良，其结实率显著提高。本文还对同源四倍体水稻各产量性状间的相关性进行了分析,结果结实率与花粉育性、穗着粒数、穗实粒数极显著相关；理论产量与花粉育性、有效穗数、穗着粒数、穗实粒数、结实率及千粒重极显著相关。i2、用(CT)n 微卫星标记和PCR-Acc Ι分子标记对40 份同源四倍体和14 份二倍体水稻Wx 基因进行研究。结果，(CT)n 微卫星标记检测，Wx 基因呈Wx1、Wx2 和Wx3 3 种多态性；PCR-Acc Ι 检测，Wx 基因表现为G-型和T-型。测定稻米直链淀粉含量(AC)、胶稠度(GC)和糊化温度(GT)，并探讨其与Wx 基因的关系，结果，二倍体和同源四倍体水稻均存在：Wx 基因型相同，AC 差异较小，Wx 基因型不同时，AC 差异较大，Wx1 基因型品种AC 最高， Wx2 基因型品种AC 次之，Wx3 基因型品种AC 最低；基因型相同时，同源四倍体AC 低于二倍体；同源四倍体与对应二倍体间，Wx 基因型相同时，AC 差异很小；而Wx 基因型发生变异时，AC 差异很大。同时，进行相关性分析，结果二倍体和四倍体水稻均存在AC、GC 与Wx 基因密切相关；而GT 与Wx 基因相关不显著。综合分析，(CT)n微卫星标记与PCR-Acc Ι 分子标记检测的相关系数为0.842，呈极显著正相关，可以将其结合起来进行同源四倍体新品种的选育和改良。3、利用RAPD 技术，对同源四倍体和二倍体水稻的遗传差异进行分析。17条引物在同源四倍体中扩增出178 条带（PPB＝81.5%），在二倍体中扩增出173条带（PPB＝76.3%）；同源四倍体和二倍体的Shannon 遗传多样性指数分别为0.4848 和0.4679，多态信息量分别为0.3301 和0.3216，遗传距离分别为0.3572和0.3460；同源四倍体与其对应二倍体间遗传距离为0.1914-0.4633，平均为0.2914。表明同源四倍体的遗传多样性高于二倍体，且同源四倍体与其二倍体之间存在较大的遗传差异，这些将为水稻品种改良和新品种选育提供科学依据。上述产量、品质性状及遗传差异分析的结果，不仅有利于加快同源四倍体水稻的遗传改良进程，而且为进一步研究、利用同源四倍体水稻奠定了初步基础。 AbstractStudy on genetic diversity, yield characters and quality traitsof autotetraploid and diploid riceLiu Yuhua (Botany)Directed by Associate Prof. Tu ShengbinIn this study, diploid and autotetraploid rice, which were cultivated in ChengduInstitute of Biology were analyzed in genetic diversity, yield characters and qualitytraits.In the study, 2 diploid and 29 autotetraploid rice(2n=4x=48) materials, including4 preliminary and 25 advanced generation, were investigated for yield characters.Compared with diploid check, the pollen fertility and seed set of autotetraploiddeclined dramatically, especially in preliminary generation. F-test indicated that therewere remarkable differences among different varieties, showing that autotetraploidmaterials had strong potential for improvement. From 1996 to 2005, someautotetraploid rice had been selected and improved. T-test showed that seed setincreased obviously. The relationships among yield characters of autotetraploid ricewere analyzed. Seed set was strongly correlated with pollen fertility, total grainnumber per panicle and productive grain number per panicle; theoretical yield wasstrongly correlated with pollen fertility, productive panicle number per plant, totalgrain number per panicle, productive grain number, seed set and 1000-grain weight.Wx genotypes of 40 autotetraploid rice and 14 diploid rice were tested by usingthe (CT)n microsatellite marker and a cleaved amplified polymorphic sequence(CAPS) molecular marker named PCR-Acc Ι. Three microsatellite alleles wereproduced, i.e. Wx1, Wx2 and Wx3 both in autotetraploid and in diploid rice.Comparatively, PCR- Acc Ι molecular marker produced two genotypes, G-type andT-type for both autotetraploid and diploid rice. In this study, amylose content (AC), gel of consistency (GC) and gelatinization temperature (GT) of rice grain weremeasured and their relationships with Wx alleles were analyzed. The results showedthat variation of AC between autotetraploid and diploid rice was small when they hadthe same Wx genotype. However, variation of AC turned to be large when the Wxgenotypes were different. Actually, AC met the maximum value in Wx1 varieties andWx2 varieties the middle and Wx3 varieties the minimum. And AC was lower inautotetraploid than in diploid. Correlation analysis was done in this experiment. ACand GC of rice grain were probably controlled by Wx gene or other gene whoselocation was strictly linked to Wx gene, while GT of rice was not. The correlationcoefficient between Wx genotypes which revealed by (CT)n microsatellite marker andPCR-Acc Ι molecular marker was 0.842 with significant level. That revealed aconsistent result between the two types of markers. So it was possible to utilize boththe two types of markers to select and promote germplasm of autotetraploid rice.RAPD molecular markers were used to analyze the genetic diversity betweendiploid and autotetraploid rice. 178 repeatable bands were detected through 17 RAPDprimers with percentage of polymorphic bands was 81.5% in autotetraploid rice while173 repeatable bands were detected with percentage of polymorphic bands was 76.3%in diploid rice. According to the measurement of Shannon index, polymorphicinformation content and genetic distance, genetic diversity of autotetraploid was on ahigher level, genetic variation between autotetraploid and diploid rice was relativelyhigh. All these contributed to the genetic selection and improvement in rice breeding.As mentioned above, the results are not only helpful to promote the process ofrice improvement, but also to confirm the basic for further study of autotetraploid rice.

在盐胁迫下外源维生素浸种对小麦发芽及幼苗生长的影响

维生素(Vitamin)又称维他命，为“万年青”产品，是维持人体生命健康必需的一类低分子有机化合物质。维生素对人体健康的作用人们研究很多, 维生素可以增强人体对感染的抵抗力，降低出生缺陷及降低癌症和心脏病发病率等，一旦缺乏，肌体代谢就会失去平衡，免疫力下降，各种疾病，病毒就会趁虚而入；而维生素对作物影响的研究却很少。目前为止，尚无对用维生素浸种的方法来研究外源维生素是否对小麦种子萌发及幼苗生长起调节作用的报道，且对其在小麦抗逆性方面影响的研究甚少，对盐的胁迫抗性研究尚未有人报道。小麦（Triticum aestivum L.）属于拒盐的淡土性作物。盐害不利于小麦生长，严重影响小麦的产量和品质。本研究采用4 种不同维生素VB1、VC、VB6、VPP，分别对供试小麦品种川育12（红皮）、川育16（白皮）小麦浸种后，在一般自然条件下和逆境（盐胁迫条件）下，进行试验。探讨在正常情况下与在不同盐浓度条件下，各维生素及盐浓度对小麦发芽及幼苗生长的影响，并且比较两种不同皮色的小麦在相同盐胁迫条件下的差异表现，同时研究维生素处理的特异性，且哪种维生素对盐害缓解作用最佳。研究结果表明：在无盐胁迫（自然）条件下，对用4 种不同维生素VB1、VC、VB6、VPP 浸种小麦川育12、川育16 后的种子萌发及幼苗生长（幼苗的根长、根重、苗高、苗鲜重）的研究结果表明：4 种外源维生素浸种均对小麦发芽有调节作用，都能提高其最终发芽率。但是提高幅度有所差异。用VB6 浸种后的小麦提高幅度最多，VC 次之，VPP 提高幅度最小。同时，4 种外源维生素浸种对小麦种子的出芽速度及芽后长势也有一定的影响。VB6、VC 处理的小麦种子出芽速度最快，萌发后长势最好；VB1 出芽速度相对较慢，VPP 最慢,但都大于对照；VB1 处理长势略高于对照，VPP 处理的小麦长势则低于对照。从整体来看，VB6、VC处理促进效应明显， VB1 次之，而VPP 在某些方面无效甚至产生负效应。此外，相同的维生素处理对不同的品种的种子萌发、生长效果也存在差异，各种维生素作用于川育12 的效应均强于对川育16。进一步对幼苗根系TTC 还原力及幼苗叶片中硝酸还原酶活性进行测定、分析。研究发现：并非所有种类的维生素对幼苗根系TTC 还原力及幼苗叶片中硝酸还原酶活性的提高都有帮助。幼苗根系TTC 还原力在不同维生素处理下存在显著差异，而与小麦品种关系甚微。经VB6、VC 处理后，根系TTC 还原力测定值均显著高于对照，VB1 不明显，VPP 则略低于对照。VB6、VC 处理的幼苗叶片中硝酸还原酶的含量大于对照，VB1 与对照相差无几，而VPP 处理的川育12 幼苗叶片中的硝酸还原酶活性比对照CK 略高，而在川育16 中则略比对照CK 有所下降，呈现出抑制效应。综上结果表明：VB6、VC 具有促进种子发芽，幼苗生长及根系生长的作用，是较好的促生长剂；VPP 具有抑制作用，是较好的抑制剂，可进一步研究、开发利用。在盐胁迫条件下，对用4 种不同维生素VB1、VC、VB6、VPP 浸种川育12、川育16 后的种子萌发及幼苗生长（幼苗的根长、根重、苗高、苗鲜重）的研究结果表明：在不同盐浓度胁迫条件下， 各处理的种子萌发及幼苗生长均受到不同程度的抑制。随着盐浓度的增加, 发芽率、发芽指数和活力指数成下降趋势；幼苗的根长、根重、苗高、苗鲜重不断降低。4 种维生素处理间也表现出较大差异。VB6、VC 在每个处理中均保持对盐害的缓解作用，VB6 较VC 更易于促进发芽及幼苗生长。最终发芽率高，根系多、长、重，苗高高、重。而VB1、VPP 则表现出抑制作用。在高盐浓度150mM 时，4 种维生素浸种后的种子，其最终发芽率均不能达到40％，但VB6、VC 处理最终发芽率、苗重、根重均高于对照，VPP 最终发芽率、苗重、根重均低于对照。进一步对幼苗根系TTC 还原力及幼苗叶片中脯氨酸含量进行测定、分析。研究发现：不同盐浓度，不同维生素处理、不同品种间存在差异。随着盐浓度的增加（75mM，100mM，150mM），幼苗根系TTC 还原力活性成下降趋势，幼苗叶片中脯氨酸的积累量成上升趋势。VB6 处理脯氨酸含量增加最为明显，VC 次之，VPP 与对照接近，其变化幅度最小。经VB6、VC 处理后的幼苗根系还原强度，在不同盐浓度下，测定值均显著高于对照，VB1 不明显，VPP 则低于对照，产生负效应。此外，品种间表现不尽相同，相同的维生素处理，相同的盐浓度对不同的品种的种子萌发、生长效果也存在差异， 4 种维生素对川育16 的作用均强于川育12，但其影响趋势是一致的。说明VB6、VC 具有耐（抗）盐性，可以促进种子发芽和幼苗生长，是较好的耐（抗）盐拌种剂。 Vitamin is one kind of necessary low molecular compound for humans tosustain health and life. Lots of Studies have been done on the effectc of the vitaminsfor people. Vitamin can help people improve the body's natural resistance to disease,Drop the rate of birth defects、cacers and the incidence of the heart diseases. Ifpeople have less of them, the metabolism of the organism may throw off balance,immunity may drop off, and catch disease; Though the effects for Vitamin to thecrops are limited. up to now, there’s no one use soking seeds of wheats with vitaminsas a method, to study on how the effects will happen on the wheat seed germinationand seedling growth, and there are only few reserches on antireversion force forwheats ,none for the antireversion force in Sault stress condition.Wheat（Triticum aestivum L.）is sensitive to the salt, so the salt damage will doharm to wheat’s growth, it will have an unfavorable impact on the output and thequality of wheat.On this reaserch, we Soaking CHY12(red)、CHY16 (white) wheat seeds withVitamin C, B1, PP, B6 (50mg/L) as a pretreatment first. Then under two condition: one is in the normal environment the other is in different Salinity, we begin ourexperiments. Then disscuss on if the vitamin and salinity affect the wheat seedgermination and seedling growth, and what is the different between the two of them,the result shows that:Under the normal condition, after soaking seeds with VB1、VC、VB6、Vpp，we study on the their seed germination and the seeding growth(the root length andweights, The seedling heights and weights), it shows that all of those four kinds ofvitamin can adjust the seed germination, but different in The growth rate. VB6 isbest for increase, VC comes second,VPP is the worst. Meanwhile, those four vitaminalso have effect on the speed of the sprouting of the wheat. VB6、Vc can faster theseed germination most, and the seedlings are all doing well; VB1 do little effects onthe budding, Vpp is the worst, but all treatments are better than CK; but in Vi, VB1some what above the CK, while VPP lower than that. On the whole, the acceleratingeffect of VB6、VC are obvious, VB1 takes second place, but VPP in some aspects arenoneffective even have negative effect. Furthermore, different kind of seeds with thesame vitamin may different in seed germination and seedling growth, four vitaminson CHY16 is better than CHY12.More studies on TTC reductive capacity of roots and the activity of nitratereductase in the leaves, the reasult shows not all the vitamin can help the seedlings toimprove the TTC reductive capacity and the activity of nitrate reductase. TTCreductive capacity in different treatments shows significant differences,but notcorrelate to the variety of the wheat. The TTC reductive capacity of VB6、Vctreatments are all higher than CK, VB1 is nearly the same as CK, VPP is a littlelower than CK. Through the study of acivity of nitrate reductase, it shows that,VB6、VC are higher than CK ,VB1 is nearly the same as CK also, VPP is a little higher inthe CK of CHY12 but lower in CHY16. Through all the results above: VB6、Vc helpthe wheat seed germination, seedling growth and the growth of roots, is theperfectable factor of stimulating the growth; Vpp is a inhibition, that’ll be furtherreserch,and well develop and utilize in the future.Under the different Salinity condition, after soaking seeds with VB1、VC、VB6、Vpp，we study on the their seed germination and the seeding growth(the root lengthand weights, The seedling heights and weights), it shows that: under differentsalinity, the seed germination and the seedling growth of any treatment are inhibited.With the increase of the concentration, the germination rate, Vi、Gi all had fallen; theroot length and weight, the seedling heights and weights steadily sank down. There are also have pronounced difference between all treatments with four differentvitamins.VB6、VC in all treatments are alleviative the salt damage, VB6 is easier tocause to put forth buds than VC, and it’s quantitative value is the highest in theultimate germination rate, in root and seedlings’ hight and weight. Though the VPP、VB1 are seems to inhibite its growth. Under the high concentration150mM Nacl, theultimate germination rate in all treatments are below the 40%, but VB6、VC’squantitative values in any experiments are higher than CK,while VPP lower thanCK.Then we study on the TTC reductive capacity of roots and the content of Polinein leaves, the result shows that between the different salinity, different vitamintreatments, different varieties of the wheat have discrepancy.along with theincreasing concentraion of the salinity（75mM，100mM，150mM），TTC reductivecapacity of roots decreases, the accumulation of the content of Poline in leaves havean upward trend. The increase of VB6’s treatment are obviously, VC comessecond,VPP is nearly come up with CK, changes a little. In TTC reductive capacity of roots’s reserch, VB6、VC are higher than CK at any time,VB1 is not palpable,VPP is lower than CK, makes negative affect on wheat. In addition, varieties of thewheats are remain different, no matter it shows promoting or inhibiting, all fourvitamins have moreobvious effects on CHY16 than CHY12, but the tendency of theeffection are the same. It is say that VB6、VC can help wheat to standwith the saultwell, and promot in growth，they are the better reagent to mix with the seed.

小麦高分子量谷蛋白新亚基基因1Dx5’的克隆与分子标记

小麦是世界第一大粮食作物, 而HMW- GS 是直接影响小麦品质的重要因子。我国小麦面粉的烘烤品质普遍较差, 这与我国品种缺少优质的HMW- GS 有关，因此创造与发掘新的优质谷蛋白亚基编码基因，并开展相关生化、农学、分子生物学等方面研究、探讨优质的分子机理，对于培育优质小麦新品种具有重要意义。W958是我们培育的种间远缘杂交（T.durum Desf. ×T.aestivum L）优良品系，该品系在1D染色体上具有父母本没有的新型亚基，由于此亚基在SDS- PAGE电泳中具有和1Dx5亚基一样的电泳迁移率, 因此我们将该亚基命名为1Dx5’亚基。为了进一步从分子水平确证该亚基为新亚基和在育种中利用该亚基，本研究对该亚基的遗传规律、基因分子结构、品质特性和农艺性状等进行了分析。结果表明1Dx5’亚基在品质上与1Dx5亚基一样优质，对于品质的贡献大于1Dx2亚基。1Dx5’亚基具有特异的遗传规律，在分离群体中，此亚基占有极大的比例，该特性十分有利于将其导入高产小麦遗传背景中。此外，本研究扩增出了1Dx5’亚基基因的启动子区域、N－端区域和部分中间重复区域，并比较了1Dx5’和传统的1Dx5、1Dx2亚基在此区域氨基酸序列。结果进一步证明了1Dx5’是一个新的基因。通过蛋白质结构模拟分析，认为1Dx5’亚基的优良特性可能是由于1Dx5’亚基的的中部重复区域能形成分子间较强的氢键，加大了分子间的相互作用，使1Dx5’亚基的面团具有优良的品质，这为1Dx5’亚基的应用提供了理论基础。同时，本研究还设计用于区分1Dx5’和1Dx5等位基因的分子标记，解决了利用SDS-PAGE生化标记难以将二者区分的问题。Wheat is one of the major crops utilized worldwide. Nevertheless, due to the lackof the superior HMW- GS, the wheat quality in China is not satisfying. Therefore,identification and characterization of the superior HMW- GS will lay good foundation to the wheat breeding.W958 is a new bread wheat line developed by interspecific cross (T.durum Desf.×T.aestivum L). It contains a novel HMW- GS. We have designated such subunit as1Dx5’ here for its unique character. To confirm that such subunit is innovative andbeneficial for wheat breeding program on the molecular level, we have investigated itin terms of inheritance, DNA and protein sequence, dough property and agronomictrait associated with it. The result shows that 1Dx5’is as superior as 1Dx5 in terms of dough property.In addition, we have cloned the promoter, N- terminal as well as partial centralrepetitive domain of the allele coding for this subunit. Comparison of the amino acidsequence of 1Dx5’ with that of 1Dx5 and 1Dx2 showed that the superior quality of1Dx5’ subunit may result from the degree of conservation of the repetitive sequencesby which the glutenin polymers interact via inter-chain hydrogen bonds formedbetween the subunit repetitive domains with longer subunits forming more stableinteractions. In addition, we have developed two dominant molecular markers tofacilitate the discrimination of 1Dx5’ and 1Dx5 which could no be achieved by SDS-PAGE.

小麦抗禾谷孢囊线虫（ Heterodera avenae）相关基因的研究

禾谷孢囊线虫严重影响禾谷类作物的产量，在小麦中由禾谷孢囊线虫引起的产量损失可达30-100%。尤其在澳大利亚、欧洲、印度和中东危害严重，目前禾谷孢囊线虫已成为危害我国作物的主要病源。控制禾谷孢囊线虫的方法主要有：作物轮作、杀线虫剂、寄主抗性等等，其中基因工程方法培育抗线虫小麦品种被认为是最经济有效的方法。分离抗禾谷类孢囊线虫基因对揭示抗性基因结构与功能及其表达调控具有重要意义。 尽管小麦是重要的粮食作物，在小麦中已发现的抗禾谷孢囊线虫的基因很少，而比其近缘属如节节麦、易变山羊草、偏凸山羊草中含有丰富的抗源。目前已鉴定出禾谷孢囊线虫抗性位点Cre，并发现了9个禾谷孢囊线虫抗性基因(Cre1,2, 3, 4, 5, 6, 7, 8, and R) ，其中只有Cre1和Cre8直接从普通小麦中获得。从节节麦中获得的Cre3基因能最有效的控制线虫数量，其次是Cre1和Cre8。这些基因的克隆对于了解禾谷孢囊线虫抗性机制及进一步的育种应用都是非常关键的。然而，目前为止仅有Cre3基因通过图位克隆的方法从节节麦中被分离得到。该基因已被克隆得到的多数线虫抗性基因一样均属于核苷酸结合位点区（NBS）－亮氨酸重复序列区（LRR）基因家族。目前，已有很多抗性基因被分离，这些已知的NBS-LRR类抗性基因的保守序列为应用PCR的方法克隆新的抗性基因提供了可能。 因此本课题的目的是采用保守区同源克隆、3′RACE 和5′RACE 等方法从抗禾谷孢囊线虫小麦-易变山羊草小片段易位系E10 中克隆小麦抗禾谷孢囊线虫基因全序列，进而通过半定量PCR 和荧光定量PCR 研究该基因的表达模式。同时通过mRNA 差别显示技术和任意引物PCR（RAP-PCR）技术分离克隆植物禾谷孢囊线虫抗性基因及其相关基因，为阐明植物抗病性分子机制以及改良作物抗病性和作物育种提供基础，为通过分子标记辅助育种和基因工程方法实现高效、定向转移抗病基因到优良小麦品种奠定了重要的理论和物质基础。主要研究结果： 1． 本实验根据此前从抗禾谷孢囊线虫材料E-10 扩增得到的与来自节节麦的抗禾谷孢囊线虫Cre3 基因及其他的NBS-LRR 类抗性基因的NBS 和LRR 保守区序列设计了两对特异性引物，从E10 中扩增到532bp 和1175bp 的两个目标条带，它们有一个32bp 的共同序列，连接构成总长为1675bp 的NBS-LRR 编码区（命名为RCCN）。根据RCCN设计引物，利用NBS-LRR区序列设计引物，通过5′RACE 和3′RACE 技术采用3′-Full RACE Core Set(TaKaRa)和5'-Full RACE Kit (TaKaRa)试剂盒，反转录后通过嵌套引物GSP1 和GSP2 分别进行两轮基因特异性扩增，分别将NBS_LRR 区向5′端和3′端延伸了1173bp 和449bp，并包含了起始密码子和终止密码子。根据拼接的得到的序列重新设计引物扩增进行全基因扩增的结果与上面获得的一致。拼接后得到全长2775 bp 的基因序列（记作CreZ, GenBank 号：EU327996）。CreZ 基因包括完整的开放阅读框，全长2775 bp，编码924个氨基酸。序列分析表明它与已知的禾谷孢囊线虫抗性基因Cre3的一致性很高，并且它与已经报到的NBS-LRR 类疾病抗性基因有着相同的保守结构域。推测CreZ基因可能是一个新的NBS-LRR 类禾谷孢囊线虫抗性基因，该基因的获得为通过基因工程途径培育抗禾谷孢囊线虫小麦新品种奠定了基础，并为抗禾谷孢囊线虫基因的调控表达研究提供了参考。 2． 通过半定量PCR和SYBR Green荧光定量PCR技术对CreZ基因的相对表达模式进行了研究。以α-tubulin 2作为参照，采用半定量PCR 分析CreZ 基因在不同接种时期1d, 5d, 10, 15d 的E-10的根和叶的的表达情况。在内参扩增一致的条件下，CreZ 在E-10的根部随着侵染时间的增加表达量有明显的增加，在没有侵染的E-10的根部其表达量没有明显变化，而在叶中没有检测表达，说明该基因只在抗性材料的根部表达。SYBR Green定量PCR分析接种前后E10根部基因CreZ基因的表达水平为检测CreZ基因的表达建立了一套灵敏、可靠的SYBRGreen I 荧光定量PCR 检测方法。接种禾谷孢囊线虫后E10根内CreZ基因的相对表达水平显著高于接种前。随接种时间的延长持续增加，最终CreZ基因的相对表达量达到未接种的对照植株的10.95倍。小麦禾谷孢囊线虫抗性基因CreZ的表达量与胁迫呈正相关，表明其与小麦的的禾谷孢囊线虫抗性密切相关，推测CreZ基因可能是一个新的禾谷孢囊线虫候选抗性基因。 3． 针对小麦基因组庞大、重复序列较多，禾谷孢囊线虫抗性基因及其相关基因的片断难以有效克隆的问题，通过mRNA 差别显示技术及RAP-PCR 技术分离克隆植物禾谷孢囊线虫抗性及其相关基因。试验最终得到154 条差异表达条带，将回收得到的差异条带的二次PCR 扩增产物经纯化后点到带正电的尼龙膜上，进行反向Northern 杂交筛选，最终筛选得到102 个阳性差异点。将其中81 个进行测序，并将序列提交到Genbank 中的dbEST 数据库，分别获得登录号(FE192210 -FE192265，FE193048- FE193074 )。序列比对分析发现，其中26 个序列与已知功能的基因序列同源；有28 条EST 序列在已有核酸数据库中未找到同源已知基因和EST，属新的ESTs 序列；另外27 个EST 序列与已知核酸数据库中的ESTs 具有一定相似性，但功能未知。其所得ESTs 序列补充了Genbank ESTs 数据库，为今后进一步开展抗禾谷类孢囊线虫基因研究工作打下了基础。结合本试验功能基因的相关信息，对小麦接种禾谷孢囊线虫后产生的抗性机制进行了探讨。接种禾谷孢囊线虫后植物在mRNA 水平上的应答是相当复杂的，同时植物的抗病机制是一个复杂的过程，涉及到多个代谢途径的相互作用。 The cereal cyst nematode (CCN), Heterodera avenae Woll, causes severe yieldreductions in cereal crops. The losses caused by CCN can be up to 30-100% in somewheat fields. At present, cereal cyst nematode has become the major disease sourcein China and it also damaged heavily in Australia, Europe, India and Middle East.The damage caused by CCN can be mitigated through several methods, includingcrop rotation, nematicide application, cultural practice, host resistance, and others.Of these methods, incorporating resistance genes into wheat cultivars and breedingresistant lines is considered to be the most cost-effective control measure forreducing nematode populations. Although wheat is an economically important crop around the world, far fewergenes resistant to CCN were found in wheat than were detected in its relatives, suchas Aegilops taucchi, Aegilops variabilis and Aegilops ventricosa. Cloning these genesis essential for understanding the mechanism of this resistance and for furtherapplication in breeding. Because of the huge genome and high repeat sequencescontent, the efficient methods to clone genes from cereal crops, are still lacking. A resistance locus, Cre, has been identified and 9 genes resistant to CCN (designatedCre1, 2, 3, 4, 5, 6, 7, 8, and R) have been described, in which Cre1 and Cre8 werederived directly from common wheat. The Cre3 locus, which was derived from Ae.tauschii, has the greatest impact on reducing the number of female cysts, followed byCre1 and Cre8. Cloning these genes is essential for understanding the mechanism ofthis resistance and for further application in breeding. However, to this point, only Cre3, a NBS-LRR disease resistance gene, has been obtained through mappingcloning in Ae. tauschii. The majority of nematode resistance genes cloned so far belong to a super familywhich contains highly conserved nucleotide-binding sites (NBS) and leucine-richrepeat (LRR) domains. To date, many NBS-LRR resistance genes have been isolated.The conserved sequences of these recognized NBS-LRR resistance genes provide thepossibility to isolate novel resistance genes using a PCR-based strategy. The aim of the present study was to clone the resistance gene of CCN fromWheat/Aegilops variabilis small fragment chromosome translocation line E10 whichis resistant to CCN and investigate the espression profiles of this gene withsemi-quantitative PCR and real-time PCR. Another purpose of this study is cloningthe relational resistance gene for CCN by mRNA differential display PCR andRAP-PCR. These works will offer a foundation for disease defence of crop andbreeding and directional transferring resistance gene into wheat with geneengineering. Primary results as following: 1．According to the conversed motif of NBS and LRR region of cereal cystnematode resistance gene Cre3 from wild wheat (Triticum tauschlii) and the knownNBS-LRR group resistance genes, we designed two pairs of specific primers for NBSand LRR region respectively. One band of approximately 530bp was amplified usingthe specific primers for conversed NBS region and one band of approximately 1175bpwas amplified with the specific primers for conversed LRR region. After sequencing,we found that these two sequences included 32bp common nucleotide having 1675bpin total, which was registered as RCCN in the Genbank. Based on the conservedregions of known resistance genes, a NBS-LRR type CCN resistance gene analog wasisolated from the CCN resistant line E-10 of the wheat near isogenic lines (NILs), by5′RACE and 3′ RACE.designated as CreZ (GenBank accession number: EU327996) .It contained a comlete ORF of 2775 bp and encoded 924 amino acids. Sequencecomparison indicated that it shared 92% nucleotide and 87% amino acid identitieswith those of the known CCN-resistance gene Cre3 and it had the same characteristic of the conserved motifs as other established NBS-LRR disease resistance genes. 2． Usingα-tubulin 2 as exoteric reference, semi-quantitative PCR and real-timePCR analysis were conducted. The expression profiling of CreZ indicated that it wasspecifically expressed in the roots of resistant plants and its relative expression levelincreased sharply when the plants were inoculated with cereal cyst nematodes. therelative expression level of the 15days-infected E10 is the 10.95 times as that ofuninfected E10,ultimately. It was inferred that the CreZ gene be a novel potentialresistance gene to CCN. 3．We cloned the relational resistance gene for CCN by mRNA differentialdisplay PCR and arbitrarily primed PCR fingerprinting of RNA from wheat whichpossess huge and high repeat sequence content genomes. Total 154 differentialexpression bands were separated and second amplified by PCR. The products werenylon membrane. The 102 positive clones were filtrated by reverse northern dot blotand 81 of those were sent to sequence. The EST sequences were submitted toGenbank (Genbank accession: FE192210 - FE192265, FE193048 - FE193074). Thesequences alignment analysis indicated 26 of them were identical with known genes;28 were not found identical sequence in nucleic acid database; another 27 ests wereidentical with some known ests, but their functions were not clear. These ESTsenriched Genbank ESTs database and offered foundation for further research ofresistance gene of CCN.

簇毛麦（ Dasypyrum villosum）矮秆突变体的研究

株高是农作物的重要农艺性状之一，适度矮化有利于农作物的耐肥、抗倒、高产等。20世纪50年代，以日本的赤小麦为矮源的半矮秆小麦的培育和推广，使得世界粮食产量显著增长，被誉为“绿色革命”。迄今为止，已报到的麦类矮秆、半矮秆基因已达70多个，但由于某些矮源极度矮化或者矮化的同时伴随不利的农艺性状，使得真正运用于育种实践的矮源较少。因此，发掘和鉴定新的控制麦类作物株高的基因，开展株高基因定位、克隆及作用机理等方面的研究，对实现麦类作物株高的定向改良，具有重要的理论意义和应用价值。簇毛麦(Dasypyrum villosum，2n＝14，VV)是禾本科簇毛麦属一年生二倍体异花授粉植物，为栽培小麦的近缘属。本课题组在不同来源的簇毛麦杂交后代中发现了一株自然突变产生的矮秆突变体。观察分析了该突变体的生物学特性，对矮秆性状进行了遗传分析，对茎节细胞长度、花粉的活力进行了细胞学观察，考察了该突变体内源赤霉素含量及不同浓度外施赤霉素对突变体的作用，分析了赤霉素生物合成途径中的内根贝壳杉烯氧化酶（KO）和赤霉素20氧化酶(GA20ox)的转录水平，对赤霉素20氧化酶和赤霉素3-β羟化酶(GA3ox)进行了克隆和序列分析，并对GA20ox进行了原核表达和表达的组织特异性研究。主要研究结果如下：1. 该突变体与对照植株在苗期无差异，在拔节后期才表现出植株矮小，相对对照植株，节间伸长明显受到抑制，叶鞘长度基本不变。在成熟期，对照植株的平均株高为110cm，而突变株的平均株高为32cm，仅为对照植株的1/3 左右。除了株高变矮以外，在成熟后期，突变株还表现一定程度的早衰和雄性不育。I2-KI染色法观察花粉活力结果表明，对照植株花粉90%以上都是有活力的，而突变植株的花粉仅20%左右有活力。2. 突变株与对照植株的杂交F1代均表现正常株高，表明该突变性状为隐性突变。F1代植株相互授粉得到的168株F2代植株中，株高出现分离，正常株高（株高高于80cm）与矮秆植株（株高矮于40cm）的株数比为130：38，经卡方检验，其分离比符合3：1的分离比，因此推测该突变体属于单基因的隐性突变。3. 用ELISA方法检测突变株和对照植株的幼嫩种子中内源性生物活性赤霉素（GA1＋3）含量，结果表明突变株的赤霉素含量为36 ng/ml，而对照植株的赤霉素含量为900 ng/ml。对突变株外施赤霉素，发现矮秆突变株的株高和花粉育性均可得到恢复。这些结果表明该突变株为赤霉素缺陷型突变。4. 用荧光定量PCR方法比较突变株与对照植株中内根贝壳杉烯氧化酶和赤霉素20氧化酶的转录水平，结果表明突变株的KO转录水平比对照植株分别提高了6倍（苗期）和16倍（成熟期），突变株的GA20ox转录水平与对照植株在苗期无明显差异，在成熟期突变株较对照植株则提高了10倍左右。这些结果表明该矮秆突变体与赤霉素的生物合成途径密切相关，而且极有可能在赤霉素的生物合成途径早期就发生了改变。5. 以簇毛麦总基因组为模板，同源克隆了GenBank登录号为EU142950，RT-PCR分离克隆了簇毛麦的GA3ox基因cDNA全长序列，分析结果表明该cDNA全长1206bp，含完整编码区1104bp，推测该序列编码蛋白含368个氨基酸残基，分子量为40.063KD，等电点为6.27。预测的氨基酸序列含有双加氧酶的活性结构，在酶活性中心2个Fe离子结合的氨基酸残基非常保守。该序列与小麦、大麦和水稻的GA3ox基因一致性分别为98%、96%、86%。基因组序列与cDNA序列在外显子部分一致，在478-715bp和879-1019bp处分别含238bp和140bp的内含子。6. 通过RT-PCR技术克隆了簇毛麦的GA20ox基因全长，命名为DvGA20ox，GenBank登录号为EU142949。该基因全长1080个碱基，编码359个氨基酸，具有典型的植物GA20ox基因结构。该基因编码的蛋白质与小麦、大麦、黑麦草等GA20ox蛋白的同源性分别为98%，97% 和91%。该序列重组到原核表达载体pET-32a(+)上，将获得的重组子pET-32a(+)-DvGA20ox转化大肠杆菌BL21pLysS后用IPTG进行诱导表达。SDS-PAGE分析表明，DvGA20ox基因在大肠杆菌中获得了高效表达，融合蛋白分子量为55kDa。定量PCR分析表明，该基因在簇毛麦不同器官中的表达差异明显：叶片中表达水平最高，根部表达水平次之，茎部和穗中表达较弱。在外施赤霉素后，该基因的表达水平在两小时以后急剧下降，表明该基因的表达受自身的反馈调节。本研究结果认为，(1)该簇毛麦矮秆突变体为单基因的隐性突变；(2)该矮秆突变体为赤霉素敏感突变，内源赤霉素含量检测表明突变体的内源性赤霉素含量仅为对照植株的1/30；(3)荧光定量PCR结果表明突变株的赤霉素生物合成途径的关键酶基因表达水平比对照植株高，而且突变植株的赤霉素生物合成改变很可能发生在赤霉素生物合成途径的早期；(4)GA20ox有表达的组织特异性，且受到自身产物的反馈调节。 Plant height is an impotrant agronomic trait of triticeae crops.Semi-dwarf cropcultivars, including those of wheat, maize and rice, have significantly increased grainproduction that has been known as “green revolution”. The new dwarf varieties couldraise the harvest Index at the expense of straw biomass, and, at the sametime, improvelodging resistance and responsiveness to nitrogen fertilizer. Moreover, dwarf traits ofplant are crucial for elucidating mechanisms for plant growth and development aswell. In many plant species, various dwarf mutants have been isolated and theirmodles of inheritance and physiology also have been widely investigated.The causesfor their dwarf phenotypes were found to be associated with plant hormones,especially, gibberellins GAs.Dasypyrum villosum Candargy (syn.Haynaldia villosa) is a cross-pollinating,diploid (2n = 2x = 14) annual species that belongs to the tribe Triticeae. It is native toSouthern Europe and West Asia, especially the Caucasuses, and grows underconditions unfavorable to most cultivated crops. The genome of D. villosum,designated V by Sears, is considered an important donor of genes to wheat for improving powdery mildew resistance, take-all, eyespot, and plant and seed storageprotein content. A spontaneous dwarf mutant was found in D. villosum populations.The biological character and modles of inheritance of this dwarf mutant are studied.The cell length of stem cell is observed. The influence of extraneous gibberellin tothe dwarf mutant is also examined; the transcript level of key enzyme of gibberellinbiosynthesis pathway in mutant and control plants is compared. GA3ox and GA20oxare cloned and its expression pattern is researched.1. The dwarf mutant showed no difference with control plants at seedlingstage.At mature stage, the average height of control plants were 110cm and the dwarfplants were 33cm. The height of the mutant plant was only one third of the normalplants due to the shortened internodes. Cytology observation showed that theelongation of stem epidermal and the parenchyma cells were reduced. The dwarfmutant also shows partly male sterile. Pollen viability test indicates that more than80% of the pollen of the mutant is not viable.2. The inheritance modle of this dwarf mutant is studied. All The F1 plantsshowed normal phenotype indicating that the dwarfism is controlled by recessivealleles. Among the 168 F2 plants, there are 130 normal plants and 30 dwarf plants, thesegregation proportion accord with Mendel’s 3:1 segregation. We therefore proposethat this dwarf phenotype is controlled by a single recessive gene.3. Quantitative analyses of endogenous GA1+3 in the young seeds indicated thatthe content of GA1+3 was 36ng/ml in mutant plants and 900ng/ml in normal plants.The endogenous bioactive GA1+3 in mutant plants are only about 1/30 of that innormal plants. In addition, exogenously supplied GA3 could considerably restore themutant plant to normal phenotype. These results showed that this mutant wasdefective in the GA biosynthesis.4. More than ten enzymes are involved in GA biosynthesis. KO catalyzes thefirst cytochrome P450-mediated step in the gibberellin biosynthetic pathway and themutant of KO lead to a gibberellin-responsive dwarf mutant. GA20ox catalyze therate-limited steps so that their transcript level will influence the endogenous GAbiosynthesis and modifies plant architecture. The relative expression levels of genesencoding KO and GA20ox were quantified by real time PCR to assess whether thechanges in GA content correlated with the expression of GA metabolism genes andwhere the mutant occurred during the GA biosynthesis pathway. In mutant plants,the transcript levels of KO increased about 6-fold and 16-fold at the seedling stage and elongating stage respectively comparing with the normal plants. For theseedlings, there was no notable difference in the expression of GA20ox betweenmutant and normal plants. At the elongating stage, GA20ox transcript increased 10times in mutant plants, suggesting that the GA biosynthesis pathway in mutant plantshad changed from the early steps rather than the late steps.5. A full length cDNA of D. villosum gibberellin 3β-hydroxylase homology(designated as DvGA3ox) was isolated and consisted of 1206bp containing an openreading frame of 1104bp encoding 368 predicted amino acid residues. Identityanalysis showed that the gibberellin 3β-hydroxylase nucleotide sequence shared 98%,96% and 86% homology with that of wheat, barley and rice. The predicted peptidecontained the active-site Fe of known gibberellin 3β-hydroxylase and the regionhomologous to wheat, barley and Arabidopsis. The genomic clone of gibberellin3β-hydroxylase has two introns.6. The full-length cDNA of D. villosum gibberellin 20 oxidase (designated asDvGA20ox) was isolated and consisted of 1080-bp and encoded 359 amino acidresidues with a calculated mol wt of 42.46 KD. Comparative and bio-informaticsanalyses revealed that DvGA20ox had close similarity with GA20ox from otherspecies and contained a conserved LPWKET and NYYPXCQKP regions. Tissueexpression pattern analysis revealed DvGA20ox expressed in all the tissues that wereexamined and the highest expression of DvGA20ox in expanding leaves followed byroots. Heterologous expression of this cDNA clone in Escherichia coli gave a fusionprotein that about 55KD. Transcript levels of DvGA20ox dramatically reduced twohours after application of biologically active GA3, suggesting that the biosynthesis ofthis enzymes might be under feedback control.

利用02428h中隐性高杆基因消除不育系包颈现象的可行性研究

梗稻广亲和种质02428h中倒一节间伸长的性状由一对隐性基因(h基因)所控制，本实验证明了将h基因导入保持系与雄性不育系中均能得到遗传表达，促进倒一节的伸长。h基因与可育胞质及野败不育胞质作用后分别使穗伸出度达15cm,1.2cm，而相应珍汕97B及珍汕97A穗伸出度仅分别为1.7cm，-9.8cm。即h基因使穗伸出度分别提高8.8倍及1.2倍。因此，利用h基因有可能充分消除雄性不育系的包颈现象。The character of greatly elongated uppermost internode of wide compatible japonica rice 02428h iscontrolled by a recessive gene(b gene). The study shows that h gene could get genetic expression in maintaince andmale-sterile line(MS-line) and make the uppermost internode elonged. H gene interacts with the fertile and wild abortive male sterile cytoplasm make panicie neck exsert to 15cm and 1.2cm, while panicle neck exsertions of parents zhenshan 97B and A were 1.7cm and -9.8cm. So, h gene makes panicle necks exsert 8.8 and 1.2 times repectively. Its is possible to introduce h gene into male-sterile line so as to eliminate panicle endosure.

绞股蓝离体培养中影响皂甙产量的几个因素研究

绞股蓝(Gynostemma pentaphyllum)系葫芦科绞股蓝属植物，药用价值广泛，但其野生资源日趋减少，绞股蓝主要药效成分为绞股 蓝皂甙。利用组织和细胞培养生产绞股蓝皂甙是合理开发利用和保护绞股蓝资源的可能途径之一。本文对绞股蓝组织培养中培养基 的蔗糖和激素的组成以及各种胁迫条件：渗透压、重金属离子、真菌诱导物等对皂苷产量的影响进行了初步研究。其中，渗透压、 重金属离子、真菌诱导物对绞股蓝愈伤组织皂甙产量的影响尚未见报道。1. 蔗糖对绞股蓝愈伤组织之生长影响显著，2,4-D对绞股 蓝愈伤组织皂甙含量、产量影响显著。增加蔗糖用量，减少2，4-D的用量可提高皂甙产量。2. Mn++ 用量的提高抑制绞股蓝愈伤组 织的生长，但可促进皂甙含量、产量的提高。Mn++用量提高至MS培养基的20-30倍时可使皂甙产量增加近一倍，而提高Cu++浓度的 作用不明显。3. 甘露醇用量增加抑制绞股蓝愈伤组织的生长，但可使皂甙含量、产量提高。0.680mol·l-1甘露醇可使皂甙产量提 高83%，而Nacl较大抑制愈伤组织的生长并使皂甙产量降低。4. 米曲霉粗提物对绞股蓝愈伤组织生长先略微促进，然后抑制，而根 霉粗提物则使愈伤组织生长受抑制；两者对皂甙含量、产量的作用相似：在较低浓度范围内升高，然后下降。米曲霉粗提物可提高 产量一倍，根霉粗提物可提高42%。这些结果为高产细胞系的筛选和生长、生产培养条件的优化积累了资料。在综述部分，对植物 细胞培养中组织和器官分化、细胞结构变化、生化水平的变化与次生物合成和积累的关系作了讨论。Gynostemma pentaphyllum blongs to Gynostemma, Cucurbitaccae. It has a wide medical use, but its wild resource is threatened by people's excessive use. Its effective medical components are gypenosides. For reasonable use and protect its resource, it is a possible way to product gypenosides by plant tissue and cell culture. This paper has a primary study on the components of sucrose and hormones and a variety of stress conditions: osmostic pressure, heavy metal ion, fungal elicitors in the medium for the calli culture. The effects of osmostic pressure, heavy metal ion and fungal elicitors on the calli of Gynostemma pentaphyllum have not been reported. 1. Sucrose had a significant effect on the growth of the calli, 2,4 D had notable effects on the gypenosides content and production of the calli. Increased the concentration of sucrose and decreased the concentration of 2,4 D improved the production of gypenosides. 2. Increased the concentration of Mn++ inhibited the growth of the calli, but improved the content and production of gypenosides. The optimum concentration was 20-30 times as MS medium which improved the production 100%. Increased the concentration of Cu++ had not a notable effect. 3. Increased the concentration of mannitol inhibited the growth of the calli, but improved the content and production of gypenosides. The optimum concentration was 0.680mol·l-1 which improved the production 83%. Nacl apparently inhibited the growth of the calli and decreased the production of gypenosides. 4. The crude preparation of Aspergillus oryzae inhibited the growth of the calli that in low concentration. The crude praparation of Rhizopus formosensis inhibited the growth of the calli throughout. Their effects on the content and production of gypenosides are alike, but the former is higher than the latter. On the optimum concentration, each crude preparation improved the production 100% (Aspergillus oryzae), 42%(Rhizopus formosensis). These results has accumulated some informantion on the select of high yield cell strains and choose the best culture conditons for the growth and gypenosides product of the calli. In the review, it is discussed that the differentiation on tissue-organal, cellular and biochemical levels related to the synthesis and accumulation of secondary metabolites in plant culuture.

ACHT操作引起黑麦(Secale cereale)染色体的畸变和重建

用ACHT处理黑麦萌动种子，对修复前后材料的观察和分析结果表明：1. ACHT操作引起染色体数目变化和染色体断裂损失。在一定 条件和范围内，不同处理引起的这种变化具有显著差异，条件越剧烈，染色体数目变化的范围和频率愈大，断片发生的数量和频率 也愈高，同时修复前后染色体数目的变化范围和频率与断片发生的数量和频率以及它们的修复频率均表现明显的相关性。2. ACHT 操作引起染色体畸变的多样性。经ACHT处理后，胚根细胞染色体有4种断裂方式，包括着丝粒断裂、次溢痕断裂、长臂断裂和短臂 断裂等，其中着丝断裂频率最高；产生6种断片类型，包括有着丝粒和端粒的、有着丝粒而无端粒的、有部分着丝粒和端粒的、有 部分着丝粒而无端粒的、只有端粒的、既无着丝粒也无端粒的断片等。3. ACHT操作引起遗传结构重建的多样性。经ACHT处理后， 对修复24-72小时材料进行核型比较(按Stebbins 和 Levan 标准)和随体分析，处理细胞在染色体数目、大小、形态、位置等方面 均发生显著变化，说明ACHT处理使这些细胞的染色体结构和染色体组型发生了深刻变化。进一步通过Giemsa C— 带分析，观察到 多种重建染色体类型，包括易位型染色体、附加型染色体、无着丝粒染色体、化染色体、增加的m染色体以及某些带型特异的染色 体等。4. RAPD 分析从分子水平上验证了ACHT能有效地引起遗传结构的改变。所用10种引物对处理和对照材料基因组DNA的扩增产 物在条带数目、条带位置及带型特征等方面均有明显差异，其中4种引物出现条带减少，6种引物出现条带增加，后者还包括一个带 位移动。这说明两种材料的基因组DNA具有明显的RAPD反应多态性差异。This paper descripes some results draw on the basis of the observation and analysis on the rye before and after repaired through treating its budding seeds by ACHT in contrast to without ACHT: 1. ACHT manipulation caused the number variation and breakage damage of rye chromosome. Within certain conditions and timits, this phenomenon caused by different treats had signifcant difference: the more the treatment condition is drastie, the more the chageable range and frequence of rye chromosomae number, and so is the produced fragments. Meanwhile, there existed striking relationship among the changeable range and frequence of rye chromosome, the produced number and frequence of fragments and repairing frequence. 2. ACHT manipulafion engendered the diversify of rye chromosomal aberration. Four breakage patterns and six sorts of fragment were observed by watching the chromosome of the rye radicle treated by ACHT, including centric breakage (occuring in the highest frequence), secondary constriction breakage, long arm breakage and short arm breakage to the former, Comprising that with both centromere and telomere, that with centromere and without telomere, that with partial centromere and with telomere, that with partfial ceetromere and without telomere, that only with telomere and that neither with centromere nor with telomere, etc. 3. ACHT manipulation engendered the diversify or rye genetic structs reconstruction. Karureotype analysis(according to Stebbins and Levan) and satellite anaeysis were carried out to rye radicle through 24-72-hour-long recoverage after ACHT manipulation, which showed remarkable change happened on the rye chromosomal number、shape、arm ration and pattern, etc. and also on the satellite number、size、shape and location etc. Those indicated that ACHT manipulation engendered violent changes to rye chromatin structure and chromosome type. Further Giemsa C-banding analysis showed many types of reconstructed chromosome, such as translocation、addition、without centromere、st and other chromosome. 4. RAPD analysis checked the validity of ACHT on changing genetic structure of rye on the level of molecular biology. The treated and recovered rye has different amplifying band pattern by using IO valid arbitary primers selected from 40 comparing with the control.