荞麦(Fagopyrum tataricum Gaertn.和F. esculentum Moench.)对紫外线B辐射的响应研究

近二十多年来，基于对臭氧层衰减、紫外线B（UV-B）增强的担心，研究者希望了解到紫外线辐射对不同作物的影响情况，增强UV-B辐射条件下是否对作物的生长发育、产量质量构成威胁。在本试验中，我们首先探讨了双子叶作物黄瓜（Cucumis sativus）和大豆（Glycine max）对不同紫外波段的生物效应[分别为B-UVA（315-400 nm），N-UVA（315-340 nm），B-UVB（275-400 nm）和N-UVB（290-340 nm），UV-（>400nm）作对照]。我们观察到所有的UV波段处理都使黄瓜和大豆的生长受到抑制，并且细胞受到不同程度的氧化伤害；UV波段处理的作用效果与不同波段的紫外有效生物辐射剂量有关。处理差异在UV-B波段内部和UV-A波段内部同样存在。植物生长UV辐射公式（BSWF）能很好的预测本试验UV-B波段内的平均植物效应，但不能预测UV-A波段的植物效应。短波UV-A的生物作用强于长波UV-A。光合色素的变化与UV波谱差异和种间差异有关。在高的紫外/可见光背景下，UV-A处理同UV-B同样导致光合色素的降低，但黄瓜类胡萝卜素/叶绿素比例升高。与其他研究者的试验结果比较后，我们认为紫外线B辐射的生物效应一致性很高，但紫外线A波段的生物学效应存在较大争议。因此我们在本试验的基础上仅进行荞麦[苦荞(Fagopyrum tataricum Gaertn.)和甜荞(Fagopyrum esculentum Moench.)]对紫外线B波段的响应研究。 我们对苦荞品种-圆籽荞进行了连续两个生长季节的大田半控制试验以观察UV-B辐射对苦荞生长、发育、产量及叶片色素的影响；试验小区进行降低UV-B、近充足UV-B和增强UV-B辐射处理。我们的试验表明在不同强度UV-B辐射下苦荞的生长、地上部生物量积累及最终产量都有所下降，但苦荞的发育加快；当前条件下的日光紫外线B辐射对植物生长和产量也造成负面影响。植物光合色素被日光及增强UV-B辐射降低；UV化合物及卢丁含量在中低剂量的UV-B辐射强度下显著升高，但在高剂量的增强UV-B辐射下短期升高后迅速下降。我们的试验表明苦荞是一个对UV-B高度敏感的作物。苦荞对UV-B的敏感性与UV-B剂量、外界环境因素及生长季节有关。 单个苦荞品种的试验结果使我们认识到外界UV-B辐射已经对苦荞生长发育构成逆境条件，未来全球气候变化条件下增强紫外线B辐射可能使其处于更不利的生长环境中。因此我们有进行了多个种群进行UV-B响应观察并筛选耐性种群。我们对15个苦荞种群进行增强UV-B辐射处理（6.30 kJ m2 UV-BBE，模拟当地25%的臭氧衰减），我们观察苦荞UV-B辐射效应存在显著的种内差异，UV-B辐射对多数种群具有抑制作用，但对一些种群还有刺激作用。我们采用主成分分析方法与作物UV-B响应指数（RI）来评价苦荞作物UV-B辐射耐性。我们发现作物的UV-B耐性不仅与其原产地背景UV-B强度有关，而且与作物相对生长效率、次生代谢产物含量（如卢丁）及其他因素有关。我们观察到苦荞伸展叶总叶绿素变化与UV-B耐性成正相关；室内苦荞幼苗的UV-B辐射致死试验表明：苦荞种群死亡率与其UV-B耐性成负相关。 此外，我们对甜荞的UV-B辐射响应也进行了初步研究。选取美姑甜荞、巧家甜荞和云龙甜荞进行5个梯度的增强UV-B辐射室外模拟试验。我们观察到UV-B辐射显著降低了甜荞的生长、生物量及产量；并严重影响了甜荞的生殖生长，降低了花序数、种子数和结实率；并且UV-B辐射对甜荞的抑制作用存在显著的剂量效应。三种甜荞品种存在显著的种内差异，其中美姑品种UV-B耐性最强，且膜脂受UV-B辐射氧化伤害最小，这与该品种UV-B辐射下较高的GR酶活性、APX酶活性和PPO酶活性、以及含量更高的抗坏血酸有关。甜荞的次生代谢也受到增强UV-B辐射的影响，其香豆酰类化合物在UV-B辐射下升高显著，而槲皮素含量也在高剂量UV-B辐射下有所增加；卢丁含量依赖UV-B辐射剂量而变化，中低剂量UV-B辐射下其卢丁含量逐渐升高，但在高剂量辐射下逐渐下降。 通过对生长在高海拔地区的荞麦作物(苦荞和甜荞)进行的室外研究，我们认识到作物不同品种存在很大的耐性差异，这就为UV-B耐性育种创造了有利条件。进一步加大荞麦种质资源筛选力度并深入认识荞麦抗性机理，在此基础上通过杂交或其他基因融合手段培育抗性品种，对高剂量UV-B辐射地区的荞麦产量的提高将起到重要推动作用，并使荞麦生产能有效应对未来全球气候变化条件下UV-B辐射可能升高的威胁。 During last few decades, due to concern of ozone layer depletion and enhancement of ultraviolet B radiation（UV-B, 280-315 nm), the agronomist want to know the responses of different crop species to UV-B. In the first experiment of our study, the effect of different UV band [B-UVA（315-400 nm), N-UVA（315-340 nm), B-UVB（275-400 nm), N-UVB（290-340 nm）and UV-（>400nm, as control)] on the cucumber（Cucumis sativus）and soybean（Glycine max）were investigated in growth room. Spectra-dependent differences in growth and oxidation indices existed within UV-A bands as well as UV-B bands. The general biological effects of different band were UV- ＜ B-UVA＜ N-UVA＜N-UVB＜B-UVB. The plant growth biologically spectra weighting function（BSWF）matched well with average plant response in UV-B region, but not in UV-A region. Shorter UV-A wavelength imposed more negative impact than longer UV-A wavelength did in both species. The effect on photosynthetic pigment was related to different UV bands and different species. The photosynthetic pigment content was decreased by UV-A spectra as well as UV-B spectra. In comparison with the results of previous studies, we found that the wavelength-dependent biological effect of ultraviolet B radiation has high consistency, but the biological effect of ultraviolet-A radiation was inconsistent. We narrow our following study on the effect of ultraviolet B radiation on the buckwheat（tartary buckwheat and common buckwheat). The tartary buckwheat（Fagopyrum tataricum Gaertn.）cultivars Yuanziqiao was grown in the sheltered field plots for two consecutive seasons under reduced, near-ambient and two supplemental levels of UV-B radiation. The crop growth, photosynthetic pigments, total biomass, final seed yield and thousand-grain weight were decreased by near-ambient and enhanced UV-B radiation, while crop development was promoted by enhanced UV-B radiation. Leaf rutin concentration and UV-B absorbing compound was generally increased by UV-B with the exception of 8.50 kJ m-2 day-1 supplemental levels. Our results showed that tartary buckwheat is a potentially UV-B sensitive species. Study on one cultivars showed that ambient solar radiation had present a stress to tartary buckwheat. This makes it necessary to observe the UV-B response of many cultivars and screen tolerant cultivars. Fifteen populations of tartary buckwheat were experienced enhanced UV-B radiation simulating 25% depletion of the stratospheric ozone layer in Kunming region, and plant responses in growth, morphology and productivity were observed. Principal components analysis（PCA）was used to evaluate overall sensitivity of plant response to UV-B as well as response index. The different populations exhibited significant differences in responses to UV-B. The photosynthetic pigments of young seedlings were also affected significantly under field condition. On the other hand, the healthy seedlings of different populations were exposed to the high level of UV-B radiation in growth chambers to determine the plant lethality rate. The plant tolerance evaluated by multivariate analysis was positively related to total plant chlorophyll change, but negatively related to lethality rate. In other hand, the UV-B responses of the other important cultivated buckwheat species, common buckwheat（Fagopyrum esculentum Moench.), were also studied preliminarily. Three widespread cultivated variety（Meigu, Qiaojia and Yunlong cultivars）were provided with five level of enhanced UV-B radiation outdoors. We observed that the crop growth, development and production were significantly decreased, and reproductive production, like anthotaxy number, seed number and seed setting ratio, was also decreased. Dose-dependent inhibition effect caused by enhanced UV-B radiation also existed in common buckwheat. Significant intraspecific difference existed in those three cultivars. The Meigu cultivars with dwarfed growth and lower production have highest UV-B tolerance as well as lowest damage in cell membrane, this could be associated with profound enhancements of glutathione reductase（GR）activity, ascorbate peroxidase activity and polyphenol oxidase activity as well as higher ascorbic acid concentration. The secondary metabolism was also affected by UV-B radiation, with profound elevation of coumarin compound and moderate increase of quercetin concentration. Rutin concentration was peaked in 5kJ m-2 UV-B. The contrasting effect of UV-B radiation on different populations indicated that there existed abundant genetic resources for selecting tolerant populations of common and tartary buckwheat. Much effort needed be pose on screening of buckwheat germplasm and clarification of mechanism of buckwheat tolerance to UV-B. On this base the tolerant cultivars could be bred by hybridization and other gene transfusion method, this would help increase buckwheat yield in high ambient UV-B region and counteract the effect of possible enhanced UV-B radiation in future.

不同小麦品种（系）中主要矿质元素的含量比较及与品质的关系

采用微波消解、电感耦合高频等离子体原子发射光谱（ICP-AES）的方法,对62份不同小麦品种（系）中锌、铁、铜、钙、钠和钾的含量进行了测定。同时利用红外线品质测定仪对主要品质指标粗蛋白、湿面筋、沉降值进行了测定。结果表明，不同小麦品种（系）中各种矿质元素的含量存在差异，2006年小麦品种中铁含量变幅为18.55-58.19 ug/g，平均为30.83ug/g ，最高与最低的相差39.64ug/g；锌含量变幅为5.70-25.80 ug/g，平均为15.13ug/g ，最高与最低相差20.10ug/g。2008年小麦品种（系）中铁含量变幅为16.68-52.25 ug/g，平均为30.10ug/g，最高与最低相差35.58ug/g；锌含量变幅为12.29-33.47 ug/g，平均为21.11ug/g，最高与最低相差21.18ug/g；钙含量变幅为167.53-348.80ug/g，平均为248.59ug/g，最高与最低相差192.59ug/g；铜含量变幅为2.32-5.83 ug/g，平均为2.98ug/g，最高与最低的相差3.61ug/g；钾含量变幅为1822.71-4414.91 ug/g，平均为2617.87ug/g，最高与最低的相差2634.72ug/g；钠含量变幅为10.25-39.82 ug/g，平均为23.05ug/g，最高与最低的相差29.57ug/g。 两年不同小麦品种（系）中矿质元素的含量分析结果表明：铁、铜、钙、钠和钾含量年际变化不明显,说明小麦对铁、铜、钙、钠和钾的吸收较稳定；锌含量变化较大，可能受环境的影响比较大。分析各矿质元素含量与粗蛋白、湿面筋、沉降值及元素之间的相关关系，结果表明，锌含量与粗蛋白含量呈极显著正相关关系，相关系数为0.317，与湿面筋含量之间呈显著正相关，相关系数达到0.246；铁含量与粗蛋白含量呈显著的正相关关系，相关系数是0.262；铜、钙、钠和钾含量与粗蛋白含量、湿面筋和沉降值之间存在正相关，但不显著，其中钠与沉降值之间为负相关。表明施锌或铁对提高小麦粗蛋白和湿面筋有显著效应，其余矿质元素有促进作用但不明显。 利用RAPD分子标记技术对川育23、41058、川育20及其父母本进行分析，力图从分子水平找到小麦矿质元素含量之间的差异性，琼脂糖电泳结果表明不同的小麦品种（系）间扩增出了差异条带。 以上研究结果，将对筛选“微量营养强化型”小麦新材料，选育“微量营养强化型”小麦新品种奠定基础。 62 different wheat cultivars was digested with HNO3 in a tightly closed vessel heated under micro-wave,then contents of zinc，iron,copper，calcium，sodium and potassium were determined by inductively coupled plasma-atomic emission spectroscopy(ICP-AES)．The main indexes of wheat quality such as total protein、wet glu and sedimentation volume were detected by Infratec 1255 Food & Feed Analyzer at the same time.The obtained results showed that variation for all of the mineral elements concentrations among different cultivars were observed .In 2006, the amplitude variation of the iron content was 18.55-58.19 ug/g,the average value was 30.83ug/g,and 39.64ug/g between the highest-content cultivar and the lowest one; the amplitude variation of the zinc content was 5.70-25.8 ug/g,the average value was 15.13ug/g,and 20.10ug/g between the highest-content cultivar and the lowest one.In 2008, the amplitude variation of the iron content was 16.68-52.25 ug/g,the average value was 30.10ug/g,and 35.58ug/g between the highest-content cultivar and the lowest one; the amplitude variation of the zinc content was 12.29-33.47 ug/g,the average value was 21.11ug/g,and 21.18ug/g between the highest-content cultivar and the lowest one; the amplitude variation of the calcium content was 167.53-348.80ug/g,the average value was 248.59ug/g,and 192.59ug/g between the highest-content cultivar and the lowest one; the amplitude variation of the copper content was 2.32-5.83 ug/g,the average value was 2.98ug/g,and 3.61ug/g between the highest-content cultivar and the lowest one; the amplitude variation of the potassium content was 1822.71-4414.91 ug/g,the average value was 2617.87ug/g,and 2634.72ug/g between the highest-content cultivar and the lowest one; the amplitude variation of the sodium content was 10.25-39.82 ug/g,the average value was 23.05ug/g,and 29.57ug/g between the highest-content cultivar and the lowest one. Analysis was made on the annual variation of mineral elements content in different Wheat cultivars ,the result shows:there is no obvious difference of iron ,copper ,sodium、calcium and potassium concentrations in wheat cultivars, suggesting the absorption of the iron, copper, sodium、calcium and potassium by wheat are relatively steady ,but zinc concentrations change obviously ,maybe influenced heavily by environment . The correlation between mineral elements 、mineral elements and total protein、mineral elements and sedimentation volume as well as mineral elements and wet glut were analysed in this paper, the result showed that there was significant positive correlation between zinc content and total protein (the correlation coefficient is 0.317), positive correlation between zinc content and wet glu (the correlation coefficient is 0.246), positive correlation between iron content and total protein (the correlation coefficient is 0.262). there was positive but not obvious correlation between the contents of copper, calcium, sodium or potassium and total protein, wet glut or sedimentation volume,among which was negative correlation between sodium and sedimentation volume.It was indicated zinc or iron fertilization has prominent effects in improving the total protein in wheat, the rest mineral elements have Non- obvious facilitation. The study then forecasted the genetic difference of different wheat by the molecular marker of RAPD in order to find differences in molecular level. Chuanyu23、41058、chuanyu20 as well as their male and female parents were analysed by RAPD markers,Agarose gel electrophoresis of DNA revealed the appearance of differential bands . The above-mentioned results of this study establish the foundation to screening the new materials of wheat of " strengthening type of micro- nutrition ", and to breeding the new wheat cultivars of" strengthening type of micro- nutrition ".

青稞B组醇溶蛋白基因与上游调控区的克隆及基因表达

高等植物种子胚乳贮藏蛋白是种子发芽时的主要氮源，也是人类和动物食用植物蛋白的主要来源。大麦种子胚乳贮藏蛋白主要是醇溶蛋白（hordeins），占大麦胚乳总蛋白的50–60%。根据大麦醇溶蛋白的大小和组成特点，大麦醇溶蛋白被划分为三种类型：富硫蛋白亚类(B，γ-hordeins)、贫硫蛋白亚类（C-hordeins)以及高分子量蛋白亚类(D-hordeins)。B组和C组醇溶蛋白是大麦胚乳的两类主要贮藏蛋白，它们分别占大麦总醇溶蛋白成分的70–80%和10–12%。遗传分析表明，大麦B、C、D和γ-组醇溶蛋白分别是由位于大麦第五染色体1H（5）上的Hor2、Hor1、Hor3和Hor5位点编码。Hor2位点编码大量分子量相同但组成不同的B组醇溶蛋白（B-hordein）。B-hordein的种类、数量和分布是影响大麦酿造、食用及饲养品质的重要因素之一。为深入了解B-hordein基因家族的结构和染色体组织，探明Hor2位点基因表达的发育调控机制，最终达到改良禾谷类作物籽粒品质的目的，本研究以青藏高原青稞为材料，采用同源克隆法，分别克隆B-hordein基因和启动子，通过原核生物表达验证B-hordein基因功能，并利用实时定量PCR探索B-hordein基因表达时空关系，取得如下研究结果： 1. 以具有特殊B组醇溶蛋白亚基组成的9份青藏高原青稞为材料，根据GenBank中三个B-hordein基因序列（GenBank No. X03103, X53690和X53691）设计一对引物，通过PCR扩增，获得23个B-hordein基因克隆并对其进行了序列分析。核苷酸序列分析表明，所有克隆均包含完整的开放阅读框。有11个克隆都存在一个框内终止密码子，推测这11个克隆可能是假基因。推测的氨基酸序列分析表明，所有大麦B-hordein具有相似的蛋白质基本结构，均包括一个高度保守的信号肽、中间重复区以及C-端结构域。不同大麦种重复区内重复基元的数目有较大差异。青稞材料Z07–2和Z26的B-hordeins仅具有12个重复基元结构，更接近于野生大麦。这些重复基元数目的差异导致了重复区序列长度和结构的变异。这种现象极可能是由于醇溶谷蛋白基因在进化过程中染色体的不平衡交换或复制滑动所造成的。对所克隆基因和禾本科代表性醇溶谷蛋白基因进行聚类分析，结果表明所有来自栽培大麦的B-hordeins聚类成一个亚家族，来自野生大麦的B-hordeins以及普通小麦的LMW-GS聚类成另外一个亚家族，表明这两个亚家族的成员存在显著差异。此外，我们发现B-hordein基因推测的C-末端序列具有一些有规律的特征：即具有相同C-末端序列的B-hordein基因在系统发生树中聚类为同一个亚组（除BXQ053，BZ09-1，BZ26-5分别单独聚为一类外）。这个特征将有助于我们对所有B组醇溶蛋白基因家族成员进行分类，避免了在SDS-PAGE电泳图谱上仅依靠大小分类的局限性。 2. 根据上述克隆的青稞B-hordein基因的5’端序列设计三条基因特异的反向引物，以青稞Z09和Z26的基因组DNA为模板，采用SON-PCR和TAIL-PCR技术分离克隆出8个B-hordein基因的上游调控序列（命名为Z09P和Z26P）。序列分析表明，推测的TATA box位于–80 bp，CAAT–like box位于–140 bp处。此外，Z09P和Z26P中有六个序列在–300 bp处均存在一个由高度保守的EM基序和类GCN4基序构成的胚乳盒(Endosperm Box，EB)，在约–560 bp处存在一个胚乳盒类似结构。而Z09P-2和Z26P-3不存在保守的胚乳盒或其类似结构，预示着这两个启动子所调控的基因表达可能受不同类型反式作用因子的调节，推测该启动子对基因的表达调控具有多样性。 3. 将B-hordein基因的开放阅读框定向克隆到表达载体pET-30a中，将其导入大肠杆菌表达菌株BL21中进行外源基因的诱导表达以验证所克隆基因的功能。结果表明仅含重组子pET-BZ07-2和pET-BZ26-5的BL21细菌有目的表达蛋白产生。在诱导3 h时的蛋白表达量最高；3 mM IPTG诱导的蛋白表达量要高于1 mM IPTG诱导的表达量。这为分离纯化B-hordein蛋白以及进一步研究其对大麦籽粒品质的影响奠定基础。 4. 根据从青稞Z09和Z26中分离克隆的B-hordein基因序列设计一对基因特异的引物，同时，选择大麦α-微管蛋白基因（GenBank no. U40042）为看家基因并设计特异引物，利用实时荧光定量PCR检测了青稞籽粒4个胚乳发育时间段的B-hordein基因表达，荧光定量结果显示：两份材料中B-hordein基因的表达量均随发育过程的进行而逐渐升高。Z09中B-hordein基因在开花后7天开始转录，而Z26开花4天后就有低水平B-hordein的表达，这表明Z26中B-hordein基因可能比Z09表达的较早或者Z09中B-hordein基因表达水平较低以致于不能被检测到。此外，在4个不同的胚乳发育时期中，Z26中B-hordein基因的表达量均高于Z09材料。在开花12天到18天的过程中，Z09和Z26中B-hordein基因的表达水平有一个急剧性的升高。这说明在不同胚乳发育时期，Hor2位点的B-hordein等位基因变异体存在mRNA的差异表达。 Seed endosperm storage proteins in higher plants are the main resources of nitrogen for germinating and plant proteins for human and animals. Barley prolamins (also called hordeins) are the major storage proteins in the endosperm and account for 50–60% of total proteins. Hordeins are classically divided into three groups: sulphur-rich (B, γ-hordeins), sulphur-poor (C-hordeins) and high molecular weight (HMW, D-hordeins) hordeins based on the size and composition. B-hordeins and C-hordeins are two major groups and each respectively account for about 70-80% and 10-12% of the total hordein fraction in barley endosperm. Genetic analysis showed that B-, C-, C-, γ-hordeins are encoded by Hor2, Hor1, Hor3 and Hor5 locus on the chromosome 1H (5). Hor2 locus is rich in alleles that encode numerous heterogeneous B-hordein polypeptides. It is reported that B-hordein species, quantity and distribution are significant factors affecting malting, food and feed quality of barley. To understand comprehensively the structure and organization of B-hordein gene family in hull-less barley and explore the developmental control mechanisms of Hor2 locus gene expression and eventually to better exploitation in crop grain quality improvement, we isolated and cloned B-hordein genes and promotors of hull-less barley from Qinghai-Tibet Plateau by PCR, and testified their expression founction in bacteria expression system and explore their spatial and temporal expression pattern by quantitative real time PCR. Our results are as followed, 1. Twenty-three copies of B-hordein gene were cloned from nine hull-less barley cultivars of Qinghai-Tibet Plateau with special B-hordein subunits and molecularly characterized by PCR, based on three B-hordein genes published previously (GenBank No. X03103, X53690 and X53691). DNA sequences analyses confirmed that the six clones all contained a full-length coding region of the barley B-hordein genes. Eleven clones all contain an in-frame stop codon and they are probably pseudogenes. The analysis of deduced amino acid sequences of the genes shows that they have similar structures including signal peptide domain, central repetitive domain, and C-terminal domain. The number of the repeats was largerly variable and resulted in polypeptides in different sizes or structures among the genes. Twelve such repeated motifs were found in Z07–2 and Z26, and they are close to those of the wild barleys, and it is most probably caused by unequal crossing-over and/or slippage during replication as suggested for the evolution of other prolamins. The relatedness of prolamin genes of barley and wheat was assessed in the phylogenetic tree based on their polypeptides comparison. Our phylogenetic analysis suggested that the predicted B-hordeins of cultivated barley formed a subfamily, while the B-hordeins of wild barleys and the two most similar sequences of LMW-GS of T. aestivum formed another subfamily. This result indicated that the members of the two subfamilys have a distinctive difference. In addition, we found the B-hordeins with identical C-terminal end sequences were clustered into a same subgroup (except BXQ053，BZ09-1 and BZ26-5 as a sole group, respectively), so we believe that B-hordein gene subfamilies possibly can be classified on the basis of the conserved C-terminal end sequences of predicted polypeptide and without the limit of SDS-PAGE protein banding patterns. 2. The specific primers were designed according to the published sequences of barley B-hordein genes from Z09 and Z26. Using total DNA isolated from them as the templates, eight clones (designated Z09Pand Z26P) of upstream sequences of the known B-hordein genes was obtained by TAIL-PCR and SON-PCR. Sequences analysis shows that the putative TATA box was present at position –80 bp and CAAT-like box at position –140 bp. Besides, a putative Endosperm Box including an Endosperm Motif (EM) and a GCN4-Like Motif was found at position –300 bp in six clones, and another Endosperm-like box was found at positon –560 bp. While the Endosperm Box or Endosperm-like box was not found in Z09P-2 and Z26P-3. This may indicate that gene expression drived by the two promtors was probably controlled by different trans-acting factors and the genetic control mechanism of corresponding gene expression may be diverse. 3. The B-hordein genic region coding for the mature peptide was cloned into expression vector pET-30a and transformed into bacterial strain BL21 for identifying gene expression fountion. Protein SDS–PAGE analysis showed that only the transformed lysate with the pET-BZ07-2 and pET-BZ26-5 constructs produced proteins related to B-group hordeins of barley, and the mounts of proteins induced by 3 mM IPTG and 3 h were higher than other conditions. This established a base for isolating and putifying B-hordein and further exploring their effects on barley grain quality. 4. The gene-specific primers of B-hordein genes from Z09 and Z26 were used for the quantification of B-hordein gene expression. The α-tubulin gene from Hordeum vulgare subsp. vulgare (GenBank accession number U40042) was used as a control gene. The result shows the transcription of the B-hordein genes in Z09 was found 7 days after flowering, while the transcription of the B-hordein genes in Z26 was found 4 days after flowering, but at a very low level, and it suggested that the B-hordein genes in Z26 probably expressed earlier than those in Z09, or the B-hordein genes in Z09 expressed at so a lower level than Z26 that it can not detected. In addition, B-hordein genes in Z26 accession showed higher expression levels than those in Z09 in four developing stages. Furthermore, a progressive increase in the expression levels of the B-hordein genes between 12 and 18 days after anthesis was observed in both Z09 and Z26. It implies that the B-hordein allelic variants encoded by Hor2 locus exist the differential expression in mRNA levels of during barley endosperm development.

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.

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.

大赖草总DNA转化小麦引起的HMW-GS基因变化及大赖草HMW-GS基因的克隆

应用花粉管通道技术将新疆大赖草总DNA导入小麦，用高重序列分析方法，已为大赖草总DNA转入小麦提供了初步的分子证据。在转 化后代中选育出稳定遗传的大穗变异株系，分析表明，这些转化株中蛋白质含量明显增高(13%-17%)。对基因供体新疆大赖草、受 体春麦761、转化株的高分子量谷蛋白亚基(HMW-GS)进行了SDS-PAGE分析，发现这些转化株中HMW-GS发生了很大变化，并在此基础 上，用来自小麦基因组的四对特异引物，以PCR方法扩增供体、受体以及转化株的1Ax、1Bx、1Dx及1Ay、1By、1Dy型HMW-GS全基因 ，比较他们扩增产物的差异，结果表明，受体与转化株在HMW-GS基因1Ax、1Bx位点上的扩增产物差异不大，在HMW-GS基因位点1Dx 和y型基因上的扩增产物有较大差异，说明了受体在基因位点1Dx、1Ay、1By和1Dy上可能发生了多位点插入，可能由于这些基因位 点上的插入引起了转化株的高分子量谷蛋白亚基(HMW-GS)的变化，这就再一次为大赖草总DNA导入提供了直接的分子证据。虽然大 赖草总DNA导入提高了小麦蛋白质的含量，改变了HMW-GS的组成，部分改变了品质评分，但我们感到这些转化株在品质改良方面仍 有很大余地，如何更好地利用新疆大赖草蛋白质的优良特性及避免总DNA导入给转化株带来的不良性状，一个大赖草HMW-GS基因正 被分离及克隆，并准备将其利用于未来的品质育种当中。Total DNA of Leymus racemosus had been transformed into wheat through pollen tube pathway. Analysis of the repeated gene sequence had provided an elementary proof. Some variant cultivars with big ear were screened from their offsprings, and their protein content increased greatly from 13% (receptor)to 17%(transformed). The result from SDS-PAGE analysis of high-molecular-weight glutenin subunits(HMW- GS) respectively in donor(Xinjiang Leymus racemosus), receptor(spring wheat cultivar 761)and transformed wheats, showed the HMW-GS composition changed in the transformed plants. On the basis of the research, Four special pairs primers from wheat(T.aestivum L.) genome were used to amplify complete coding regions of HMW-GS genes on 1Ax、1Bx 、1Dx and 1Ay、1By、1Dy loci of donor、receptor and the big ear transformed cultivars. By comparing amplified PCR products. Faint differences were found among receptor and transformed cultivar's 1Ax、1Bx PCR amplifed products and apparent differences on those of 1Dx、y-typePCR product. We gueseed that there may be some DNA inserts in 1Dx 、1By、1Dy loci resulted in the changes of the HMW-GS among transformed cultivars. This provides second direct molecular witness to the exogenous DNA introduction. Even though the transformed plants have higher protein content, changed HMW-GS composition, partially improved process quality, there still leave much work to improve quality. In order to make full use of the excellent property of Leymus racemosus protein and avoid the disadvantages introducced by total DNA transformation, a HMW-GS gene of Leymus racemosus was being isolated and cloned.

中国青藏高原青稞抗穗发芽资源评价与α-淀粉酶基因的克隆及表达

穗发芽（PHS，preharvest sprouting）是影响禾本科作物生产的重要的灾害之一。收获时期如遇潮湿天气容易导致穗发芽发生。发生穗发芽的种子内部水解酶（主要是α-淀粉酶）活性急剧升高，胚乳贮藏物质开始降解，造成作物产量和品质严重降低。因此，选育低穗发芽风险的品种是当前作物育种工作中面临的重要任务。 青稞(Hordeum vulgare ssp. vulgare)主要分布于青藏高原，自古以来就是青藏高原人民的主要粮食。近年来，由于青稞丰富的营养成分和特有的保健品质、在燃料工业中的潜力以及在啤酒酿造工业中的利用前景，在发达国家日趋受到重视，掀起综合研究利用的热潮。我国拥有占全世界2/3 以上的青稞资源，具有发展青稞产业的得天独厚的条件。然而，由于青稞收获期间恰逢青藏高原雨季来临，常有穗发芽灾害发生，使青稞生产损失巨大。目前对青稞穗发芽研究很少，适用于育种的穗发芽抗性材料相对缺乏，不能很好的满足青稞穗发芽抗性育种的需要。本研究以青藏高原青稞为材料，对其穗发芽抗性的评价指标和体系进行构建，同时筛选青稞抗穗发芽品种并对其抗性进行评价，还利用分子生物学手段对青稞穗发芽抗性的分子机理进行了初步探讨。主要研究结果如下： 1. 本试验以来自于我国青藏高原地区的青稞为材料，对休眠性测定的温度范围进行探讨，并对各种穗发芽抗性测定方法的对青稞的适用性进行评测。通过探讨温度对13 个不同基因型的青稞籽粒发芽和休眠性表达的影响，对筛选青稞抗穗发芽资源的温度条件进行探索，并初步分析了其休眠性表达的机理。在10，15，20，25，30℃的黑暗条件下，选用新收获的13 个青稞品种为材料进行籽粒发芽实验，以发芽指数（GI）评价其休眠性。结果发现，不同品种对温度敏感性不同，其中温度不敏感品种，在各温度条件下均表现很低的休眠性；而温度敏感品种，其休眠性表达受低温抑制，受高温诱导。15℃至25℃是进行青稞休眠性鉴定的较适宜的温度范围。通过对供试材料发芽后的α-淀粉酶活性，发现温度对青稞种子的休眠性表达的影响至少在一定程度上表现在对α-淀粉酶活性的调控上。随后，对分别在马尔康和成都进行种植的34 份青稞穗发芽指数（SI），穗发芽率（SR），籽粒发芽指数（GI）和α-淀粉酶活性（AA）进行了测定和分析，发现它们均受基因型×栽培地点的极显著影响，且四个参数之间具有一定相关性。GI 参数由于其变异系数较低，在不同栽培地点稳定性好，且操作简便，是较可靠和理想的穗发芽评价参数。SI 参数可作为辅助，区别籽粒休眠性相似的材料（基因型）或全面评价材料（基因型）的穗发芽抗性特征。AA 参数稳定性较差，并且检测方法复杂，因此不建议在育种及大量材料筛选和评价时使用。此外，青稞穗发芽抗性受环境影响较大，评价时应考虑到尽可能多的抗性影响因素及其在不同栽培条件下的变异。 2. 对来自青藏高原的青稞穗发芽抗性特征及其与其它农艺性状间的关系进行研究。通过测定穗发芽指数（SI）、籽粒发芽指数（GI）和α-淀粉酶活性（AA），表明113 份青稞材料的穗发芽抗性具有显著差异。SI、GI 和AA 参数的变幅分别为1.00~8.86、0.01~0.97 和0.00~2.76，其均值分别为4.72、0.63 和1.22。根据SI 参数，六个基因型，包括‘XQ9-5’，‘XQ33-9’，‘XQ37-5’，‘XQ42-9’，‘XQ45-7’和‘JCL’被鉴定为抗性品种。综合SI、GI 和AA 参数，可以发现青稞的穗发芽抗性机制包含颖壳等穗部结构的抗性和种子自身的抗性（即种子休眠性），且供试材料中未发现较强的胚休眠品种，除‘XQ45-7’外，所有品种在发芽第四天均能检测出α-淀粉酶活性。穗部结构和种子休眠的抗性机制因基因型不同而不同，在穗发芽抗性中可单独作用或共同作用。农家品种和西藏群体分别比栽培品种和四川群体的穗发芽抗性强，而在不同籽粒颜色的青稞中未发现明显差异。相关性检验发现，青稞的穗发芽抗性，主要是种子休眠性，与百粒重、开花期、成熟期、穗长、芒长和剑叶长呈显著负相关关系，与株高相关性不显著。农艺性状可以作为穗发芽抗性材料选育中的辅助指标。本试验为青稞穗发芽抗性育种研究提供了必要的理论基础和可供使用的亲本材料。 3. α-淀粉酶是由多基因家族编码的蛋白质，在植物种子萌发时高度表达，与植物种子的萌发能力密切相关。在大麦种子发芽时，高等电点α-淀粉酶的活性远大于低等电点的α-淀粉酶。为了研究不同穗发芽抗性青稞品种中编码高等电点α-淀粉酶Amy1 基因结构与抗性间的关系，我们以筛选得到的抗性品种‘XQ32-5’（TR1）、‘XQ37-5’（TR2）、‘XQ45-7’（TR3），易感品种‘97-15’（TS1）、‘9657’（TS2）以及强休眠大麦品种‘SAMSON’（SAM）为材料，对其Amy1 基因的编码区序列进行克隆和结构分析，并对它们推导的氨基酸序列进行比较。结果显示，青稞Amy1 基因具有三个外显子、两个内含子，编码区中有13 个核苷酸变异位点，均位于2、3 号外显子，2 个变异位点位于2 号外显子。SAM 和TS1 分别在2 号外显子相应位置有5 个相同的碱基(GAACT)的插入片段。相应α-淀粉酶氨基酸序列推导发现，所有核苷酸变异中有8 个导致相应氨基酸残基的改变，其余位点为同义突变。青稞Amy1 基因编码区序列品种间相似度高达99%以上，部分序列变异可能与其穗发芽抗性有关。随后，我们又通过SYBR Green 荧光定量技术对该基因在不同发芽时间（1d~7d）的相对表达水平进行了差异性检测。结果发现，7 天内不能检测到SAM 的Amy1 基因表达，5 个青稞品种间的Amy1 基因的相对表达量均随着发芽时间延长而上升，但上升方式有所不同。弱抗品种该基因表达更早，转录本增加速率更大，且在4~5 天可达到平台期。发芽7 天中，抗性品种总转录水平明显低于易感品种。本研究结果表明，青稞Amy1 基因的转录水平是与其穗发芽抗性高度相关。 我国青藏高原青稞，尤其是农家品种的穗发芽抗性具有丰富的变异，蕴藏着穗发芽抗性育种的宝贵资源。本研究为青稞穗发芽抗性育种建立了合理抗性评价体系，筛选出可供育种使用的特殊材料，阐明了农艺性状可辅助穗发芽抗性育种，同时还对穗发芽抗性与α-淀粉酶基因的结构和表达关系进行分析，为青稞穗发芽抗性资源筛选奠定了基础。 Preharvest sprouting (PHS) is a serious problem in crop production. It often takes place when encountering damp, cold conditions at harvest time and results in the decrease of grain quality and great loss of yield by triggering the synthesis of endosperm degrading enzymes (mostly the α-amylase). Therefore, PHS is regarded as an important criterion for crop breeding. In order to minimize the risk of PHS, resistant genotypes are highly required. Hulless barley (Hordeum vulgare ssp. vulgare) is the staple food crop in Qinghai-Tibetan Plateau from of old, where is one of the origin and genetic diversity centers of hulless barley. Recently, interest in hulless barley has been sparked throughout the world due to the demonstrations of its great potential in health food industry and fuel alcohol production. Indeed, hulless barley can also be utilized to produce good quality malt if the appropriate malting conditions are used. In China, overcast and rainy conditions often occur at maturity of hulless barley and cause an adverse on its production and application. PHS resistant genotypes, therefore, are highly required for the hulless barley breeding programs. However, few investigations have been made so far on this issue. The objectives of this study were: 1) to assessment of methods used in testing preharvest sprouting resistance in hulless barley; 2) to evaluate the variability and characteristics of PHS resistance of hulless barley from Qinghai-Tibet Plateau in China; 3) to select potential parents for PHS resistance breeding; 4) to primarily study on the molecular mechanism of PHS resistance of hulless barley. Our results are as followed: 1. We investigated the temperature effects on seed germination and seed dormancy expression of hulless barley, discussed appropriate temperature range for screening of PHS resistant varieties, and analyzed the mechanism of seed dormancy expression of hulless barley. The dormancy level of 13 hulless barley were evaluated by GI (germination index) values calculating by seed germination tests at temperature of 10，15，20，25，30℃ in darkness. There were great differences in temperature sensitivity among these accessions. The insensitive accessions showed low dormancy at any temperature while the dormancy expression of sensitive accessions could be restrained by low temperature and induced by high temperature. The temperature range of 15℃ to 25℃ was workable for estimating of dormancy level of hulless barley according to our data. Analysis of α-amylase activity showed that the temperature effects on seed germination and the expression of seed dormancy be achieved probable via regulating of α-amylase activity. Furthermore, we evaluated the differences in sprouting index (SI), sprouting rate (SR), germination index (GI) and α-amylase activity (AA) between Maerkang and Chengdu among 34 accessions of hulless barley from Qinghai-Tibetan Plateau in China. These PHS sprouting parameters were significantly affected by accession×location, and they had correlation between each other. GI was the most reliable parameter because of its low CV value, good repeatability and simple operation. SI could assist in differentiating between accessions of similar dormancy or overall evaluation of the resistance. AA was bad in repeatability and had relatively complex testing method, therefore, not appropriate for breeding and evaluation and screening of PHS resistant materials. Besides, since PHS resistance of hulless barley was greatly influenced by its growth environment, possibly much influencing factors and variations between cultivated conditions should be considered. 2. In this study, large variation was found among 113 genotypes of hulless barley (Hordeum vulgare ssp.vulgare) from Qinghai-Tibetan Plateau in China, based on the sprouting index (SI), germination index (GI) and α-amylase activity (AA) which derived from sprouting test of intact spikes, germination test of threshed seeds and determination of α-amylase activity, respectively. The range of SI, GI and AA was 1.00~8.86, 0.01~0.97 and 0.00~2.76，the mean was 4.72, 0.63 and 1.22 espectively. Six resistant genotypes, including ‘XQ9-5’, ‘XQ33-9’, ‘XQ37-5’, ‘XQ42-9’, ‘XQ45-7’ and ‘JCL’, were identified based on SI. Integrating the three parameters, it was clear that both hulls and seeds involved in PHS resistance in intact spikes of hulless barley and there was no long-existent embryo dormancy found among the test genotypes. All the genotypes, except ‘XQ45-7’, had detectable α-amylase activity on the 4th day after germination. There was PHS resistance imposed by the hull and seed per se and the two factors can act together or independent of each other. Besides, landraces or Tibet hulless barley had a wider variation and relatively more PHS resistance when compared with cultivars or Sichuan hulless barley. No significant difference was found among hulless barley of different seed colors. The correlation analysis showed PHS resistance was negatively related to hundred grain weight, days to flowering, days to maturity, spike length, awn length and flag length but not related to plant height. This study provides essential information and several donor parents for breeding of resistance to PHS. 3. Alpha-amylase isozymes are encoded by a family of multigenes. They highly express in germinating seeds and is closely related to seed germination ability. In barley germinating seeds, the activity of high pI α-amylase is much higher than low pI α-amylase. The aim of this study was to determine the relationship between preharvest sprouting resistance of hulless barley and the gene structure of Amy1 gene which encodes high pI α-amylase. The coding region and cDNA of Amy1 gene of three resistant accessions, including ‘XQ32-5’ (TR1), ‘XQ37-5’ (TR2), ‘XQ45-7’ (TR3), two susceptible accessions ‘97-15’ (TS1), ‘9657’ (TS2) and one highly dormant barley accession ‘SAMSON’ (SAM) was cloned. Analysis of their DNA sequences revealed there were three exons and two introns in Amy1 gene. Thirteen variable sites were in exon2 and exon3, 2 variable sites were in intron2. SAM and TS1 had a GAACT insert segment in the same site in intron2. Only 8 variable sites caused the change of amino acid residues. There were 99% of similarity between the tested hulless barley and some of the variable sites might be related with preharvest sprouting resistance. Then, we investigated the expression level of Amy1 gene in the 7-day germination test. Results of quantitative real-time PCR indicated that the relative expression trends of Amy1 gene were the same but had significant differences in the increase fashion between hulless barleys and no detectable expression was found in SAM. Susceptible accessions had earlier expression and faster increase and reached the maximum on day 4 ~ day 5. Besides, total transcripts level was found lower in resistant accessions than susceptible accessions. This study indicated that α-amylase activity was highly related to the transcription level of Amy1 gene which not correlated to missense mutation sites. In conclusion, hulless barley, especially the landraces from Qinghai-Tibetan Plateau in China possesses high degree of variation in PHS performance, which indicates the potential of Tibetan hulless barley as a good source for breeding of resistance to PHS. This study provides several donor parents for breeding of resistance to PHS. Our results also demonstrate that agronomic traits may be used as assistants for PHS resistance selection in hulless barley. Besides, analysis of high pI α-amylase coding gene Amy1 revealed the relative high expression of was Amy1 one of the mainly reason of different PHS resistance level in hulless barley.

小麦中微量元素锌铁硒的含量的分析

本研究应用微波消解ICP-AES 法对62 个小麦品种及3 个地区土壤的锌铁硒含量进行了分析测定，发现不同小麦品种中微量元素含量差异很大，姊妹系间也存在差异。含铁量最高与最低的小麦品种铁含量相差29.68mg/kg。含锌量最高与最低的小麦品种锌含量相差46.70 mg/kg。含硒量最高与最低的小麦品种硒含量相差0.056 mg/kg。对不同地点的小麦及土壤中锌铁硒含量进行方差分析，发现双流和西昌两地种植小麦的铁含量和硒含量均有显著差异，西昌和荣县种植的锌含量有显著差异。在3 个地点中双流种植小麦硒含量最高，西昌种植小麦的铁和锌含量最高。 通过对小麦微量元素含量与土壤中微量元素含量进行了相关性分析，结果表明：小麦中的锌铁含量与土壤中的锌铁含量呈显著正相关，土壤中铁与锌含量呈极显著正相关，小麦中铁与锌含量也呈极显著正相关。随着土壤微量元素锌铁的提高，小麦中的锌铁元素含量同时提高，而且小麦对两种元素的吸收互相促进。土壤中的硒含量与锌铁含量呈负相关。小麦中硒含量也与锌铁含量也呈负相关。说明锌和铁与硒互相拮抗。小麦硒含量与土壤硒含量呈正相关，但不显著。表明土壤硒含量可以影响小麦硒含量，但不是决定因素，小麦硒含量与小麦自身因素有关。 对姊妹系G290（高硒含量）和G289（低硒含量）进行抗重金属胁迫和抗旱性实验发现，高硒品种G290的抗逆性优于低硒品种G289。 利用RAPD 技术对7 个姊妹系进行遗传差异分析发现，高硒材料G290出现了特异条带，分别标为1、2、3、4，其他姊妹系品种中未发现特异条带，回收4 条特异条带并连接转化，得到目的片段1、2、3 的重组子，进行测序。NCBI 中结果显示没有找到植物中的同源序列，说明特异序列可能是未发现的基因片段，推测可能与小麦硒含量有关，有待进一步研究。 以上研究结果，对小麦营养研究及功能性小麦的筛选和栽培具有指导作用。 In this study, we determinated the contents of zinc, iron, selenium in 62 wheat cultivars and soil samples of three regions by method of microwave digestion/ ICPAES,found that there was great difference of zinc, iron, selenium contents in different wheat cultivars as well as different sister lines. Iron content difference was 29.68 mg/kg between the highest-iron-content cultivar and the lowest one, and zinc content difference was 46.70 mg/kg , selenium content difference was 0.056 mg/kg. Anova analysis was made on contents of zinc, iron, selenium in wheat and soil samples of different locations, significant differences of Fe and Se contents were found between wheat in Shuangliu and Xichang, significant difference of Zn content was found between wheat in Xichang and Rongxian. Se content in wheat of Shuangliu was highest, Fe and Zn contents in wheat of Xichang were highest. Relativity analysis was made on three trace elements in Wheat and in soil, the result showed that there was significant positive correlation of zinc, iron content between in Wheat and in soil, as well as between Fe and Zn both in wheat and in soil. With the improving of Zn, Fe contents in soil, contents of Zn and Fe in wheat increased and absorption of Zn and Fe in wheat will mutual promote. Negative correlation of Se and Zn contents was found in wheat and soil, but not significant, that meant the antagonism of Se and Zn. Positive correlation of Se content in wheat and soil was found. High selenium content G290 and low selenium content G289 in sister lines were selected for heavy metal stress and drought resistance experiments, the result showed that the resistance of high-selenium-content cultivar was better than low selenium one. Analysis on genetic difference was made by RAPD, and specific bands were selected, marked 1,2,3,4, no more specific bands were found in other sister lines.4 bands were recovered, ligated to T-vector and transformed E.coli. Three recombinant plasmids were obtained and sequenced. NCBI Blast showed there was no homology with other plants. It implied that these fragments probably be new genes and maybe were related to selenium in wheat. It needs further research. This paper would be useful for the study of wheat nutrition as well as selection and cultivation of functional wheat.

Effects of ion beam irradiation on adventitious shoot regeneration from in vitro leaf explants of Saintpaulia ionahta

The effects of 960 MeV carbon ion beam and 8 MeV X-ray irradiation on adventitious shoots from in vitro leaf explants of two different Saintpaulia ionahta (Mauve and Indikon) cultivars were studied with regard to tissue increase, shoots differentiation and morphology changes in the shoots. The experimental results showed that the survival fraction of shoot formation for the Mauve and Indikon irradiated with the carbon ion beam at 20 Gy were 0.715 and 0.600, respectively, while those for both the cultivars exposed to the Xray irradiation at the same dose were 1.000. Relative biological effectiveness (RBE) of Mauve with respect to X-ray was about two. Secondly, the percentage of regenerating explants with malformed shoots in all Mauve regenerating explants irradiated with carbon ion beam at 20 Gy accounted for 49.6%, while that irradiated with the same dose of X-ray irradiation was only 4.7%; as for Saintpatdia ionahta Indikon irradiated with 20 Gy carbon ion beam, the percentage was 43.3%, which was higher than that of X-ray irradiation. Last, many chlorophyll deficient and other varieties of mutants were obtained in this study. Based on the results above, it can be concluded that the effect of mutation induction by carbon ion beam irradiation on the leaf explants of Saintpaulia ionahta is better than that by X-ray irradiation; and the optimal mutagenic dose varies from 20 Gy to 25 Gy for carbon ion beam irradiation.

黄土高原旱作小麦化感表达在根际土中的时空异质性研究

采用盆栽试验基于统计学方法,对4种不同品种小麦在4个连续生长期根系表层土壤(0~20cm)化感潜势时空异质性进行了研究。结果表明:不同品种小麦之间根系土壤化感潜势差异极显著(P=0.01),随生育期变化,化感潜力的变异与品种有关,一般15cm为普通小麦耕层土壤化感潜力的转折位点。变异函数分析显示,4种小麦根系土壤化感潜势变化是独立、随机、异质性的。"碧玛1号"、"丰产3号"、"宁冬1号"、"小偃22号"变异函数理论模型分别为线形模型、球型模型、指数模型和高斯模型。其中"小偃22号"的化感背景值和化感潜力空间结构比随生育期增大,导致0~20cm表层土壤化感表达具有高度的空间异质性。4种普通小麦的化感表达均存在时空异质性,其中"宁冬1号"的表层土壤化感表达具有很好的分形特征,其空间分布格局的变异存在尺度依赖。"宁冬1号"化感潜势在表层土壤的空间分布趋于离散表达,化感潜力的有效纵向半径和有效延深半径分别为5cm和14cm。这种时空变异格局可能与根系发育特征、根系翻转运动及土壤环境有关,化感实施过程可能为熵增过程。根系表层土壤化感潜势时空异质性的研究可为监测化感作用实施,定位有效化感物质和合理利用土壤化感潜势提供理论...

小麦抗旱生态分类中适合性聚类方法的研究

探索了适合于小麦品种抗旱生态分类的聚类方法 .选用 2 1个农艺性状和 15个冬小麦品种 (系 ) ,在聚类分析的各环节上 ,通过采用不同的策略 ,大规模进行了各种分类结果的比较 .结果表明 ,在与专家经验分类接近程度上 ,数据转换方法中 ,原始数据法依次大于普通相关阵基础上的方差极大正交旋转法、Promax斜交旋转法、主成份法 ;相似性度量上 ,欧氏距离大于马氏距离 ;聚类方式上 ,对应分析法和模糊聚类法大于最短距离法、最长距离法、类平均法 ;所有可组合的方法中 ,以对应分析法和直接用原始数据的模糊聚类法的分类结果最接近专家经验分类 .结合各方法理论上优缺点的分析与检验 ,认为这两种方法也是较理想的方法 .

根际沉积及其在植物-土壤碳循环中的作用

植物根际沉积是一种重要的植物与土壤交换的界面过程,在土壤碳周转方面具有重要的作用;根际碳的沉积也是联系植物、土壤及微生物的桥梁.本文就近年来关于根际沉积中碳平衡、碳循环等相关研究,阐述了根际碳沉积的机制,探讨了相关试验中存在的问题,以及不同植物品种、种类和生育期根际沉积的差异和根际沉积物与土壤呼吸的关系,指出了根际沉积在植物-土壤体系中碳循环的重要作用.在此基础上,提出了未来的研究领域及方向.

A biofuel cell harvesting energy from glucose-air and fruit juice-air

The membraneless biofuel cell (BFC) is facile prepared based on glucose oxidase and laccase as anodic and cathodic catalyst, respectively, by using 1,1'-dicarboxyferrocene as the mediators of both anode and cathode. The BFC can work by taking glucose as fuel in air-saturated solution, in which air serves as the oxidizer of the cathode. More interestingly, the fruit juice containing glucose, e.g. grape, banana or orange juice as the fuels substituting for glucose can make the BFC work. The BFC shows several advantages which have not been reported to our knowledge: (1) it is membraneless BFC which can work with same mediator on both anode and cathode; (2) fruit juice can act as fuels of BFCs substituting for usually used glucose; (3) especially, the orange juice can greatly enhance the power output rather than that of glucose, grape or banana juice. Besides, the facile and simple preparation procedure and easy accessibility of fruit juice as well as air being whenever and everywhere imply that our system has promising potential for the development and practical application of BFCs.