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.

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.

Cytological and Molecular Identification of Alien Chromatin in Giant Spike Wheat Germplasm

Alien chromosomes of twelve giant spike wheat germplasm lines were identified by C-banding, genomic in situ hybridization (GISH), sequence characterized amplified region (SCAR), and random amplified polymorphic DNA (RAPD). All lines showed a chromosome number of 2n = 42, five of them carried both a pair of wheat-rye (Triticum aestivum-Secale cereal) 1BL/1RS translocation chromosomes and a pair of Agropyron intermedium (Ai) chromosomes, three carried a pair of Ai chromosomes only, three others carried a pair of 1BL/1RS chromosomes only, and one carried neither 1BL/1BS nor Ai chromosome. Further identification revealed that the identical Ai chromosome in these germplasm lines substituted the chromosome 2D of common wheat (Triticum aestivum L.), designated as 2Ai. The genetic implication and further utilization of 2Ai in wheat improvement were also discussed.

Isolation and characterization of a novel variant of HMW glutenin subunit gene from the St genome of Pseudoroegneria stipifolia

The x- and y-type high molecular weight (HMW) glutenin subunits are conserved seed storage proteins in wheat and related species. Here we describe investigations on the HMW glutenin subunits from several Pseudoroegneria accessions. The electrophoretic mobilities of the HMW glutenin subunits from Pd. stipifolia, Pd tauri and Pd strigosa were much faster than those of orthologous wheat subunits, indicating that their protein size may be smaller than that of wheat subunits. The coding sequence of the Glu-1St1 subunit (encoded by the Pseudoroegneria stipifolia accession PI325181) was isolated, and found to represent the native open reading frame (ORF) by in vitro expression. The deduced amino acid sequence of Glu-1St1 matched with that determined from the native subunit by mass spectrometric analysis. The domain organization in Glu-1St1 showed high similarity with that of typical HMW glutenin subunits. However, Glu-1St1 exhibited several distinct characteristics. First, the length of its repetitive domain was substantially smaller than that of conventional subunits, which explains its much faster electrophoretic mobility in SDS-PAGE. Second, although the N-terminal domain of Glu-1St1 resembled that of y-type subunit, its C-terminal domain was more similar to that of x-type subunit. Third, the N- and C-terminat domains of Glu-1St1 shared conserved features with those of barley D-hordein, but the repeat motifs and the organization of its repetitive domain were more similar to those of HMW glutenin subunits than to D-hordein. We conclude that Glu-1St1 is a novel variant of HMW glutenin subunits. The analysis of Glu-1St1 may provide new insight into the evolution of HMW glutenin subunits in Triticeae species. (C) 2007 Elsevier Ltd. All rights reserved.

中国粮食生产的区域演进研究

Grain is one of the primary material conditions of the human survival and the grain production concerns the stability and development of the society directly. The regional patterns influence greatly on the grain production and the rational production distribution the regional comparative advantages and promotes grain production. This thesis starts with summarizing of the characteristics of changes and the overall trend of regional pattern of grain production of our country since 1949. Then it carries on network analyses to the factors, which influences the evolvement of regional grain production patterns of our country. And finally it gives some proposals to the grain production distribution in the future. The main content includes: Firstly, Reviewing the regional evolvement of grain production in our country, and analyzing the changes of the regional pattern of grain production of our country on the provincial scale and county scale separately, since 1949, especially since the reform and opening up policy. The main grain production areas are acting an important position in ensuring the national grain security, so this thesis analyses the main matter of the main grain production areas, forecasts the grain production situation in the future, and selects the Northeastern main grain production areas as the typical area to carry on the positive research. Secondly, this thesis analyzes the origin causes from two respects of natural and social economy of the regional evolvement pattern of grain production in China. Thirdly, based on the summarizing to the status of the regional pattern of the grain production, this thesis proposes the precept of the grain production distribution in the future in our country. Therefore, the areas of three major cereal crops, rice, wheat and corn, are confirmed on the basis of the comparative advantages. Finally, this thesis puts forward the security system of guaranteeing the grain production progressing steady in China. According to the above analysis, some conclusions have been achieved as follows: (1) The grain gross production gets on extricating itself from awkward position frequently while fluctuating greatly annually since 1949 in China. (2) Since the reform, its traditional regional pattern of grain production, the most of which was concentrated in the south area, has changed rapidly. China's center of gravity of grain production has shifted from the south to the north, and on the belts of latitude, the grain production has represented a trend of focusing to the middle area in China. (3) The main grain production areas play a very important role in ensuring China's food security. With their relative severe situation of the problems of agriculture, rural area and peasant, China has carried out a series of measures, which aim at improving the food-producing conditions of the main grain production areas, and enhancing the grain yields there. Under this condition, a forecast of the producing amount of the main grain production areas under the nation's self-supplying rate of over 95% shows that the increasing provision production in these areas can meet the demand of the country. (4) The natural and social economic factors influence together on the changes of the grain production regional pattern. Along with the state system transition and progress of agricultural science and technology, the regional pattern of grain production is affected heavier by the agricultural policy and technological elements. (5) The grain production will be concentrated to the middle province in the future, which economic development level being medium-sized; According to crop allocation, although the rice superiority production area located in the South, its comparative advantage index is little in some degree. Meanwhile, the wheat and corn superiority production areas are in the North mainly and its scale superiority and production level advantage are all comparatively obviously.