【作者】 陈军方；

【导师】 任正隆； 贾继增；

【作者基本信息】 四川农业大学 ， 作物遗传育种， 2003， 博士

【摘要】 太谷核不育小麦是我国1972年发现的一份珍贵小麦材料，其独特之处主要表现在：其不育性受显性单基因控制，雄性不育彻底，至今未发现一例自交结实现象；雄性不育稳定，不受环境条件的影响；不育性只受显性基因ms2控制，不受遗传背景的影响。它是世界上发现的第一份小麦显性核不育材料，具有巨大的经济价值和理论研究价值。在此基础上育成的矮败小麦是具有矮秆基因标记的显性核不育材料，其显性不育基因ms2与显性矮秆基因Rht10紧密连锁，二者之间的连锁交换率不超过0.18％。本研究所用材料为以中国春为轮回亲本连续回交十二代的矮败中国春小麦近等基因系，该近等基因系的高杆可育及矮杆不育株除在育性、株高上存在差异外，其它遗传背景相同，是进行各项分子生物学研究的好材料。 利用拟南芥中已克隆的雄性核不育基因Ms2和水稻中假定雄性不育蛋白的保守区域，设计一对简并引物MSP，在太谷核不育小麦可育株及不育株花药中扩增出了一条134bp的片段，序列分析发现该片段在可育及不育花药cDNA间没有差异。该序列电子延伸得到一个长为1，604bp的小麦Contig，序列比较发现，该拼接Contig推测编码的氨基酸序列包含有一段由200个氨基酸组成的雄性不育保守区，与拟南芥核不育蛋白Ms2和水稻假定雄性不育蛋白同源性分别为88％和98％。与拟南芥雄性不育蛋白Ms2相同，推测的雄性不育保守区也含有一个微体结合信号序列：Gly-Arg-Ala。RT-PCR分析表明该拼接Contig只在小麦可育花药中表达，而在败育花药、叶片和根中不表达，说明该雄性不育同源基因属于花药发育特异基因，为太谷核不育基因ms2的一个下游基因。本研究表明基于同源序列的定位候选克隆策略与电子克隆技术相结合可大大加快基因克隆的速度。 本研究首先试图寻找与太谷核不育基因紧密连锁的分子标记，以矮败小麦的高杆可育及矮杆不育DNA为亲本，分别筛选了已定位在小麦4D染色体上的11对SSR引物和4个RFLP探针，另外还筛选了大麦、节节麦上的4个RFLP探针，结果这11对SSR引物和8个RFLP探针在可育及不育小麦DNA间均无差异。分别选取小麦4D染色体短臂上两个顶端区域内的4条EST序列设计4对PCR引物，在矮败小麦可育及不育基因组DNA中进行扩增，结果4对来自EST序列的PCR引物在矮败小麦中有较强的扩增带，但在可育及不育材料间均不表现多二衣 根据比较基因组的共线性原理，推断水稻第三染色体长臂上m:7-bcj区域与小麦4D染色体短臂上的msZ一Rht了O区域具有可能的共线性。其中水稻BAC克隆AC087797含有的赤霉酸反应迟钝基因伪，GAI与小麦4DS上的矮杆基因RhtIO高度同源，利用该BAC克隆中的22个假定基因设计22对PCR引物，在矮败小麦可育及不育基因组DNA中进行扩增，有扩增产物的16对引物在可育与不育基因组DNA间扩增条带均无差异。用可育及不育花药cDNA为探针与水稻22个基因做反向Northern杂交，表明水稻BAC克隆AC087797中的赤霉酸反应迟钝基因OsG月I在小麦花药中表达，推断小麦矮杆基因RhtIO在小麦花药中也表达。 在己有的SSR引物中，本研究未发现有与太谷核不育基因msZ连锁的标记，为了寻找与m对紧密连锁的SSR标记，本研究在从EST中开发新的SSR引物方面进行了尝试。小麦EST数量的迅速增加为开发新的SSR标记提供了宝贵的数据资源，从国际小麦族EST协作网(ITEC)上公布的1 0，380条EST序列中检索到444条含有SSR的EST序列，检出率为4.1%。其中含二核营酸重复单元和三核普酸重复单元的SSR一ESTs分别为34条(7.7%)和347条(78%)。利用这些小麦SSR一ESTs序列共设计135对cSSR引物，在矮败小麦近等基因系的高杆可育株和矮杆不育株基因组DNA间进行筛选，其中82对cSSR引物在小麦上有扩增产物，占所设计引物总数的61%，但这82对引物在两个亲本间均不表现差异。利用小麦一套缺体一四体系列，32对cSSR引物分别被定位到小麦除ZD、4B和4D外的18条染色体上，丰富了SSR引物来源。 为探讨太谷核不育小麦败育机制，本文利用。DNA一AFLP技术，以太谷核不育小麦减数分裂开始前的可育株及不育株花药mRNA为材料，对太谷核不育小麦败育发生过程中基因的差异表达进行了分析。结果表明，在花药减数分裂开始前，可育花药与不育花药中基因的表达存在很大差异。在所统计的144对引物组合扩增结果中，在gobp至622bp之间的扩增条带数共为2712条，平均每对引物了扩增出18.8条带纹。在所筛选的469对AFLP引物组合中合在可育株与不育株间表现多态，筛选出232条差异片段。共有193对引物组在反向Northem印迹鉴定的50条差异片段中共有30条为阳性克隆。对30条阳性克隆进行了序列测定和同源性比对分析，其中22条序列与GenBank中己知功能基因同源，17条序列在可育花药中特异表达或表达量高，5条序列在败育花药中特异表达或表达量高。24条序列与小麦EST同源，这些序列绝大多数来源于小麦减数分裂期花药的cDNA文库。其中1条可育花药特异表达序列与大麦参与成花的MADS一box保守结构域同源性高达94%，而该序列在败育花药中不表达。 总之，本研究从小麦EST中共开发新的EST一SSRS引物82对，其中32对定位到了小麦具体更多还原

【Abstract】 The Taigu Genie Male-Sterile Wheat is one of the most characteristic male sterile plants discovered up to the present, which is great valuable in genetic improvement by its stable inheritance, complete male abortion, and high cross-fertilize rate. Dwarfing-Sterile Wheat is a dwarf marked male sterile material, carrying the dominant gene ms2 for male sterility and dominant dwarf gene Rht10. The two genes are closely linked together on the short arm of chromosome 4D, with a crossing-over value as small as 0.18%. The material used in this study was the Dwarfing-Sterile Chinese Spring separated individual. In our research, several techniques such as RT-PCR, SSR, RFLP, cDNA-AFLP and in silic cloning were used to explore the co-separate marker of male sterile gene, clon the male sterile homology gene and to investigate the difference in gene expression between sterile and fertile anthers development of TGMSW. One 134bp fragment was amplified in male sterile and fertile wheat anthers using one pair of degenerated primer MSP designed based on conserved domain of MS2 gene in Arabidopsis thaliana and putative male sterile protein of Oryza sativa, and one l,604bp Contig was extended by in silico cloning based on the 134bp fragment. The comparison of sequence indicated that the putative amino acid encoded by the Contig include a 200bp conserved domain of male sterility, and this Contig expressed in male fertile wheat anthers, no expression in male sterile anthers, leaves and roots. This research demonstrated that the male steile homology sequence is specific to wheat anther development and the combination of candidate gene clon strategy based on the homologue sequence and in silico cloning technique can accelerated the gene clon.To find molecular markers tightly linked to the male sterility gene ms2 of wheat, 11 SSR markers on 4D chromosome, 8 RFLP probes, 4 PCR primers designed from EST on the short arm of chromosome 4D and 22 PCR primers designed from the BAG clon AC087797, which were located on the chromosome 3 of rice, were screened between the genomic DNA of male sterile and fertile plants of TGMSW. In the result,none polymorphism were found between the genomic DNA of male sterile and fertile plants of TGMSW. Reverse Northern blotting result indicated that the gene OsGAI of rice express in wheat anthers.The growing availability of EST sequences in wheat provides a potentially valuable source of new SSR markers. In this study, 444 SSR-ESTs were identified from 10,380 ESTs in the International Triticeae EST Cooperative database(ITEC), representing 4.1% of the total number of ESTs. 34 dinucleotide repeats and 347 trinucleotide repeats were found in these SSR-ESTs, representing 7.7% and 78% of the total. SSR-ESTs, respectively. 135 cSSR primers were designed to sequences flanking SSRs from 175 selected SSR-ESTs, of which 82 primers were efficient in wheat and 32 cSSR primers were located on 18 wheat chromosomes except 2D, 4B and 4D. In order to investigate the difference in gene expression between sterile and fertile anther development of Taigu Genie Male-Sterile(TGMSW), cDNA-AFLP was employed to compare the gene expression in pre-meiotic anthers between the male sterile and fertile plants of TGMSW NILs. In this study. 469 Pstl/Msel primer combinations were analysed in cDNA of male sterile and fertile anthers, as a result, a mean of 18.8 amplification products were detected from the above cDNA templates and 232 differentially expressed transcript derived fragments (TDFs) have been identified. Fifty differentially amplified PCR prodcuts specific to either male fertile or sterile anthers were excised from polyacrylamide gels, reamplified and directly sequenced following purification. Of the fifty sequenced fragments, thirty were confirmed by reverse northern hybridazation. Sequence analysis of the cloned PCR products revealed that twenty-two clones showed significant similarity to known genes in GenBank, of which seventeen sequences were specific to or shows high expression in male fertile anthers, Five sequences were spe更多还原