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以Ms2 近等基因系处于减数分裂期的可育小穗cDNA 作为驱动因子(driver),以同一时期的不育小穗cDNA作为测验因子(tester)进行缩减杂交(SSH),将扩增后的缩减杂交产物进行克隆,构建了一个包含882 个重组克隆的SSH 文库。分别以可育小穗和不育小穗的cDNA 为探针与SSH 文库克隆进行反式Northern 杂交,结果显示接近90% 的克隆在不育小穗中呈上调表达。对文库中21 个克隆插入片段的序列相似性分析表明其中有18 个与来源于穗部或减数分裂期的花药cDNA 同源。13 个克隆的编码产物与已知功能的蛋白质同源,其中5 个参与碳代谢活动,4 个参与胞内分子的运输,2 个蛋白产物参与染色体的构成及染色体的结构变化,1 个是生长素抑制蛋白,1 个是转录因子。用中国春缺体四体材料对9 个克隆以Ms2 近等基因系处于减数分裂期的可育小穗cDNA 作为驱动因子(driver),以同一时期的不育小穗cDNA作为测验因子(tester)进行缩减杂交(SSH),将扩增后的缩减杂交产物进行克隆,构建了一个包含882 个重组克隆的SSH 文库。分别以可育小穗和不育小穗的cDNA 为探针与SSH 文库克隆进行反式Northern 杂交,结果显示接近90% 的克隆在不育小穗中呈上调表达。对文库中21 个克隆插入片段的序列相似性分析表明其中有18 个与来源于穗部或减数分裂期的花药cDNA 同源。13 个克隆的编码产物与已知功能的蛋白质同源,其中5 个参与碳代谢活动,4 个参与胞内分子的运输,2 个蛋白产物参与染色体的构成及染色体的结构变化,1 个是生长素抑制蛋白,1 个是转录因子。用中国春缺体四体材料对9 个克隆进行了染色体定位,其中一个克隆定位于第四染色体同源群,与Ms2 所在的染色体同属一个同源群。通过搜索水稻的同源BAC (bacterial artificialchromosome)和PAC (P1 artificial chromosome)克隆,推测另外11 个克隆的染色体位置,其中4 个克隆可能位于第四染色体同源群。用RNA点杂交对11 个克隆进行表达谱分析,其中8 个克隆在不育株的小穗和花药中呈上调表达。
In this study, spermatogenic spikelet cDNA of Ms2 near-isogenic line at meiosis stage was used as a driver and SSH of sterile spikelet cDNA of the same period was used as a tester. After amplification Was cloned to construct a SSH library containing 882 recombinant clones. Transgenic Northern hybridization was carried out using SSH cDNA clones from cDNAs of fertile spikelets and sterile spikelets respectively. The results showed that nearly 90% of clones were up-regulated in sterile spikelets. Sequence similarity analysis of 21 cloned insertions in the library showed that 18 of them were homologous to anther cDNA derived from panicles or meiosis. Thirteen cloned products were homologous to proteins of known function, five of which were involved in carbon metabolism, four involved intracellular trafficking of molecules, two protein products involved in the formation of chromosomes and structural changes in chromosomes, and one was growth Supressin, a transcription factor. Nine clones were used as driver of Chinese melanogaster (GenBank accession No.0) with the spikelet cDNA of Ms2 near-isogenic line at meiosis as driver, and the spikelet cDNA of the same period as tester (SSH). The cloned shortened hybridization product was cloned to construct a SSH library containing 882 recombinant clones. Transgenic Northern hybridization was carried out using SSH cDNA clones from cDNAs of fertile spikelets and sterile spikelets respectively. The results showed that nearly 90% of clones were up-regulated in sterile spikelets. Sequence similarity analysis of 21 cloned insertions in the library showed that 18 of them were homologous to anther cDNA derived from panicles or meiosis. Thirteen cloned products were homologous to proteins of known function, five of which were involved in carbon metabolism, four involved intracellular trafficking of molecules, two protein products involved in the formation of chromosomes and structural changes in chromosomes, and one was growth Supressin, a transcription factor. Chromosome mapping was carried out on nine clones using Chinese spring four-body material. One of the clones was located on the fourth chromosome homologous group, which belongs to the same homologous group as the chromosome in which Ms2 locates. The chromosome positions of the other 11 clones were inferred by searching for the clones of bacterial artificial chromosomes and PAC (artificial artificial insects, BACs) in rice, of which 4 were probably located on the fourth chromosome homologue. Eleven clones were analyzed by RNA dot blotting, of which eight were up-regulated in spikelets and anthers of sterile plants.