通过小麦Ms2近等基因系的抑制缩减杂交分析揭示小穗和花药中差异表达基因

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.

Suppression Subtractive Hybridization Analysis of Ms2 Near-isogenic Lines of Wheat Reveals Genes Differentially Expressed in Spikelets and Anthers

The dominant male sterility gene Ms2 inwheat has been widely used in recurrent selection andvariety improvement. Identification of genes associ-ated with the male sterility in Ms2-carrying wheat willhelp us understand how Ms2 functions. Using a pairof isogenic lines of Ms2, subtractive hybridization wasconducted with cDNA from bulked spikelets atmeiophase of sterile plants as the tester and cDNA fromthe same tissues of fertile plants as the driver. Twomajor bands at 270 bp and 450 bp were obtained bysuppression PCR (polymerase chain reaction) of thesubtractive cDNA. A total of 882 recombinants fromPCR product cloning were isolated for reverse North-ern analysis. The results demonstrated that up to 90%of the inserts in the library were up-regulated in thesterile spikelets. Twenty-one unique inserts from thislibrary were sequenced. Similarity search showed thateighteen of them were homologous to ESTs (expressionsequence tags) derived from spike or anther tissues atmeiophase. The chromosome locations of nine of theESTs were determined using C.S. (Chinese spring)nulli-tetrasomic lines, one of which was assigned tochromosome group 4 that includes chromosome 4Dwhere Ms2 is located. In addition, four additional ESTscould also be assigned to this group according to theirhomology to BACs (bacterial artificial chromosomes)or PAC (P1 artificial chromosomes) of rice chromo-some 3. The expression patterns of eight of the insertsexamined displayed increased expression in spikeletsand anthers of the sterile plants.