Zhang Zheng-Zhi, Ma Zheng-Qiang, Liu Da-Jun
National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China.
Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao. 2005 Apr;31(2):175-82.
The dominant male sterility gene Ms2 in wheat has been widely used in recurrent selection and variety improvement. Identification of genes associated with the male sterility in Ms2-carrying wheat will help us understand how Ms2 functions. Using a pair of isogenic lines of Ms2, subtractive hybridization was conducted with cDNA from bulked spikelets at meiophase of sterile plants as the tester and cDNA from the same tissues of fertile plants as the driver. Two major bands at 270 bp and 450 bp were obtained by suppression PCR (polymerase chain reaction) of the subtractive cDNA. A total of 882 recombinants from PCR product cloning were isolated for reverse Northern analysis. The results demonstrated that up to 90% of the inserts in the library were up-regulated in the sterile spikelets. Twenty-one unique inserts from this library were sequenced. Similarity search showed that eighteen of them were homologous to ESTs (expression sequence tags) derived from spike or anther tissues at meiophase. The chromosome locations of nine of the ESTs were determined using C.S. (Chinese spring) nulli-tetrasomic lines, one of which was assigned to chromosome group 4 that includes chromosome 4D where Ms2 is located. In addition, four additional ESTs could also be assigned to this group according to their homology to BACs (bacterial artificial chromosomes) or PAC (P1 artificial chromosomes) of rice chromosome 3. The expression patterns of eight of the inserts examined displayed increased expression in spikelets and anthers of the sterile plants.
小麦中显性雄性不育基因Ms2已广泛应用于轮回选择和品种改良。鉴定携带Ms2的小麦中与雄性不育相关的基因将有助于我们了解Ms2的功能。利用一对Ms2等基因系，以不育植株减数分裂期小穗混合cDNA为测试者，可育植株相同组织的cDNA为驱动者进行消减杂交。通过对消减cDNA进行抑制PCR（聚合酶链反应）获得了270 bp和450 bp的两条主要条带。从PCR产物克隆中分离出882个重组子用于反向Northern分析。结果表明，文库中高达90%的插入片段在不育小穗中上调表达。对该文库中的21个独特插入片段进行了测序。相似性搜索表明，其中18个与减数分裂期穗或花药组织来源 的EST（表达序列标签）同源。利用中国春缺体-四体 系确定了9个ESTs的染色体位置，其中一个被定位到包括Ms2所在的4D染色体的第4染色体组。此外，根据它们与水稻第3染色体的BAC（细菌人工染色体）或PAC（P1人工染色体）的同源性，另外4个ESTs也可定位到该组。所检测的8个插入片段在不育植株的小穗和花药中表达增加。
