CAJ | 학술논문

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.
이Ms2근등기인계처우감수분렬기적가육소수cDNA작위구동인자(driver),이동일시기적불육소수cDNA작위측험인자(tester)진행축감잡교(SSH),장확증후적축감잡교산물진행극륭,구건료일개포함882개중조극륭적SSH문고.분별이가육소수화불육소수적cDNA위탐침여SSH문고극륭진행반식Northern잡교,결과현시접근90%적극륭재불육소수중정상조표체.대문고중21개극륭삽입편단적서렬상사성분석표명기중유18개여래원우수부혹감수분렬기적화약cDNA동원.13개극륭적편마산물여이지공능적단백질동원,기중5개삼여탄대사활동,4개삼여포내분자적운수,2개단백산물삼여염색체적구성급염색체적결구변화,1개시생장소억제단백,1개시전록인자.용중국춘결체사체재료대9개극륭진행료염색체정위,기중일개극륭정위우제사염색체동원군,여Ms2소재적염색체동속일개동원군.통과수색수도적동원BAC(bacterial artificialchromosome)화PAC(P1 artificial chromosome)극륭,추측령외11개극륭적염색체위치,기중4개극륭가능위우제사염색체동원군.용RNA점잡교대11개극륭진행표체보분석,기중8개극륭재불육주적소수화화약중정상조표체.
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 4Dwhere 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.