棉花晋A细胞质雄性不育系及其保持系线粒体基因组文库的构建  

BAC Library Construction for Mitochondrial Genome of JA Cytoplasmic Male Sterile and Maintainer Lines in Cotton

作　　者：黄晋玲[1] 胡建斌[1] 张锐[1] 郭三堆[1] 

机构地区：[1]中国农业科学院生物技术研究所

出　　处：《中国农业科学》2008年第6期1603-1610,共8页Scientia Agricultura Sinica

基　　金：国家“863”项目(AA211050)

摘　　要：【目的】构建棉花晋A细胞质雄性不育系及其保持系的线粒体BAC文库,为研究棉花线粒体基因组结构以及棉花细胞质雄性不育的形成机理提供基础。【方法】在借鉴其它作物的线粒体基因组BAC文库构建方法的基础上,对棉花线粒体DNA的提取及其BAC文库的构建进行探索和优化,构建了棉花晋A细胞质雄性不育系及其保持系的线粒体BAC文库,并采用同源克隆的方法克隆棉花线粒体的功能基因。【结果】构建的不育系和保持系的线粒体基因组文库分别包括2600个克隆,插入DNA片段大小在10.3kb和37.5kb之间,平均分别为22.29kb和21.36kb。重组子的覆盖率分别达到棉花线粒体基因组的79倍和76倍。获得了3个棉花线粒体功能基因序列,通过比较,证明晋A不育系与其保持系的这3个基因序列无差异;以这3个基因为探针筛选文库,均获得阳性克隆。【结论】分别构建了棉花晋A细胞质雄性不育系及其保持系线粒体基因组的BAC文库。获得了晋A不育系与其保持系的orfB,coxⅠ和nad4L3个基因的全序列,通过比较,证明晋A不育系与其保持系在orfB、coxⅠ和nad4L基因全序列上无差异。【Objective】The experiment was carried out to construct mitochondrial genome BAC libraries of cotton JA cytoplasmic male sterile （JA CMS） and its maintainer lines （JB）. 【Method】Based on the methods used for constructing mitochondrial genome libraries of other crops,a high-efficient technology system of BAC library construction was established. This system consists of isolation of mitochondrion,preparation of high-molecular weight （HMW） mtDNA,partial digestion of megabase DNA,selection of digested fragment and highly-efficient transformation. A set of primers were designed according to the conserved sequence of the orfB,coxⅠ and nad4L coding region in other plants,and subsequently used to amplify these mitochondrial genes in cotton. The PCR products were cloned into vectors and further sequenced in both direction. 【Result】Two mitochondrial genome BAC libraries were constructed using cotton JA CMS and JB. Each library contains about 2 600 clones. The average insert size was 22.29 kb for JA CMS library and 21.36 kb for JB,ranging from 10.3 kb to 37.5 kb. The two libraries covered cotton mitochondrial genome by 79 and 76 folds,respectively. The genes orfB,coxⅠ and nad4L were amplified using mitochondrial DNA as template,and their sequences were no different between JA CMS and JB. The coding sequences of mitochondrial genes orfB,coxⅠ and nad4L cloned were used as probes to screen the two libraries,and the positive clones were identified by colony in situ hybridization. 【Conclusion】Two mitochondrial genome BAC libraries of cotton JA CMS and JB were constructed respectively. The obtained full-length DNA sequence of orfB,coxⅠ and nad4L from JA CMS and JB showed no difference.

关 键 词：棉花 细胞质雄性不育 线粒体基因组 细菌人工染色体(BAC)文库 

分 类 号：S562[农业科学—作物学] Q943[生物学—植物学]