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

 【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2 600个克隆，插入DNA片段大小在10.3 kb和37.5 kb之间，平均分别为22.29 kb和21.36 kb。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nad4L 3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。

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

Abstract: 【Objective】Two kinds mitochondrial genome BAC library were constructed using JA cytoplasmic male sterile in cotton and its maintainer lines. 【Method】According to many methods of crops, a high-efficient technology system of BAC library construction was established. This system contains that isolation of mitochondrion, preparation of high-molecular weight（HMW）mtDNA, partial digestion of megabase DNA with a restriction enzyme, selection of digested fragment and transformation with high efficiency. We designed a set of primers based on the conserved sequences of the orfB、coxⅠ and nad4L coding region in other plants. These genes locus were subjected to PCR analysis using these primers. The PCR products using mitochondrial cotton DNA as a template were further sequenced after cloning.【Result】Two kinds mitochondrial genome BAC library were constructed using JA cytoplasmic male sterile in cotton and its maintainer lines. Each library consisted of about 2600 clones with average insert size 22.29kb(or 21.36kb), ranging from 10.3kb to 37.5kb. Three genes were colned from mitochondrial genome of cotton, and It was testified that orfB, coxⅠ and nad4L were conversed between Jin-A cytoplasmic male sterile and its maintainer lines. With mitochondrial genes orfB, coxⅠ and nad4L as probes, We obtained all positive clones from two BAC library by colony in situ hybridization.【Conclusion】It was calculated that each library contains approximate 79 and 76 times of cotton mitochondrial genome and provide 100% possibility to isolate any interested cotton genes or sequences in each library.