Bacterial Artificial Chromosome Library Construction And Character For Mitochondrial Genome Of Cytoplasmic Male Sterility From Cotton

The cotton is one of the main industrial crops in our country, With the textile industry and the lives of the people level enhancement, China already become the biggest cotton producer country and expense country in the world.With increasing unceasingly to cotton's demand, in order to meet people's need, the cultivation of high production, the high quality cotton new variety to solve the question is an inevitable way. Now, the world cotton breeding main research direction using the cotton cytoplasmic male-sterile line cultivation cotton hybrid variety. The massive research indicate that cytoplasmic male sterility has the close relation with the mitochondrial DNA variation. Therefore, in order to research cytoplasmic male sterility with mitochondrial DNA between relations further, this research will construct cotton mitochondrial DNA bacterial artificial chromosome library, and has carried on the preliminary appraisal to the library, male sterility builds the solid foundation for a more thorough understanding and explanation cytoplasm with the mitochondrion between relations.This research toke the upland cotton (Gossypium hirsutum) cytoplasmic male-sterile line P30A etiolation seedling as the material. Reference rice, maize and other crops'mitochondrial DNA bacterial artificial chromosome library construction methods, and unifies cotton itself to include the gossipol, tannin easily to oxidize materials'particularity, established a high quality mitochondrial DNA's BAC library system. This system contained three very important conditions: the high molecular weight mtDNA extraction,dephosphorization effect good carrier preparation, transformed efficiency high electrocompeten cell preparation.In the process of separating mitochondrion and its DNA, the mitochondrion which obtained using discontinuous sucrose gradient centrifugation ,after the fluoroscope examination indicated that its integrity was good, did not have pollutions such as chloroplast and cell nucleus and so on. We obtained mitochondrial DNA using the embedding decomposition method. And examine the purity using the digesting with HindⅢand BamHⅠ, PCR applifiction. The results indicated that the mitochondrial DNA purity was high, the quality was good; the dephosphorization effect was good. Carries on the examination to prepare the electrocompeten cell transformation efficiency, indicated its had achieved 1.22×1010cfu/μg, namely was more than 1010cfu/μg. Further indicated that, the above three conditions completely conform to the construction cotton mitochondrial DNA bacterial artificial chromosome library requirement.This research used establish BAC library construction technology system, and successfully constructed upland cotton cytoplasmic male-sterile line P30A mitochondrial DNA bacterial artificial chromosome library. This library contained about 3840 colonies ,average insert fragment approximately 15.5kb, the reorganization rate is more than 93% approximately, empty clones rate is less than 6.23%., the stability is good, the coverage fraction surpasses 80 times. In addition, directs the thing using the mitochondrial DNA to carry on the PCR examination to the establishment 10 mix poors, in 10 mix poors all included the positive clone. It was proved that the BAC library had certain representation, the library quality was high, for the following fertility gene and the understanding mitochondrial DNA structure, the analysis function gene, the mitochondrial DNA sequence measured the grade had built the technical platform.