Construction Of BAC Library For Mitochondrial Genome Of JA Sterile And Maintainter Lines In Cotton And Analysis Of Functional Genes

Cytoplasmic male sterility (CMS) is a widespread phenomenon observed in＞150 flowering plant species. CMS is a maternally inherited trait and is often associated with unusual open reading frames (ORFs) found in mitochondrial genomes. Two kinds mitochondrial genome BAC library were constructed using JA cytoplasmic male sterile and its maintainer lines. The library construction of mitochondrial genome is a necessary step to clone gene and research the structure of the genome.The results can be summarized as follow:1. 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.2. The sterile line and maintainer line of JA-type cotton were used as materials, and BAC libraries of their genome were constructed.(1) Each library consisted of about 2500 clones with average insert size 22.29kb(or 21.36kb), ranging from 10.3kb to 37.5kb. 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. The deep coverage and the large insert size of the libraries will facilitate physical mapping, isolation, and cloning of cotton mitochondrial genes.(2) The BAC clone could be amplified by culturing for 100 generations without changing the structure of BAC clone.3. We designed a set of primers based on the conserved sequences of the orfB,coxⅠ,atpA,nad4L and atp6 coding region. 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.4. It was testified that orfB, coxⅠand nad4L were conversed between Jin-A cytoplasmic male sterile and its maintainer lines.5. We obtained all positive clones from two BAC library by colony in situ hybridization of orfB, coxⅠ, atpA, nad4L and atp6. 6. It is documented that plant mtDNAs contain many repeated sequence and sequences of foreign origin. Chloroplast DNA (cpDNA) sequences and sequences of nuclear origin are found in cotton mitochondrial genome.