Cloning And Sequence Analyzing Of Fragments Associated With MtDNA In Cytoplasm Male-sterile Line Of Cotton

Cotton is the main economic crop in our country. Using the cytoplasmic male-sterility (CMS) of cotton to produce hybrids is one of the major objectives in the present world cotton breeding, and making larger benefits of socity and economic. Because the forming mechanism CMS is closely associated with mtDNA in high plant, the cloning mitochondrial gene in cotton cytoplasmic male sterility line will be the important method to further research the structure of the mitochondrial genome in CMS cotton and has an important value for enrichment of cotton CMS mechanism.In this study, we use etiolated cotton for our mertrerile to extract mtDNA. Electrophoresis detection showed that the mitochondrial DNA were quite pure, no degeneration and could be used for PCR analysis. After analyzing the alignment of many other plant mitochondrial gene(orfB,cox1, nad4L,atp6,cox3,cob,nad9,rps12,atp9,matr and atpA), 11 pairs of primers(ob,c1,n4,a6,c3,cb,n9,rp,a9,mr and aa) were used for amplifing the gene of mtDNA in P53A by means of PCR technique. The expected products were obtained through 11 pairs of primers. Some of clones were selected randomly to be sequenced.The sequence were compared with those in NCB1/Blast/nr database and NCB1/Blast/protein database and were analyzed with DNAstar. The results are as following: ob sequence showed more than 92% similarity ratios to the sequence of orfB gene in other plant, and showed more than 88% similarity ratios to the sequence of ORFB; c1 sequence showed more than 96% similarity ratios to the sequence of cox1 gene in other plant, and showed more than 98% similarity ratios to the sequence of cytochrome oxidase subunit 1; n4 sequence showed more than 96% similarity ratios to the sequence of nad4L gene in other plant, and showed more than 93% similarity ratios to the sequence of NADH dehydrogenase subunit 4L; a6 sequence showed more than 94% similarity ratios to the sequence of atp6 gene in other plant, and showed more than 93% similarity ratios to the sequence of ATP synthase protein 6; c3 sequence showed more than 94% similarity ratios to the sequence of cox3 gene in other plant, and showed more than 96% similarity ratios to the sequence of cytochrome oxidase subunit 3; cb sequence showed more than 98% similarity ratios to the sequence of cob gene in other plant, and showed more than 97% similarity ratios to the sequence of cytochrome b; n9 sequence showed more than 98% similarity ratios to the sequence of nad9 gene in other plant, and showed more than 97% similarity ratios to the sequence of NADH dehydrogenase subunit 9; rp sequence showed more than 97% similarity ratios to the sequence of rps 12 gene in other plant, and showed more than 94% similarity ratios to the sequence of ribosomal protein S12; a9 sequence showed more than 95% similarity ratios to the sequence of atp9 gene in other plant, and showed more than 93% similarity ratios to the sequence of ATP synthase protein 9. Howerery, mr sequence and aa sequence have very low similarity ratios, we think they maybe were fake clone.These suggested that 9 sequences were mitochondrial gene and 2 sequence were fake clone in 11 sequence, ob,c3,n4 sequence were whole CDS sequence. And we found all have, except for rp sequence, that same as in theory that plant mitochondrial genome largely inclueding repeat sequence especially invert repeat sequence.