Isolation And Sequence Analysis Of Coxâ…¡, Coxâ…¢ Genes In Cytoplasmic Male Sterility Soybean

Cytoplasmic male sterility (CMS) in plant is a phenomenon of failing to produce functionalpollen in the course of sexual propagation. It is a frequently encountered phenotype in floweringplants. In 1921, CMS was first observed in flax, and so far, it has been found in maize, broomcorn,rice, wheat, rape and sunflower. Now, the CMS has been observed in about 200 kinds of plant andplays an important role in heterosis application, which could lead to manpower saving, reducingseeds production cost, seeds quality and accelerating the process of Crop genetic improvement.Great success has been achieved in the development of hybrid varieties in several major crops,including rice and maize. Due to lacking of genetic male sterile resources or mutants, soybean isone of a few crop species, in which heterosis has not been exploited until in 1993 Huan Sun et al.found the soybean CMS line after about twenty years' work, which promoted the research andapplication in this area, however, there is still no work was reported on molecule mechanism untillnow.At present, the researches about the mechanism of CMS are all focusing on mitochondrialgenome analysis.As we known, in mitochdrial genomes, the genes that coding protein arecytochrome oxidase subunitⅠ,Ⅱ,Ⅲ, FO-F1ATPase complex, ribosome protein subunit rps4,rps13,rps14 and NADH-coenzyme Q oxidoreductase complex. In addition, there are threerRNA(5S,18S and 26S rRNA)genes in mitochondrial genome. Morever, there are someunidentified ORFs and URFs, and large number of non-coding sequences in mitochondrial genome.Mitochondrial cytochrome oxidase subunitⅡ,subunitⅢare the important enzyme systemmembers of reducing oxygen into water in plant respiraton. Now, in CMS maize,petunia,rice,wheat,grain sorghum and cole et al., CoxⅠ,CoxⅡ,CoxⅢ,atp6,atp9,atpA and cob, whichare associated with mitochondrial energy metabolism, have obvious discrepancy according to thesterile line in gene coding sequence,gene organization or copy numbers and have been proved tohave some relationshipwith CMS.In this paper, primers were designed according to soybean Mitochondrial cytochrome oxidasesubunitⅡ,subunitⅢsequences and the two genes were isolated and analysed from the CMSsoybean from Jilin Academy of agricultural science.The main results are as follows: 1. The soybean mitochondrial DNA purification system was established for PCR after repetitiveexperiments.2. A high-efficient and stable PCR system was set up in this paper, and the optimum PCRcomponents and reaction conditions were determined in 25μl volume as follows: dNTPsconcentration 0.1 mmol/l; rTaq enzyme concentration 1.5 U; DNA concentration 0.4 ng/μl; primerconcentration 0.8 ng/μl.3. Cytochorome oxidase subunitⅡgenes were isolated from mitochondrial DNA of 9B andliao82B and coding a protein consisting 280 amino acids. And also cytochorome oxidase subunitⅢgenes were isolated from mitochondrial DNA of 9A,9B and liao82A,liao82B, and coding aprotein consisting 213 amino acids.