RAPD Analyse Of Soybean Mitochondrial DNA Of Sterile Line And Its Maintainer

With the development of molecular biology, the research about mechanism of cytoplasmic male sterility (CMS )has made generous progress. In the mitochondria genome of maize, petunia, rice, wheat, brassica napus, sunflower and common bean, some seats of gene may relate to CMS had already been found. Compared with other crops, soybean CMS researches made little progress. Since Jilin Academy of Agricultural Sciences's Huan Sun found the first soybean CMS line since 1993, gratifying progress of studying and using was made in this field, but the report on its molecule mechanism still belonged to the blank.Three pairs cytoplasmic male sterile line and its maintainer were used in this experiment. (JLCMS1-1A, JLCMS1-1B , JLCMS1-2A, JLCMS1-2B , JLCMS1-3A, JLCMS1-3B) . We amplified mitochondria genome of soybean cytoplasmic male sterile line and with Randomly Amplified Polymorphic DNA(RAPD) technology, and wanted to find different between sterile line and its maintainer. This was the foundation for the isolation and identification of the relevant genes of CMS soybean, and offered the theoretical foundation for the molecularmechanism of announcing the soybean cytoplasm male sterility (CMS ).The quality of template DNA in RAPD analyse of the soybean mitochondria genome will be high and can't contain the pollution of proteins or nuclear DNA ,etc. Referencing some abstraction methods of other crop mitochondrial DNA, some steps were modified and obtained high quality mtDNA.Evaluation of mtDNA by gel electrophoresis,measured OD by UV spectrophotometer and well digested by the restriction endonuclease. finally its rate was indicated and the purity may satisfy PCR amplify,the soybean mtDNA samples were ready to be used as templates for random amplified polymorphic DNA(RAPD).This research has set up steady and high-efficient reflected system of RAPD at first, then JLCMS1-1A male sterile line and its maintainer line JLCMS1-1B, JLCMS1-2A male sterile line and its maintainer line JLCMS1-2B, JLCMS1-3A male sterile line and its maintainer line JLCMS1-3B mitochondrial DNA were screened by 400 RAPD single-primers from OPA to OPT and 14 paires double-primers . the optimum conditions in 25 μ L volume was as follows: dNTPs concentration 0. 1mmol/L ; Taq enzyme concentration 2U;sample DNA concentration 0.4ng/ u l;primer concentration 0.8ng/ μ L .In RAPD analyse, some peculiar strips only presented on the sterile line, and absented on its maintainer.as OPE01 , OPH07 , OPI12 , OPK04 , OPP17 and OPN03 they had one peculiar strip on sterile line,as OPD02 and OPD20 presented two strips. Mitochondrial genome of plant maybe take place mutation and make for CMS. These peculiar passage thatonly presented on the sterile line showed that mitochondrial genome of sterile line maybe occur some obtained mutions .These mutains causeed the cytoplasm male sterility of soybeans finally.In addition, RAPD analyse still showed that there were 16 primers (OPA12, OPB06, OPD11, OPD18, 0PH18, OPI06, OPI09, OPK07 , OPL05 , OPL10 , OPL13 , OPM06 , OPP09 , OPP15 , OPQ13 , OPR03) kept one or some special amplified production. These peculiar passages that only presented on its maintainer not the sterile line showed that mitochondrial genome of maintainer line maybe occur some absence mutions.The number of obeation mutions were more than absence mutions. This may reflect among the long-term evolution, maintainer line mitochondrial DNA had a strong conservation, and the sterility line mitochondrial DNA was easy to be influenced by inherent or external factor in the nucleus and cytoplasm fashion, caused to some sequences occur changes , and then losed more combination chances to the same primer. The pvs gene of the common bean caused sterile because of its absence in the sterile line. So the specical amplified productions on the maintainer line had great value for the study of cytoplasmic male sterility in soybean.In double-primer amplify , there were 4 groups (OPF03/OPF06, OPB20/OPI14, OPG13/OPP17, OPG13/OPD18)amplified results same to corresponding single-primer's results in the 14 group s double-primer, the results of the other 10 groups had difference to corresponding single-primer's results. This explained that double-primer RAPD analyse can reveal some polymorphic that single- primer RAPD analyse can not reveal.This research proved that we can measure polymorphic of mitochondrial DNA of sterile line and its maintainer with RAPD technology. We found some special amplified productions of mitochondrial DNA of soybean through RAPD,these amplifies maybe relate to CMS of soybean. These differences were studied between sterile line and its maintainer. Retrieving these difference passages, clone, sequencing and analysising the structure and function of the gene, confirming which are the sterile gene further, and establish the foundation for the mechanism of studying the cytoplasm male sterility of soybeans on the molecule level.