Analysis Of Type-k And Type-G Of Rice Mitochondrail DNA Based On AFLP

Cytoplasmic male sterility (CMS) plays an important role in plant male sterility and heterosis application. Since three-type theory on plant male sterility was put foward by Sears in 1947, CMS has been the hot point in the reseach of plant genetic.And Plant male sterility was divided into cytoplasmic male sterility (CMS), nucleus male sterility (NMS)and cytoplasmic-nucleus male sterility (CNMS) according to genotype of male sterility. Rice male sterility was controlled by nuclear gene and cytoplasmic gene and showed phenomenon of nucleo-cytoplasmic interaction. It has been proven that there was close relationship between rice male sterility and mitochondria DNA (线粒体DNA). Therefore, it is necessary to carry out research on rice mitochondria gene to elucidate molecular mechanism of rice cytoplasmic male sterility.Amplified fragment length polymorphism (AFLP) was created by Zabeau and Vos from Dutch and great progress has been made in the past years. Compared to restriction rragment rength rolymorphism (RFLP),Simple Sequence Repeat Microsatellite(SSR) and random amplified polymorphic DNA( RAPD),AFLP has many good traits such as fewer DNA template, unwanted Southern blot, easy repetition, significant polymorphism. Therefore,AFLP is an ideal molecular model and is widely used in agriculture, forest, animal husbandry, fishery and medicine field such as genetic diversity analysis, germplasm verification, gene mapping, genetic map constructing and so on.In this study, Oryza sativa L cv Type-K male sterility K17A,maintainer line K17B, F1 generation K 17 and Type-G of male sterility G46A,maintainer line G46B, F1 generation G615 were used to explore the relationship between rice mitochondria DNA and male sterility based on AFLP method. A system was established to analyze rice mitochondria DNA using AFLP method. Moreover, distinct and significant polymorphism bands were observed which give helpful information for further study. The main results were as followings:1. A kind of method for rice mitochondria DNA extraction was established based on repeated experiments. Mitochondria DNA quality was good to meet AFLP analysis and other molecular study.2.Two hundred and sixty four pair primers were used to analyze the rice mitochondria DNA of Type-K and Type-G male sterility, maintainer line and F1 generation. Missing Bands was observed in male sterility and F1 generation as compare to the absence in maintainer line. It is consistent with the hypothesis of defect sterility.3. Seven pair primers were good for PCR amplification through preliminary screening. Nine different bands were observed which probably related to Type-K male sterility while three different bands which probably related to Type-g male sterility.4. A band of about 750 bp was observed in Type-K and Type-G male sterility and F1 generation using primer combinations E10M8(EcoRⅠ-ATA×MseⅠ-AGC)as compare to the absence in maintainer line. It is consistent with the hypothesis of defect sterility.5. The specific band from rice mitochondria DNA K17A,K-17 was reclaimed and purified for sequencing. The fragment was composed of 422 base pairs. Compare with other related sequences in GenBank,no relationship was observed between the sequence and the known specific fragment of rice nucleo-cytoplasmic interaction sterility although it was relative to expression of two unknown rice functional protein.6. The bands of 900bp, 350bp, 700bp and 450bp were observed at equal pace in Type-K and Type-G male sterility, maintainer line and F1 generation rice using the same primer combinations. It means that the same relative conservatism exits in rice线粒体DNA fragment.7. Using the same primer combinations, polymorphism of Type-K male sterility, maintainer line and F1 generation rice is more significant than that of Type-G's. Type-K's polymorphism was averaged to 37.33% and Type-K's polymorphism was averaged to 32.49%.