Cloning Of The Genes Associated With Mitochondria Genome Rearrangement In Brassica And Constructing Of MSH1-RNAi Line In Brassica Juncea

Brassica is a kind of oil-bearing plants consisting of many commercial crops in our country. Cytoplasmic male sterility (CMS) is the main way to utilize heterosis in Brassica, which is closely related with the recombination of mitochondria genome. The Mitochondria DNA (mtDNA) configurations are under surveillance of nuclear genes. Thus study on these nuclear genes is essential to explore the mechanism of mtDNA and it is also significant to analyze the mechanism of CMS.MSH1, OSB1and RecA3are the critical nuclear genes which participate in regulating mitochondrial genome recombination in plants. MSH1is MUTS HOMOLOG1. It functions as the maintainer of mitochondrial genome stability by suppressing the recombination of mtDNA.In this study, we clone the MSH1genes in Brassica species containing Brassica rapa(AA), Brassica nigra (BB), Brassica oleracea (CC), Brassica napus (AACC), Brassica juncea (AABB) and Brassica carinata (BBCC) to analyze the function and characteristics. Furthermore, I construct MSH1-RNAi Line in Brassica juncea to survey the phenotypic changes and the level of mtDNA recombination. The main results are shown as follows:1. MSH1gene cloning and characteristic analyzing in Brassica:MSH1sequences of B.rapa, B.napus were download from their whole sequences database; Sequences of B.nigra and B.juncea were from the whole genome sequencing project in our lab; Sequences of B.oleracea, B.carinata were cloned by using degenerated primers method. We find that sequences of MSH1gene were highly homologous in the Brassica species by multiple sequences alignment and phylogeny evolution analysis. All the MSH1genes contain22exons and21introns. The sequence of coding region is about3.3kb in length, which coding1120amino acid. MSH1genes were also found to consist of DNA mismatch repair region, which is special for maintaining the stability of mitochondrial genome.2. RecA3and OSB1genes’cloning and characteristics in Brassica:RecA3and OSB1were know to play important roles in DNA repair, reputation and recombination in mitochondrial genome. Thus we clone the RecA3and OSB1gene in Brassica species.3. Construction of MSH1-RNAi transgenic mustard:The MSHl-RNAi vector pGEG1008was transformed into mustard by agrobacterium-mediated genetic transformation. And total8To transgenic plants were gained which are confirmed by PCR.8transgenic plants show several phenotype variations, such as:leaf variegation and yellow or branching. We checked the relative expression of MSH1by RT-qPCR and found significant reduced MSH1expression in transgenic plants compared with wild type.4. The relative expression of MSH1in T1:T1generation were gained from strict selfing of To, the separation ratio of T1with the MSH1-RNAi (T1+) and T1without the MSH1-RNAi (T1) is near3:1. The results of RT-qPCR showed that the MSH1expression in PCR positive T1plants (T1+) are significantly reduced compared with the wild type, while the expression of MSH1in PCR negative T1(T1-) are return to wild type level. Growth rate of T1generation in seedling stage is much slower and the seedling size is generally smaller than wild type. Recombination of mtDNA was observed in T1generation, which resulted in substoichiometric shifting(SSS) of nad2gene.