Functional Analysis Of BcRISP1 Gene In Non-Heading Chinese Cabbage

Cytoplasmic male sterile lines as an important material of cross breeding, the study on its molecular mechanism will be helpful for understanding the mechanism of pollen development and cytoplasmic inheritance. Cytochrome C reductase iron sulfur protein (RISP) is a subunit of the mitochondrial electron transport chain multimeric enzyme complexes which transfers an electron from a reduced quinol to cytochrome C. RISP is essential in mitochondrial respiratory chain, as an important guarantee for the mitochondria to produce energy used for organisms life activities. The research of BcRISP1 gene function in non-heading Chinese cabbage laid a good foundation on further understanding of the mechanism of cytoplasmic male sterility. We analysised the difference between sterile line and maintainer line by using suppression subtractive hybridization library of non-heading Chinese cabbage. BcRISP1 was found to be the differentially expressed between sterile line and maintainer line in non-heading Chinese cabbage. The full-length cDNA of BcRISP1 was obtained to screening the interactions genes by yeast two hybrid. To clear the relationship between expression of BcRISP1 gene and male sterility of non-heading Chinese cabbage, subcellular localization and transgenic experments were performed.The main results are as follows,1. BcRISP1 gene which was a member of ubiquinol-cytochrome C reductase iron-sulfur protein was cloned from a suppression subtractive hybridization cDNA library of non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino). The full-length cDNA of BcRISP1 was consisted of 810 bp and encoded 269 amino acids, which contained an ubiquinol cytochrome reductase transmembrane region (Ala77-Asp141) and a Rieske [2Fe-2S] domain (Val170-Lys266). The deduced amino acid sequence of the BcRISP1 cDNA revealed that it is with high homology to RISP members in other species. Quantitative reverse transcription-PCR (qRT-PCR) results showed that there was a significant increase of the BcRISP1 in sterile line at the archesporial cell stage when compared with that in maintainer line. Subcellular localization in transient expression system showed the GFP signals were mainly detected in the cytomembrane and nucleus. These findings suggested that BcRISP1 may play prominent roles in sterility of Pol CMS (cytoplasmic male sterility) in non-heading Chinese cabbage.2. The overexpression vector was constructed by Gateway technique and BcRISP1 was overexpressed in Arabidopsis thaliana by Agrobacterium tumefaciens-mediated transformation techniques. These screened transgenic plants were used for further study of BcRISPl function. Phenotypic analysis showed that the seed set was affected by the overexpression of BcRISP1, and shorter siliques with lower seed sets were observed in 35S::BcRISP1 Arabidopsis plants. qRT-PCR results showed that in 35S::BcRISP1 Arabidopsis plants the expression of mitochondrial respiratory chain related genes COXIO and RIP1 were enhanced, while the expression of QCR7 and SDH2-1 were reduced. Moreover, the reduced formation of pollen and impaired pollen tube growth was found. It implies that overexpression of BcRISP1 may reduce the fertility of transgenic Arabidopsis plants.3. The total RNA was isolated from Pol CMS flowers of non-heading Chinese cabbage and then double strand cDNA was obtained through LD-PCR approach. By using SMART technique and homologous re-organization method, we constructed the yeast two hybrid cDNA library in yeast Y187, which the conversion rate of the library was about 1.80×106 recombinants/3 μg pGADT7-Rec, the capacity of the library would be up to 1.21×107 cfμ/mL, and the recombinant rate would be 94%. Yeast two hybrid (Y2H) technique was used and the potential interaction proteins of BcRISP1, CYP81G and PIP2 was obtained successfully. The cotransformation in yeast and bimolecular fluorescence complementation (BiFC) analysis were performed to further verify the interaction relations.