Functional Confirmation Of A Male Sterility Gene BcMF10 In Chinese Cabbage (Brassica Campestris L. Ssp. Chinensis Makino)

Brassica crops is one of the vegetable crops species of the highest yield and cultivated most widely as well as is a kind of crops of the most successful in utilizing of heterosis in China. Much attention was paid to research on the plant breeding of the male sterile line and the basis for application in Brassica crops. A lot of researchers have researched on the theories and application of the plant male sterility for almost a century, and plenty of progress has been obtained in the methods of study and the basic theory obviously. The progress not only has got the advance to theory of male sterility, but also to bring along the heterosis to apply in the production with a large of economical and social benefits. However, the mechanism of the plant male sterility is not demonstrated completely, so a lot of questions remain. Along with the development of molecular biological theories and methods, it provides powerful tools to study on the molecular mechanism of plant male sterility in Brassica. While the mechanism is being researched, it can drive the heterosis to apply in the production in Brassica crops and others.A gene BcMF10 was obtained in floral bud of Chinese cabbage-pak-choi [B. campestris ssp. chinensis (L.) Makino] between A line and B line by cDNA-cAFLP technology and rapid amplification of cDNA ends (RACE) technology. The ORF of the gene is composed of 1305 base pairs, and the result of Genbank BLAST showed that it is a new gene. The result of Northern hybridization confirmed that gene BcMF10 was inhibited obviously in the male sterile line, and the gene only expressed in floral bud but not in leaf and stem. In order to find out the function of gene BcMFlO, we study further in this paper. The efficient shoot regeneration system of flowering Chinese cabbage (B. campestris ssp.chinensis (L.) Makino var. parachinensis (Bailey) Tsen et Lee) were established using petiole with cotyledon as explants and the plant expressing plasmid vector were constructed, and the explants were inoculated with Agrobacterium tumefaciens strain LBA4404 containing pB135S-BcMFl OA, pB\A9-BcMFlOA, pB135S-BcMFM & pB\A9-BcMF10\. More than 47 plantlets KanR seedlings were obtained. The experiment of the pollen germination of the transgenic flowering Chinese cabbage were also done, and the results indicated that the pollen of the transgenic flowering Chinese cabbage could not germinate normally. The primary results obtained from the research provide some clues for the study on the biological function of gene BcMFlO and molecular mechanism of the male sterility. The main results are as follow:(1) The BcMFlO gene encodes a hydrophobic stable protein which contains 434 amino acids. The sequence contains N-glycosylation site and N-myristoylation site, which relate to signal transduction, transport, protein location and adherence. So we presume that BcMFlO gene may be an important gene of cell adherence, signal transduction in the process of the development of pollen.(2) We constructed the constitutive type plant expressing plasmid vector (pBB5S-BcMF10A, pB135S-BcMF10i) and the tapetum-spccific plant expressing plasmid vector (pBlA9-BcMF10A, pBlA9-BcMF10i).(3) 47 plantlets KanR seedlings were obtained based on the efficient genetic transformation system of Yu X L et al (2002). The frequency of positive to the KanR seedlings is 85.71%, The result of Northern hybridization indicated that BcMFlO gene was inhibited obviously in the transgenic plantlets.(4) We did the experiment of the pollen germination of the transgenic Chinese cabbage-pak-choi, and the results indicated that the pollen of the transgenic Chinese cabbage could not germinate normally (31.6% - 35.4%). So we suppose that the BcMFlO gene may relate to the germination of pollen.