Functional Confirmation Of A Male Sterility Gene CYP86MF And Creation Of Artificial Male Sterile Lines In Chinese Cabbage (Brassica Campestris L.ssp.chinensis Makino)

Brassica crops is one of the vegetable & oil 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.Supported by the National Natural Science Foundation, a cytochrome P450 gene CYP86MF was obtained in floral bud of Chinese cabbage pak-choi (B. campestris ssp. chinensis (L.) Makino var. communis Tsen et Lee, syn. B. rapa ssp. chinensis (L.) Makino var. communis Tsen et Lee) between A line and B line by mRNA differential display PCR technology (DD-PCR) and rapid amplification of cDNA ends (RACE) technology. The ORF of the gene is composed of 1 578 base pairs, and the result of Genbank BLAST showed that it has 85.3% nucleotide similarity to a gene CYP86C4 encoding cytochrome P450 from Arabidopsis thaliana, and has 84.9% amino acid similarity to CYP86C4. The result of Northern hybridization confirmed that gene CYP86MF 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 CYP86MF, we study further in this paper. After the efficient shoot regeneration system of Chinese cabbage-pak-choi and flowering Chinese cabbage (B. campestris ssp. chinensis (L.) Makino \ar.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 pBI35S-AMF & pB!A9-AMF carrying antisense gene CYP86MF. More than 130 plantlets KanR seedlings, including 37 plantlets flowered, were obtained after we constructed an efficient genetic transformation system. And except for the mosaic male sterile in two plantlets and the floral organ deformed in three plantlets, the floral organs of the other 32 KanR seedlings were normal. But self-infertility was found in the transgenic plantlets even self-pollination was performed in budding period. So the experiment of the pollen germination of the transgenic Chinese cabbage were done, and the resultsindicated that the pollen of the transgenic Chinese cabbage could not germinate normally, but the frequency of the pollen germination of the non-transgenic plantlets reached 25% ~ 30%. Therefore, the expressing characteristics of gene CYP86MF were analyzed by Northern hybridization in transgenic plantlets, non-transgenic plantlets of the genie male sterile AB line. At the same time, the morphology and the development course of microsporogenesis were observed in transgenic plantlets and non-transgenic plantlets. In order to understand the biological function of gene CFP5<5A/F, the concerned indexes of plant physiology & biochemistry were compared between the transgenic plantlets and non-transgenic plantlets. The primary results obtained from the research provide some clues for the study on the biological function of gene CYP86MFand molecular mechanism of the male sterility. The main results are as follow:(1) The study has developed an efficient and fast method for plant regeneration from petiole with cotyledo...