Molecular Identification,cloning And Sequencing Of The Genic Male Sterility Related Gene Mf-CYP 450 In Chinese Cabbage(Brassica Campestris L.ssp.chinensis Makino)

The phenomenon of genie male sterility (GMS) occurs widely in plant. The genie male sterile AB line is not only the male-sterile line (A line) but the maintainer line (B line) as well, so the genie male sterile plant is very important in crossbreed and theory research. Chinese cabbage (Brassica campestris L. ssp. chinensis Makino), originated from China, is one of the crops of the highest yield and is cultivated most widely as well as is a kind of crop most successful in using of heterosis in China. Much attention was paid to the research on the breeding of the male sterile line and the basis for application in Chinese babbage pak-choi. Genie male sterile AB line of Chinese cabbage pak-choi, developed in 1980s, have been playing an active roll in the crossbreed of vegetable. Most important, the plants of AB line of Chinese cabbage pak-choi are simple in genetic background, and the male sterility was controlled only by a pair of nuclear recessive gene. Genie male sterile AB line of Chinese cabbage pak-choi held the advantage in the study on molecular mechanism of genie male sterility. The research on the morphology, cytology and physiology of the AB line was conducted in the past years in Chinese cabbage pak-choi in China, however little information could be obtained on the molecular mechanism causing the male sterility of Chinese cabbage pak-choi. Molecular biology provides a powerful tool to study in the molecular mechanism of genie male sterility in Chinese cabbage pak-choi.The progeny of Chinese cabbage genie male sterile AB line from a test cross was used in this study. In the study of molecular marker linkage to fertility, RAPD-PCR technology was used. Four hundred and twelve random primers were screened between the DNA pools of A line and B line of Chinese cabbage pak-choi. DD-PCR (differential display PCR) technology was applied in order to cloning of the genes related to male sterility. The primary results obtained from the research provide some clues for the study on the molecular mechanism of the genie male sterility. The main results are as follow:(1) It was proved that higher PCR annealing temperature could prevent the DNA fragment from non-specific amplification, so that increased the identification accuracy. As a whole, 42 癈 annealing temperature was suitable for PCR amplification of the Chinese cabbage pak-choi. A rapid, simple and low cost method of DNA extraction was established, and good quality genomic DNA was got.(2) The difference on the main characters between A line and B line of Chinese cabbage-pak-choi was compared, such as the growth status of the plants, and themorphology of anthers, and the fertility and the cytology of pollens. The results showed that the AB line of Chinese cabbage- pak-choi was reliable and stable. The protein pattern of floral bud of A line and B line was compared by SDS-PAGE. Two specific bands were obtained, one 45kD protein named A-l was shared by A line, the other 50kD protein was shared by B line. About twelve protein bands were observed in the floral bud of genie male sterile AB line in the Chinese cabbage pak-choi, and the protein types between A and B line is very similar except the two differential bands, which was clearly different from the results of the SDS-PAGE pattern of floral bud protein in Chinese cabbage-heading genie male sterile AB line. About twenty-two bands were found in B line of Chinese cabbage-heading, however only ten bands were observed in A line. The genie male sterility might be caused by different factors between these two kinds of material,(3) Cytological morphology studies were made in the development of anther and microsporogenesis in A line and B line of Chinese cabbage-pak-choi. In A line, pollen abortion happened in pollen mother cell meiosis stage. The first meiosis of the pollen mother cells happened, but the second meiosis couldn't be continued, and then the pollen mother cells spread from the center of the anther chamber. With the development of pollen mother cells, the content of the cells leaked, and in...