Cloning And Expression Analysis Of Male Sterility-Related Genes Tuba2and BcCOI1in Non-Heading Chinese Cabbage(Brassica Campestris Ssp. Chinensis Makino)

Cytoplasmic male sterility (CMS) is of tremendous importance in utilization of heterosis. Compared with the self-incompatible lines, CMS lines eliminated the high cost by artificial pollinated propagation, easily degradation of successive inbreeding, can not have the100%hybrid rate, intellectual property is not effectively protected and some others problems. Non-heading Chinese cabbage (Brassica campestris ssp. chienesis Makino), which originated from China, is one of the most widely cultivated vegetables in China, especially in the south of China. Because of the obvious heterosis, it is the ideal material to product hybrids by the male sterile line. A new OguCMS germplasm, obtained by Hou Xilin using asymmetric cell fusion technology, has a good prospect in being produced. Compared with the primary OguCMS line, the new germplasm of non-heading Chinese cabbage is characterized by no degeneration of nectarines and no seedling yellowing at low temperature. At the same time, both the rate and the degree of male sterility for the new CMS line are100%. Based on previous research, this thesis, by using RT-PCR and RACE technology, two full-length cDNA of male sterility-related genes were cloned from the new CMS line. The aim of our study is to analyze molecular biology of non-heading Chinese cabbage CMS line and its maintainer line, in order to explore the cytoplasmic male sterility mechanism in higher plants. The results were as follows:1. On the basis of one cDNA-AFLP differential expression fragment isolated from the new germplasm of non-heading Chinese cabbage(Brassica campestris ssp. chinensis Makino). The full length cDNA of the gene were cloned by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The gene named TUBA2, the protein encoded by this gene was highly homologous with that of a-tubulin protein.The Southern blot result suggested that the cloned and characterized gene TUBA2was a single copy gene and belonged to a multiple gene family (about two to four α-tubulin genes) in non-heading Chinese cabbage. The TUBA2gene was accumulated in stamens and was expressed in a significantly lower level in CMS line than that in the maintainer and all of the stages of the microsporogenesis. Its expression was increased continually in maintainer line, but it was always down-regulated in CMS.2. According to one cDNA-AFLP differential expression fragment isolated from the new germplasm of non-heading Chinese cabbage(Brassica campestris ssp. chinensis Makino), and by using RT-PCR and RACE technology, the full length cDNA of the gene named BcCOI1were cloned. The gene encompassed an open reading frame (ORF) with1791bp encoding596bp amino acid residues. The protein encoded by BcCOI1contains characteristic F-box motif and LRRs motif, indicating that it belongs to a kind of special F-box protein. Southern-blot analysis indicated that there was more than one copy of BcCOI1gene in Brassica campestris ssp. chinensis Makino. Real-time quantitative PCR analysis revealed that the expression of BcCOI1was clearly regulated by the induction of Methyl Jasmonate. Therefore, the new gene has the purative characteristics of COI1, and is probably involved in the JA responses. The discovery of BcCOI1not only provides insight in the study of the jasmonate signal transduction pathway in Brassica campestris ssp. chinensis Makino, but is also helpful in regulating crop’s Cytoplasmic Male Sterile and stress resistance.