| Brassica is a kind of crops whose heterosis is utilized prevalently in China, and great attention has been paying to its mechanism of male sterility and breeding of male sterile line. But in plant the mechanism of male sterility is still a mystery that has not been unveiled thoroughly, and there are many problems need to solve. Supported by NNSF (the National Natural Science Foundation of China), we had isolated 5 genes form the fertile B line of A/B line in common Chinese cabbage-pak-choi (B. campestris ssp. chinensis (L.) Makino var. communis Tsen et Lee), by combining mRNA differential display PCR (DD-PCR) or complementary deoxyribonucleic acid amplified fragment length polymorphism (cDNA-AFLP) with rapid amplification of cDNA ends (RACE). BcMF3 and BcMF4 were specifically expressed in middle size flower buds and large size flower buds of the fertile B line. In order to understand the biological function of the two genes, we classified the flower buds into 5 grades and then detected the expression patterns of BcMF3 and BcMF4 by Northern blot. Plant expressing plasmid vectors of antisense RNA and RNA interference (RNAi) were constructed using fragments of BcMF3 and BcMF4, and then all the vectors were used to infect Arabidopsis thaliana and flowering Chinese cabbage (B. campestris ssp. chinensis (L.) Makino var. parachinensis (Bailey) Tsen et Lee) respectively, and consequently the transgenic plants of Arabidopsis and flowering Chinese cabbage were obtained. Subsequently, the morphology of pollen grain, germination and vigor of pollen, growth of pollen tube and activity of Pectin methylesterase (PME) of positive transgenic plants were investigated. The results provided available information on exploring the function of BcMF3 and BcMF4 and the mechanism of pollen development in Cruciferae. The results are as follows:(1) The expression patterns of BcMF3 and BcMF4 were analyzed by Northern blot. The results showed that BcMF3 and BcMF4 were specifically expressed in flower buds >1.1 mm in diameter of the fertile B line, and strongly expressed in flower buds of 1.2 ~ 2.5 mm. But no expression was detected in flower buds with other sizes and rosette leaves of the fertile B line and the mixed flower buds of the sterile A line. The results suggested that BcMF3 and BcMF4 are related to pollen development.(2) Ten plant expressing plasmid vectors of antisense RNA and RNAi were constructed, and CaMV 35S promoter and A9 promoter were used in both types of vectors respectively. Then all the vectors we introduced into Agrobacterium tumefaciens strain LBA4404 by triparental mating.(3) All the above vectors were used to infect Arabidopsis thaliana by Floral Dip, and 12 transgenic plants of Arabidopsis were obtained. The results of PCR and Southern blot analysis indicated that the foreign genes were integrated into the genome of the transgenic plants, and Floral Dip is a convenient and low-cost shortcut for transformation of Arabidopsis.(4) All the above vectors were used to infect flowering Chinese cabbage via tissue culture and 148 transgenic plants were obtained. Among the transgenic plants resistant to Kanamycin, the frequency of plants positive to PCR reached 97.73%, and the ratio of positive plants in Southern blot analysis to the positive plants in PCR was also as high as 95.12%.The results indicated the foreign genes were integrated into the genome of the transgenic plants of flowering Chinese cabbage.(5) The flower organs had no difference between transgenic plants and wild type plants of Arabidopsis, but transgenic plants had obviously less amounts of pollen grains compared with the control. Averagely, 47.8%, 46.3% and 51.4% pollen grains from transgenic plants of pBI-B3, pBI-B4R and pBI-BB34 exhibited abnormal shapes respectively, and the abnormal shapes were smaller or shrunken. Approximately 31.6%, 32.7% and 25.6% of pollen grains from transgenic plants of pBl-B3, pBI-B4R and pBI-BB343 germinated, which were significantly lower than that of control plants, moreover 45.7% of pollen tubes from plants of pBI-B3 burst during germination. Of pollen grains from these transgenic plants, only a small portion of pollen tubes grew normally in the pistil. These results suggested that BcMF3 and BcMF4 play important roles during the development of pollen and pollen tube growth, and the development of cytoplasm respectively, and silencing them separately or synchronously can affect the development of pollen and pollen tube growth, and consequently result in partial pollens abortion of Arabidopsis plants.(6) In all transgenic plants of flowering Chinese cabbage from vectors with A9 promoter, phenotypes had no difference between transgenic plants and control plants. Among the transgenic plants of vectors with CaMV 35S promoter, averagely 100%, 43.7%, 100%, 45.8% and 100% of the pollen grains from some transgenic plants of pBI-B3, pBI-B4, pBI-B3R, pBI-B4R and pBI-BB34 exhibited abnormal shape, and the abnormal shapes were smaller or shrunken. Approximately, 34.1%, 24.7%, 32.3%, 23.7% and 16.7% of the pollen grains from transgenic plants of these vectors germinated normally, which were significantly lower than that of control plants, but no bursting pollen tube. Averagely, 29.7% and 36.5% pollen grains from transgenic plants of pBI-B4 and pBl-B4R had no vigor, while many of the pollen grains from transgenic plants of pBI-B3, pBI-B3R and pBI-BB34 were difficult to determine their vigor. The PME activity of anther from transgenic plants of BI-B3, pBI-BB34 and pBI-B3R decreased. Silencing efficiency of antisense RNA vectors of BcMF3 and BcMF4 were 18.7% and 30.6% lower than those of their RNAi vectors respectively. These results suggested that BcMF3 and BcMF4 play important roles during pollen grains, and silencing them separately orsynchronously can affect the development of pollen and consequently result in partial pollens aborted of Chinese cabbage plants.
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