【作者】 刘乐承；

【导师】 曹家树；

【作者基本信息】 浙江大学 ， 蔬菜学， 2006， 博士

【摘要】 芸薹属(Brassica)植物是杂种优势利用最为普遍的作物种类之一,其雄性不育的机理及雄性不育系选育的研究一直深受重视。但是,雄性不育产生的机制仍然是一个尚未完全解开之谜,还有许多问题需要深入研究。在国家自然科学基金委和浙江省科技厅的资助下,本实验室在前一阶段利用mRNA DD-PCR、cDNA-AFLP技术结合RACE技术,从普通白菜(B.campestris ssp.chinensis(L.)Makino var.communis Tsen et Lee)核雄性不育两用系中成功分离得到了与白菜核雄性不育相关基因CYP86MF、BcMF1、BcMF2、BcMF3和BcMF4。对其中的BcMF3和BcMF4基因的RT-PCR分析表明,它们都只在普通白菜两用系可育株系的中蕾和大蕾中特异表达。为进一步了解BcMF3和BcMF4基因的生物学功能,将普通白菜花蕾按大小进行更细分级,首先采用Northern杂交分析对BcMF3和BcMF4在白菜中的表达特征进行了进一步的确认:接着构建了这两个基因的组成型启动子CaMV 35S的和绒毡层组织特异性启动子A9的反义RNA和RNA干涉基因沉默植物表达载体:然后将所构建的载体,采用Floral Dip法转化拟南芥(Arabidopsis thaliana),通过组织培养途径转化与普通白菜同属于白菜亚种的菜心(B.campestris ssp.chinensis (L.)Makino var.parachinensis(Bailey)Tsen et Lee),获得了拟南芥3个载体的共12株转基因植株、菜心10个载体的共148个转基因植株株系:随后对转基因拟南芥和菜心植株的花粉形态、花粉萌发率与活力、花粉管的伸长、果胶甲酯酶的酶活性及不同载体的转化效率进行了观察和检测,取得结果如下: (1)采用Northern blot分析了BcMF3和BeMF4基因的表达特征。结果显示,BcMF3基因只在普通白菜核雄性不育两用系(‘ZUajh97-01’)可育株的直径为1.1mm以上的花蕾中特异表达,并且在1.2～2.5mm的花蕾中强表达,而在可育株<1.2mm的花蕾、可育株的叶片和不育株的混合花蕾中均不表达;BcMF4基因在可育株1.2～2.5mm的花蕾中特异表达,而在可育株<1.2mm和>2.5mm的花蕾、可育株的叶片和不育株的混合花蕾中均不表达。表明BcMF3和BcMF4基因可能与白菜的花粉发育有关,而且主要作用在花粉成熟前后。 (2)构建了BcMF3和BcMF4基因的组成型启动子CaMV 35S的和绒毡层组织特异性启动子A9的反义RNA和RNA干涉基因沉默植物表达载体共10个,并分别将它们导入了农杆菌LBA4404菌株中。 (3)将所构建的10个载体的农杆菌菌液采用Floral Dip法转化拟南芥。通过卡那霉素筛选,得到了3个具有CaMV 35S启动子的载体pBI-B3、pBI-B4R和pBI-BB34的共12株抗性转基因植株,PCR和Suothern blot分析证明外源基因成功整合到了这些转基因植株的基因组中,同时说明更多还原

【Abstract】 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.更多还原