【作者】 张豫超；

【导师】 曹家树；

【作者基本信息】 浙江大学 ， 蔬菜学， 2006， 硕士

【摘要】 芸薹属(Brassica)蔬菜是我国栽培面积最大、总产量最高、杂种优势利用最为普遍的一类蔬菜作物。其雄性不育的选育及其应用基础的研究深受人们重视。对植物雄性不育的研究已有近百年的历史,国内外学者做了大量的理论和实验的探讨,在方法和研究思路上都有了明显的进步,不仅取得了一些重要的研究进展,还带动了杂种优势在生产中的广泛应用,产生了巨大的经济效益和社会效益。但是,雄性不育的产生机制仍然是一个尚未完全解开的谜,目前对其的认识是零散和不完全的,还有许多问题需要深入研究。随着现代生物学,尤其是分子生物学理论与技术的发展,不仅为最终解决植物雄性不育机制提供了条件,而且随着这个问题的解决过程,也将大大推动芸薹属和其他农作物杂种优势的有效应用。 我们实验室在前一阶段实验中利用cDNA-AFLP技术从白菜[B.campestris ssp.chinensis(L.)Makino var.communis Tsen et Lee]核雄性不育两用系中分离得到了一个可能与白菜核雄性不育相关的基因BcMF10。该基因具有一个完整的长1305 bp的开放阅读框,经GenBank BLAST查询表明,BcMF10基因是一个未知基因,其同源基因很少,只与拟南芥中的未知蛋白At3g28980有较高的同源性,而该蛋白在其他植物中的研究也没有报道,功能作用仍然未知。Northern验证表明BcMF10基因在白菜两用系不育株中的表达受到了明显地抑制,并且该基因在花蕾中特异表达,而在叶片和茎中不表达。为进一步了解本实验室获得的BcMF10基因的生物学功能,在已有的菜心再生体系,以及构建了植物表达载体的基础上,我们采用反义技术及RNA干扰技术将可育株高表达的BcMF10基因的反义和干扰片段导入正常可育菜心[B.campestris ssp.chinensis var.parachinensis(Bailey)Tsen et Lee]中,得到了47株转化植株,由此我们进行了转基因植株的花粉萌发试验。结果如下:更多还原

【Abstract】 Brassica crops is one of the vegetable 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.A gene BcMF10 was obtained in floral bud of Chinese cabbage-pak-choi [B. campestris ssp. chinensis (L.) Makino] between A line and B line by cDNA-cAFLP technology and rapid amplification of cDNA ends (RACE) technology. The ORF of the gene is composed of 1305 base pairs, and the result of Genbank BLAST showed that it is a new gene. The result of Northern hybridization confirmed that gene BcMF10 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 BcMFlO, we study further in this paper. The efficient shoot regeneration system of flowering Chinese cabbage (B. campestris ssp.chinensis (L.) Makino var. 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 pB135S-BcMFl OA, pB\A9-BcMFlOA, pB135S-BcMFM & pB\A9-BcMF10\. More than 47 plantlets KanR seedlings were obtained. The experiment of the pollen germination of the transgenic flowering Chinese cabbage were also done, and the results indicated that the pollen of the transgenic flowering Chinese cabbage could not germinate normally. The primary results obtained from the research provide some clues for the study on the biological function of gene BcMFlO and molecular mechanism of the male sterility. The main results are as follow:(1) The BcMFlO gene encodes a hydrophobic stable protein which contains 434 amino acids. The sequence contains N-glycosylation site and N-myristoylation site, which relate to signal transduction, transport, protein location and adherence. So we presume that BcMFlO gene may be an important gene of cell adherence, signal transduction in the process of the development of pollen.(2) We constructed the constitutive type plant expressing plasmid vector (pBB5S-BcMF10A, pB135S-BcMF10i) and the tapetum-spccific plant expressing plasmid vector (pBlA9-BcMF10A, pBlA9-BcMF10i).(3) 47 plantlets KanR seedlings were obtained based on the efficient genetic transformation system of Yu X L et al (2002). The frequency of positive to the KanR seedlings is 85.71%, The result of Northern hybridization indicated that BcMFlO gene was inhibited obviously in the transgenic plantlets.(4) We did the experiment of the pollen germination of the transgenic Chinese cabbage-pak-choi, and the results indicated that the pollen of the transgenic Chinese cabbage could not germinate normally (31.6% - 35.4%). So we suppose that the BcMFlO gene may relate to the germination of pollen.更多还原