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1、 博博 士士 学学 位位 论论 文文 白菜雄性不育相关新基因白菜雄性不育相关新基因 BcMF1 的克隆和 功能验证 的克隆和 功能验证 姓姓 名名 王 华 新 指指 导导 教教 师师 曹 家 树 教 授 学学 科科 专专 业业 蔬 菜 学 研研 究究 方方 向向 植物基因组及其分子调控 所在学院所在学院 农业与生物技术学院 论文提交日期论文提交日期 2008 年 5 月 4 日 中中 国国 杭杭 州州 Cloning and Functional Confirmation of a New Male Sterility-related Gene BcMF1 in Brassica campes
2、tris L. By Huaxin WANG A Dissertation Submitted as Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy with Major in Plant Genome the new promoter sequences were isolated by TAIL PCR, secondly; the sequence characteristics of BcMF1 and its promoter were analysed and the fu
3、nction of the hypothetical protein BcMF1 was predicted, thirdly; then RNA interference (RNAi) expression vector and anti- sense expression vector of BcMF1 were constructed, and transformed into flowering chinese cabbage (B. campestris ssp. chinensis var. parachinensis Tsen et Lee ) by agrobacterium-
4、 mediated method to obtain their loss- of- function mutants for confirming the function of BcMF1 in pollen development process; homologous genes of BcMF1 were cloned from deferent species in Brassica by PCR, and the phylogenetic trees of BcMF1 were constructed based on the alignment of these homolog
5、ous genes, lastly. The major study results were as follows: (1) On the basis of cDNA- AFLP differential fragment BcMF1- A15T17 and its two RACE ends, the Abstract VIII real full- length cDNA and DNA sequences of BcMF1 were cloned by PCR. BcMF1 was 1 684- bp long in cDNA and 1 985- bp long in DNA, in
6、cluding two introns and three extrons in its DNA sequence, and its largest open reading frame consisted of 1 416 bp, encoding 471 amino acid residues. The preliminary full- length cDNA sequence obtained from earlier study in our laboratory, was 1 613 bp long, not including a 70- bp AT- rich sequence
7、 and polyT tail, and its largest open reading frame consisted of 1 344 bp, coding 447 amino acid residues. The result in this paper corrected the errors of the cDNA and deduced amino acid sequence of BcMF1 from earlier study. Homology analysis showed that the deduced protein of BcMF1 with no signifi
8、cant similarity to any known genes, had higher amino acids sequence similarity to a unknown protein family DUF1216 in Arabidopsis thaliana. BcMF1, encoding a extracellular protein in Secretory pathway, is the first expressed gene which had been reported in DUF1216 family. (2) The expression patterns
9、 of BcMF1 in the genic male sterile- fertile line Bajh97-01AB of Chinese cabbage were analyzed by Northern blot. The results showed that BcMF1 specially expressed in middle and big flower buds (1.0 mm in diameter), open flowers and stamens of the fertile line Bajh97-01B, and didnt express at any sta
10、ge of the sterility Bajh97-01A line. The results suggest that BcMF1 is a new gene related with genic male sterility in Chinese cabbage. (3) The DNA sequence of BcMF1 was amplified by PCR in the sterility line Bajh97-01A, and the promoter sequences of BcMF1 were isolated simultaneously by TAIL PCR Bc
11、MF1 from the fertile line Bajh97-01B and the sterility line Bajh97-01A.The sequence alignment was performed for finding out the molecular cause , in DNA level, why BcMF1 expressed differently between the fertile line Bajh97-01B and the sterility line Bajh97-01A. The results showed that no difference
12、 in BcMF1 and its promoter sequences was found between the fertile line Bajh97-01B and the sterility line Bajh97-01A, and the results suggested that the expression difference was not caused by sequence mutantion of BcMF1 and its promoter between the fertile line Bajh97-01B and the sterility line Baj
13、h97-01A.Thus, it was deduced that another gene or some genes might regulate the expression of BcMF1.In the promoter sequence of BcMF1, various cis- elements responding to light inducement, hormone inducement and stress condition were present, so it was predicted that the expression of BcMF1 might be
14、 influenced by light, hormone and stress condition. (4) BcMF1 Homologous genes were isolated from 7 species in Brassica by PCR, and the identification 浙江大学博士学位论文 白菜雄性不育相关新基因浙江大学博士学位论文 白菜雄性不育相关新基因 BcMF1 的克隆和功能验证的克隆和功能验证 IX ratio of nucleotide and amino acid sequence were 97.8% 99.6% and 95.3% 98.9% i
15、n 7 specises, respectively. The phylogenetic trees were produced on the basis of homologous sequences alignment of BcMF1 from different seven species in Brassica, and the evolution of homologous BcMF1 in these species was demonstrated by the phylogenetic trees. (5) It was proved that flowering Chine
16、se cabbage is fit to confirm the function of BcMF1, by cloning and expression analysis of the homologous BcMF1 in flowering Chinese cabbage. Anti- sense plant expression vector pBcMF1- AS and RNAi plant expression vector pBcMF1- RNAi of BcMF1 were constructed, and transformed into flowering Chinese cabbage by agrobacterium- mediated method. The molecular assay showed that pBcMF1- AS and pBcMF1- RNAi constructs were integrated into the genomes of BcMF1-A
