【作者】 张弢；

【导师】 曹家树；

【作者基本信息】 浙江大学 ， 蔬菜学， 2005， 博士

【摘要】 十字花科作物是我国重要的农作物之一,也是杂种优势利用最为普遍的一类作物。发掘和创建其雄性不育系,用于配制一代杂种在生产实践中具有重要意义。花粉发育相关基因的克隆和分析有助于通过反义技术等阻断与花粉发育有关基因的表达从而获得雄性不育植株。同时,由于十字花科植物形态差异大,又有许多中间过渡类型,因而很难对其中的各类物种进行细致分类和进化定位。近年来,随着分子生物学和生物信息学的发展,为从分子水平上研究十字花科植物的物种演化成为可能。本研究以BcMF2和BcMF4基因为研究对象,通过PCR扩增方法对白菜BcMF2和BcMF4基因的基因组全长和cDNA全长进行了克隆、鉴定以及蛋白质结构和功能的预测;以十字花科不同种属的植物为材料进行BcMF2和BcMF4基因同源序列克隆和鉴定,在BcMF2和BcMF4基因同源序列比对的基础上构建分子进化树;探讨不同物种BcMF2和BcMF4基因的进化关系,分析基因结构上的保守性以及在不同物种间的变异,从而了解该基因在十字花科植物中的普遍作用,为更好的研究基因的功能奠定理论基础;同时依据BcMF2和BcMF4基因的同源比对结果,对十字花科植物不同种属的系统发育关系进行了初步研究,尝试探讨BcMF2和BcMF4基因用于物种进化分析的可行性,为十字花科植物的物种进化提供新的分子证据。研究结果如下: （1） 通过PCR扩增的方法在‘矮脚黄’白菜核隐性不育两用系（A/B line）中成功克隆了花粉发育相关的多聚半乳糖醛酸酶基因BcMF2的DNA全长序列和cDNA序列。结果表明,该基因包含4个外显子和3个内含子,最大开放阅读框为1266 bp,编码421个氨基酸序列。对推导的氨基酸序列进行分析,发现该序列包含3个N-糖基化位点,4个蛋白激酶C磷酸化位点,7个酪蛋白激酶Ⅱ磷酸化位点,12个N-豆蔻酰化位点,1个多聚半乳糖醛酸酶活性位点(²⁵⁷RVTCGPGHGLSVGS)等。将BcMF2基因氨基酸序列与数据库中的其他多聚半乳糖醛酸酶基因进行同源序列比对,构建系统进化树,发现该基因具有所有多聚半乳糖醛酸酶基因特有的4个保守结构域,并且与花粉中特异表达的多聚半乳糖醛酸酶基因聚为一类,进一步证明了该基因是与花粉发育相关的多聚半乳糖醛酸酶基因。 （2） 首次对多聚半乳糖醛酸酶基因BcMF2的原核表达进行了研究,将BcMF2基因的完整开放阅读框插入到pET-32a（+）原核表达载体上,成功诱导其在大肠杆菌中的高效表达。SDS-PAGE电泳结果发现在70 kDa处有一条特异条带,除去载体上26 kDa的谷胱甘肽巯基转移酶后,与预期的目的蛋白大小一致。通过对诱导条件的优化发现,IPTG终浓度为0.4 mmol·L^-1,30℃下诱导6 h可获得最佳的表达效果。 （3） 根据白菜多聚半乳糖醛酸酶基因BcMF2的全长序列设计引物,在十字花科植物的11更多还原

【Abstract】 Cruciferae crops are one of the most important vegetable crops and also are a kind of crops of the most successful in utilizing of heterosis in China. In order to completely elucidate the mechanism of plant male sterility, we must know the genes related to plant pollen development. In recent years, an increasingly amount of research has been devoted to the molecular biology of the pollen development of flowering plant. The cloning and characterization of pollen development related genes is important to analysis the mechanism of male sterility in Cruciferae and construct male sterility line. At the same time, because of the emergence of different morphological characters and many transitional types in Cruciferae crops, there are still many ambiguities and debates on phylogentic evolutionary relationship of Cruciferae. With the development of molecular biology and bioinformatics, it is possible to identify and classify species and evaluate the evolutionary relationship in molecular level. In this study, the full length cDNA and DNA of pollen development related BcMF2 and BcMF4 gene was cloned and characterized from Chinese cabbage pak-choi (Brassica campestris L. ssp. chinensis Makino). Homologous genes with the BcMF2 and BcMF4 were cloned from 11 species in 6 genera of Cruciferae by PCR amplification, with two pairs of primers designed according to the sequence of BcMF2 and BcMF4 and Phylogenetic trees of BcMF2 and BcMF4 gene were constructed based on the alignment of their homologous genes, respectively. The results were as follows:(1) The full length DNA and cDNA of BcMF2 gene encoding pollen-specific polygalacturonase were cloned from B. campestris ssp. chinensis cv. Aijiaohuang. The largesteset open reading frame is 1266 bp and encodes a protein of 421 amino acids with a predicted molecular mass of 43.9 kDa, interrupted by three introns of 142 bp, 75 bp and 83 bp in length. Sequence analysis revealed that it has three potential N-glycosylation sites and one polygalacturonase active position (RVTCGPGHGLSVGS). Four domains which are highly conserved in the all plant and fungal PGs is present in BcMF2. Phylogenetic analysis showed that BcMF2 falls into the category of clade-C, which includes PG related to pollen. These results test that BcMF2 acts as pollen-specific polygalacturonase.(2) We firstly studied on prokaryotic expression of BcMF2. A cDNA sequence was inserted into pET-32a (+) and a recombinant plasmid expressing the full length BcMF2 fusion protein was constructed. By using IPTG inducing, overexpression of BcMF2 fusion protein in E.coli BL21 was achieved. The result showed that there is a specific band at about 70 kDa in size, which is identical withthe expected molecular weight of the recombinant protein. And by optimizing the inducing parameters, we find when the recombinant plasmid was induced at 30℃ for 6 h and the corresponding IPTG was added to the media to the final concentration of 0.4 ~ 1 mmol·L-1, Expression amount of the recombinant protein was the largest.(3) BcMF2 Homologous genes were isolated from 11 species in 6 genera of Cruciferae by PCR amplification. The Clustal X program was used to align the homologous sequences from different 13 species of Cruciferae and to produce NJ, ME and MP phylogentic trees based on distances estimated by the two-parameter method with 1000 bootstrap samples using MEGA 2.1. The result shows three kinds of phylogentic trees have the strictly accordant topologic structure. B. rapa L. and B. campestris L. ssp. pekinensis cv. Xiaoqingkou form a specific clade (BCL≥91%) and B. campestris L. ssp. pekinensis cv. Xiaoqingkou and B. campestris L. ssp. pekinensis cv. Hugangya-14 belong to different clade, which is consistent with the results from morphology and cytology.(4) By using GenBank, EMBL and DDBJ database, we downloaded 86 PG sequences from bacteria, fungi and plants. Then the homology analysis was performed and the phylogenetic tree was constructed. The amino acids sequence alignment confirmed that there are four strictly conserved sequence segments, which更多还原