【作者】 王华新；

【导师】 曹家树；

【作者基本信息】 浙江大学 ， 蔬菜学， 2008， 博士

【摘要】 芸薹属是十字花科植物的重要类群，也是我国栽培面积最大的蔬菜作物。白菜（Brassica campestrisL．ssp．chinensis Makino）是芸薹属芸薹种的一个亚种，是典型的异花授粉作物，具有显著的杂种优势，F₁代杂种的推广应用可以提高白菜的生产水平。雄性不育材料在白菜F₁代种子的生产中具有重要地位，其选育和利用日益受到人们的重视。以白菜雄性不育材料为基础，分析雄性不育相关基因的表达特征，分离雄性不育相关基因并验证基因的功能，不仅可以促进白菜雄性不育材料的选育，而且对于揭示植物雄性不育的分子机制也具有重要的理论意义。本文以‘矮脚黄’白菜隐性核雄性不育两用系为研究对象，在前人获得雄性不育相关新基因Brassica campestris Male Fertility 1（BcMF1）的cDNA拼接序列的基础上，通过PCR进一步克隆BcMF1的DNA全长和cDNA全长；通过TAIL PCR技术克隆该基因的启动子序列；对BcMF1及其启动子的序列特征以及编码蛋白的功能进行分析和预测；并在几种芸薹属植物中进行BcMF1同源基因的克隆和进化分析。为验证BcMF1的功能，构建了RNA干涉（RNAi）和反义RNA植物表达载体，在分析菜心BcMF1同源基因表达特征的基础上，通过农杆菌介导法转化菜心植株，获取BcMF1表达沉默的卡那霉素抗性植株，对转基因植株的花粉发育状态进行观察，深入分析BcMF1在白菜花粉发育中的生物学功能。本研究获得的主要结果如下：（1）根据表达差异片段BcMF-A15T17及其RACE扩增序列设计引物，PCR扩增得到BcMF1准确的cDNA全长和DNA全长序列，cDNA全长为1 684 bp，基因全长为1 985 bp，含有两个内含子和3个外显子，最大开放阅读框为1 416 bp，预测该基因可编码一个含有471个氨基酸的蛋白质。不计算70 bp的3’末端A／T丰富序列和poly T尾部结构，前人所得到的cDNA拼接序列为1613 bp，最大开放阅读框为1344 bp，预期可编码一个含有447个氨基酸的蛋白质。本文的结果校正了前人cDNA拼接序列及其预测的氨基酸序列的误差。在GeneBank中没有发现与BcMF1同源性较高的功能已知基因，BcMF1仅与拟南芥未知功能蛋白家族DUF1216的相似性较高，是第一个被报导的DUF1216表达基因，蛋白质特征分析显示BcMF1基因编码蛋白很可能是一个定位在细胞外的分泌蛋白。（2）以两用系的可育株群和不育株群为研究对象，使用Northern杂交的方法较全面地分析了BcMF1的转录表达特征，结果显示BcMF1仅在可育株系的中花蕾、大花蕾、花以及雄蕊中特异表达，在不育株系中不表达。因此，BcMF1是一个与白菜育性相关的新基因。（3）通过PCR在不育株中扩增了BcMF1序列，通过TAIL PCR在两用系可育株和不育株中同时扩增了该基因的启动子序列，从DNA水平上探讨该基因表达差异形成的分子机理。序列比对发现BcMF1及其启动子序列在可育株和不育株中没有出现变异，表明该基因的这种表达差异不是因为基因或启动子序列突变所引起的，推测BcMF1的表达受其他基因的调控。在BcMF1启动子序列中，发现了一些与光诱导响应、激素响应和胁迫响应有关的顺式作用元件，推测BcMF1的表达可能受到光条件、内外源激素以及逆境胁迫的影响（4）根据BcMF1的全长序列设计引物，在7种芸薹属蔬菜作物中克隆到BcMF1的同源序列，BcMF1同源基因在核苷酸水平和氨基酸水平相似性分别为97．8％～99．6％和95．3％～98．9％。以同源基因的多序列比对为基础，对7种芸薹属蔬菜作物的BcMF1同源基因进行系统分析，揭示了BcMF1同源基因在这些蔬菜作物中的进化关系。（5）克隆了菜心BcMF1同源基因并分析其表达特征，证明了菜心适用于白菜BcMF1的功能分析。构建了RNA干涉表达载体pBcMF1-RNAi和反义RNA表达载体pBcMF1-AS，农杆菌介导法转化菜心，获得了Kan抗性菜心植株。分子检测表明已得到了BcMF1-AS转基因植株和BcMF1-RNAi转基因植株，Northern杂交显示BcMF1的表达在转基因植株中受到抑制。对转基因植株的生长状况、花器官发育、花粉形态、花粉离体萌发率以及花粉发育过程进行观察，发现抑制BcMF1的表达可以影响菜心植株的花粉发育，因此推论BcMF1是一个与白菜花粉发育相关的新基因。（6）根据pBI121质粒多克隆位点两侧的DNA序列设计一对通用引物，以几种pBI121表达载体及其转基因植株为模板进行PCR扩增，并使用限制性内切酶BamHⅠ酶切分析来验证扩增条带的真实性，检测结果与常规鉴定方法完全一致，为各种pBI121表达载体及其转基因植株的鉴定提供了一种快速、有效的方法。更多还原

【Abstract】 Brassica is one of the most important genus of Cruciferae,and is one of the vegetable crops culturedmost widely in China.Chinese cabbage pak-choi（Brassica campestris L.ssp.chinensis Makino）,asubspecies of Brassica campestris L,is a typical allogamy plant with significant heterosis,and thepopularization and application of its F₁ hybrid seeds can improve the level of production in Chinese cabbage.The male sterility lines are very important for producing F₁ hybrid seeds in Chinese cabbage,and thebreeding and utilization of new male sterility lines are paid more attention by researchers.The isolation andfunctional confirmation of male sterility-related genes based on male sterility lines,as well as theinvestigation of behavior characteristics of these genes in chinese cabbage,might be useful to elucidate tothe possible molecular mechanism of plant male sterility,while improving breedings of male sterility lines.Earlier study in our laboratory has obtaind the preliminary full-length cDNA sequence of a newgene named Brassica campestris Male Fertility 1（BcMF1）,by assembling the 5’ end,and the 3’ end fromRACE with a transcript-derived fragment（TDF） from cDNA-AFLP.In this paper,from the genic malesterile-fertile line‘Bajh97-01AB’of Chinese cabbage taken as a research object,the real full-lengthcDNA and DNA sequences of BcMF1 were cloned by PCR amplification,firstly;the new promotersequences were isolated by TAIL PCR,secondly;the sequence characteristics of BcMF1 and its promoterwere analysed and the function of the hypothetical protein BcMF1 was predicted,thirdly;then RNAinterference（RNAi） expression vector and anti-sense expression vector of BcMF1 were constructed,andtransformed into flowering chinese cabbage（B.campestris ssp.chinensis var.parachinensis Tsen et Lee） byagrobacteriurn-mediated method to obtain their loss-of-function mutants for confirming the function ofBcMF1 in pollen development process;homologous genes of BcMF1 were cloned from deferent species inBrassica by PCR,and the phylogenetic trees of BcMF1 were constructed based on the alignment of thesehomologous 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 real full-length cDNA and DNA sequences of BcMF1 were cloned by PCR.BcMF1 was 1 684-bp long incDNA and 1 985-bp long in DNA,including two introns and three extrons in its DNA sequence,and itslargest open reading frame consisted of 1 416 bp,encoding 471 amino acid residues.The preliminaryfull-length cDNA sequence obtained from earlier study in our laboratory,was 1 613 bp long,notincluding a 70-bp AT-rich sequence 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 deducedamino acid sequence of BcMF1 from earlier study.Homology analysis showed that the deduced protein ofBcMF1 with no significant similarity to any known genes,had higher amino acids sequence similarity to aunknown protein family DUF1216 in Arabidopsis thaliana.BcMF1,encoding a extracellular protein inSecretory pathway,is the first expressed gene which had been reported in DUF1216 family.（2） The expression patterns of BcMF1 in the genic male sterile-fertile line‘Bajh97-01AB’ofChinese cabbage were analyzed by Northern blot.The results showed that BcMF1 specially expressed inmiddle and big flower buds（≥1.0 mm in diameter）,open flowers and stamens of the fertile lineBajh97-01B,and didn’t express at any stage of the sterility‘Bajh97-01A’line.The results suggest thatBcMF1 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 thepromoter sequences of BcMF1 were isolated simultaneously by TAIL PCR BcMF1 from the fertile line‘Bajh97-01B’and the sterility line‘Bajh97-01A’.The sequence alignment was performed for finding out themolecular 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 in BcMF1 and its promotersequences was found between the fertile line‘Bajh97-01B’and the sterility line‘Bajh97-01A’,andthe results suggested that the expression difference was not caused by sequence mutantion of BcMF1and its promoter between the fertile line‘Bajh97-01B’and the sterility line‘Bajh97-01A’.Thus,it wasdeduced that another gene or some genes might regulate the expression of BcMF1.In the promotersequence of BcMF1,various cis-elements responding to light inducement,hormone inducement andstress condition were present,so it was predicted that the expression of BcMF1 might be influencedby light,hormone and stress condition.（4） BcMF1 Homologous genes were isolated from 7 species in Brassica by PCR,and the identification ratio of nucleotide and amino acid sequence were 97.8%～99.6% and 95.3%～98.9% in 7 specises,respectively.The phylogenetic trees were produced on the basis of homologous sequences alignment ofBcMF1 from different seven species in Brassica,and the evolution of homologous BcMF1 in these specieswas demonstrated by the phylogenetic trees.（5） It was proved that flowering Chinese cabbage is fit to confirm the function of BcMF1,by cloningand expression analysis of the homologous BcMF1 in flowering Chinese cabbage.Anti-sense plantexpression vector pBcMF1-AS and RNAi plant expression vector pBcMF1-RNAi of BcMF1 wereconstructed,and transformed into flowering Chinese cabbage by agrobacterium-mediated method.Themolecular assay showed that pBcMF1-AS and pBcMF1-RNAi constructs were integrated into the genomesof BcMF1-AS and BcMF1-RNAi transgenic plants with Kanamycin resistance respectively,and theexpression level of BcMF1 was down-regulated in BcMF1-AS and BcMF1-RNAi transformants.Growthperformance,floral organ development,pollen morphology,pollen germination in vitro and pollendevelopment of of transgenic plants were observed,these results showed that pollen development wereinfluenced by down-regulated expression of BcMF1 in transgenic plants.These results suggest that BcMF1is a new pollen development-related gene in Chinese cabbage.（6） Recombinant plant expression vectors constructed on the basis of the the binary vector pBI121,andtheir transgenic plants,were amplified by PCR with a universal primer pair designed according to the flanksequence of multiple clone site in pBI121;Further,the PCR products with predicted sizes were verified bydigestion with the restriction enzyme BamHⅠ.The detecting results by PCR-Digestion were consistent withthese results by the traditional means,therefore a rapid and effective method to identify the expressionvectors and their transgenic plants containing pBI121 plasmid was developed in this paper.更多还原