【作者】 田爱梅；

【导师】 曹家树；

【作者基本信息】 浙江大学 ， 蔬菜学， 2008， 博士

【摘要】 十字花科作物是我国栽培面积最大,总产量最高的一类蔬菜作物。同时它也是杂种优势利用最为广泛的一类作物,其雄性不育的选育及其应用基础的研究深受人们重视。对雄性不育机理的研究可以了解小孢子的发育机制,还可以为人工创造雄性不育系提供理论依据,在生产实践和理论上都具有十分重要的意义。本研究以白菜(Brassica campestris L.ssp.chinensis Markino)减数分裂胞质分裂突变体mmc(male meiotic cytokinesis)的野生型(可育株)花粉转录组分析中发现的基因BcMF15(Brassica campestris Male Fertility 15)为研究对象,采用RACE技术扩增分离花粉发育相关的LTP基因BcMF15的cDNA和DNA序列全长,分析其序列特征并预测其蛋白质结构和功能。采用RT-PCR分析BcMF15在白菜不同发育阶段生殖器官和营养器官中的表达状况。在此基础上构建了由组成型启动子CaMV35S(Cauliflower mosaic virus 35S)驱动的BcMF15干涉基因表达载体,通过对菜心[B.campestris L.ssp.chinensis Markino var.parachinensis(Bailey)Tsen et Lee]进行遗传转化,以获得该基因功能缺失突变体,从而分析其在花粉发育进程中的生物学功能。获得的主要研究结果如下:(1)采用RACE技术从白菜可育株中成功克隆到花粉发育相关的BcMF15(Genbank登录号:EF600901)。序列分析表明该基因cDNA序列全长768 bp,最大开放阅读框312 bp,编码了103个氨基酸,该蛋白分子重量约11.36 kDa,属于一个跨膜蛋白,具有显著的疏水区。蛋白质二级结构预测结果表明:该氨基酸序列存在6个推定的结构域:包括信号肽,跨膜域,vWF结构域,C4-锌指结构域,Tryp_alpha_amyl结构域,AAI结构域。感兴趣的是AAI结构域包含在许多花粉发育关键的蛋白中。经GenBank数据库BLAST源性比对发现BcMF15序列与拟南芥((Columbia)的脂质转移相似蛋白(lipid transfer protein,LTP)有88%的相似性,且具有LTP的典型结构特征。RT-PCR对其时空表达模式进行分析发现该基因在可育系的1～5级花蕾中表达,并持续表达直到嫩角果的形成,在花茎和花茎叶等孢子体组织中未检测到BcMF15的表达,而在不育系的对应组织均没有表达,因此,推测其与花粉发育密切相关。(2)根据BcMF15 DNA全长序列设计引物,运用PCR技术扩增得到十字花科6属10个物种的LTP同源序列,序列特征分析表明:不同植物物种LTP同源氨基酸序列在N端有长度为26个氨基酸残基的保守区域,而在C端则存在一定程度的变异。对LTP基因同源序列比对分析的结果表明:在核苷酸水平上的序列相似性在80%以上,氨基酸序列相似性大于53%。说明了LTPs在十字花科中相对保守。在此基础上构建其MP(The maximum parsimony)系统进化树,结果表明:芸薹属与萝卜属关系较近,其次为荠菜属、拟南芥属和山芥属,与诸葛菜属关系最远。(3)构建了BcMF15基因含有组成型启动子CaMV35S的RNA干涉载体pBD5S-RMF15,并利用农杆菌介导的方法将构建的BcMF15基因RNA干涉植物表达载体导入菜心中,获得了菜心转BcMF15干涉基因植株。(4)对BcMF15干涉载体转化获得的Kan~R菜心植株进行了分子、形态和细胞学的检测。利用荧光定量RT-PCR技术对前期经PCR和Southern印迹杂交检测为阳性的转pBI35S-RMF15株系,以花蕾包括小蕾,中蕾,大蕾、开放的花、嫩角果的cDNA为模板,对pBI35S-RMF15菜心转基因植株和正常植株在花粉发育的不同时期的动态表达进行了研究,结果表明:pBI35S-RMF15在各级花蕾和开放花中的表达都明显低于对照,且在中、大蕾时期降低更为显著。这说明pBI35S-RMF15干涉载体已经引发了转录后沉默效应,抑制了BcMF15基因的表达。对转化植株花器官的形态学观察发现:转化植株的雄蕊表现为花丝短小,花药不饱满、发白,花粉量少。花粉萌发试验和花粉形态电镜扫描表明,pBI35S-RMF15菜心转基因植株花粉的萌发率为35.94%,明显低于相应的空载体对照(74.40%),存在显著差异。这表明转BcMF15干涉载体引发了RNAi沉默效应,抑制了BcMF15的表达,影响正常花粉结构的形成,从而使花粉形状发生改变,导致花粉畸形。树脂半薄切片和透射电镜相结合观察转基因植株花蕾的发育过程,发现在中蕾阶段-约小孢子发育单核靠边期,绒毡层提前降解,而使花粉发生败育。因此我们推测BcMF15编码的LTP基因作用于花药细胞中,通过影响花药在花粉发育过程中的脂质转运,并进一步调节脂质的合成和转化等代谢活动,从而影响绒毡层的发育,进而对小孢子发育过程中花粉壁的形成、花粉的萌发及花粉管的伸长产生影响。(5)利用TAIL-PCR方法首次克隆了BcMF15基因的启动子pBc15。序列分析表明,该序列A+T的比例为61.61%,C+G的比例为38.39%,符合启动子的碱基组成。对ATG上游的5’-UTR(5’-untranslated region)序列用Blast软件在GENEBANK数据库上作同源搜索发现,这段序列与大白菜(Brassica rapa subsp.pekinensis)基因组DNA的同源性为99%。使用三个重要的植物启动子顺式作用元件数据库对其中的功能元件进行分析发现:BcMF15基因的5′-UTR不仅含有启动子特有元件如TATAbox和35S启动子持有元件GATA box,而且还具有多个调控花粉发育特异表达的功能元件,如GGTT和GTGA元件以及番茄lat52/LeMAN5激活花粉特异表达的调节元件,POLLEN1LELAT52元件。此外还存在多个环境诱导的序列元件,说明了BcMF15基因的5’-UTR序列可能是一个花粉发育特异的调控序列,同时受光和干旱等胁迫因子的诱导。(6)成功构建了带有GUS报告基因的嵌合启动子载体pBI-pBc15,并通过基因枪将其转化到洋葱表皮细胞,进行瞬时表达,随后进行了X-Glue组织化学染色分析。结果表明BcMF15启动子能够激活GUS基因的表达,且融合蛋白初步定位在转化细胞的细胞核中。(7)采用浸花法(Floral Dip)成功地将BcMF15启动子表达载体转化到拟南芥中,对经过卡那抗性筛选和PCR检测的T1代拟南芥进行了GUS组织化学染色分析和花蕾GUS组织化学染色后的树脂切片观察,结果表明BcMF15启动子在拟南芥花药中特异表达。更多还原

【Abstract】 Brassica crops is a kind of important crops in agricultural production.The male sterile line was widely utilized in practice to produce F₁ hybrid seeds.Much attention was paid to research on the plant breeding of the male sterile line and the basis for application in Brassica crops.The research on male sterility mechanism of Brassica contributed to the understanding of microspore and pollen development process and provided theory direction to create man-made male sterile line.In this investigation a novel preferentially expressed cDNA sequence from B.campestris ssp.chinensis was isolated and characterized. The corresponding full-length cDNA and DNA was subsequently amplified by Rapid amplification of cDNA ends（RACE）,named BcMF15（Brassica campestris Male Fertility 15,GenBank accession no.EF600901）. The gene sequence was analyzed and proteins functions were predicted.Spatial and temporal expression patterns were analysed by RT-PCR during the different stages of plant development.On this base the plant RNAi expression vector of BcMF15 was constructed regulated by the constitutive promoter CaMV35S. flowering Chinese cabbage[B.campestris L.ssp.chinensis Markino var.parachinensis（Bailey）Tsen et Lee] was transformed by agrobacterium-mediated method to obtain their loss-of-function mutant.Lastly,their function in pollen development process was analyzed,The major study results as follows:（1）The full-length cDNA and DNA has been cloned from Chinese cabbage using RACE.The full-length cDNA was 768 bp.A comparison of the cDNA and DNA sequence by DNAStar software showed that BcMF15 was composed of a 312 bp open reading frame,encoding an 11.36 kDa protein of 103 amino acids.This amino acid sequence was a membrane protein with a signal peptide at the N-terminus.which has an obvious transmembrane helix.The GenBank BLAST results,showed that the BcMF15 sequence that produced significant alignments had 88%nucleotide similarity to a LTP-like mRNA sequence from Arabidopsis（Columbia）.Meanwhile,six domains were predicted in the deduced BcMF15 protein,such as the AAI domain existing in some crucial proteins of pollen development-preferential,signal peptide, transmembrane domain,vWF domain,ZnF_C4 domain,and Tryp_alpha_amyl domain.The expression of its spatial and temporal expression patterns by comparing RNA isolated from various tissues of the wild-type plant with those of the mmc mutant（ZUBcajh97-01A）by RT-PCR.The transcript of the BcMF15 was undetectable in sporophytic tissues of fertile line such as scapes and leaves of seedling.Moreover,the expression signal of BcMF15 was detected from flower buds of stage 1 to stage 5,the flower and germinal siliques In contrast,the expression signal of BcMF15 was undetectable in any tissues of the mmc mutant. This indicated that BcMF15 was a crucial gene in microspore development.（2）Gene analogues from 10 species of six genera in Cuciferae were obtained by PCR strategy using specific primers designed from the full length of BcMF15.Homologous sequences of BcMF15 comparison indicated that the similarities among the genes at nucleotide and amino acid levels were over 80%and 53%, respectively.The conserved region of amino-acid sequences of BcMF15 orthologs included the transmembrane region in N-terminal,however there was some variation in C-terminal.The maximum parsimony（MP）trees showed that Brassica was closely related to Raphanus,followed by Capsella Medic, Arahidopsis Heynh and Barbarea,most distantly related to Orychophrogmus.These results.showed in Cruciferae the BcMF15 was relative conservative in evolution,and BcMF15 may play an important role in pollen development.（3）The RNAi expression vector of BcMF15 containing cons（?）tutive promoter CaMV35S was constructed,by agrobacterium-mediated method,their flower Chinese cabbage transgenic plantlets were obtained.（4）Molecular,morphological and cytological characterization of flower Chinese cabbage transgenic Kan^R plantlets transformed from pBI35S-RMF15 was carried out Real time quantitative RT-PCR on cDNA of small,middle,big floral buds,flower and-germinal siliques to quantify gene expression in plantlets transformed from pBI35S-RMF15 and nontransgenic plantlets.The results showed the expression of BcMF15 was caught and decreased in comparison to positive CK,and the expression of BcMF15 in middle, big floral buds of pBI35S-RMF15 were sharply inhibited,which proved that BcMF15 was a pollen-expressed LTP.The morphology of flowers in flowering Chinese cabbage transgenic plants for the RNAi BcMF15 revealed there were short and white flower filaments and little pollen in plants.As far as pBI35S-RMF15 was concerned the pollen germination was 35.94%in vitro,germination ratio of pollen decreased distinctly than 74.40%in the transgenic plants containing pBI121.The comparison of microspore morphology detected by scan electron microscope in transgenic plants Containing pBI35S-RMF15,When BcMF15 was inhibited,the abnormal development of pollen would be captured.The result proved that it was essential for BcMF15 to keep normal shape of pollen.Combining resin semithin sections in light microscope with Ultrathin sections of flower buds in transmission electron microscope,the observations for pollen development reveal at late uninucleate microspore stage the tapetum degenerated ahead of schedule,and the pollen was aborted.The result indicated that the expression of BcMF15 was inhibited by pBI35S-RMF15 which resulted in germination ratio of pollen decreased and abnormal pollen.So it was presumed the LTP encoded by BcMF15 was anther-specific,it may be responsible for lipid metabolism in anther through regulating the development of tapetum,and has an effect on the formation of pollen coat,pollination and elongation of pollen tube during microspore development.（5）Promoter of BcMF15 was isolated by TAIL-PCR for the first time.The GenBank BLAST results showed that the 5’-flanking regions of BcMF15 gene,named pBC15,produced significant alignments had 99%nucleotide similarity to genomic DNA from Brassica rapa subsp,pekinensis（Accession:AC189225）. The sequence was abundant A/T,which consistent to the composition of bases in promoter sequence.A number of potential regulatory motifs corresponding to known cis-regulatory signals of eukaryotic genes were found to be present in the sequence.The cloned Sequence analysis showed that TATA box elements were found in the sequence,GATA motif in CaMV 35S promoter,also some pollen-specific function elements were present in promoter of BcMF15,such as GGTT and GTGA motif,which were pollen-specific enhancer sequences and shared regulatory elements;POLLEN1LELAT52 motif,which was One of two co-dependent regulatory elements responsible for pollen specific activation of tomato lat52_gene.Some elements to respond to environment were also found in the 5’-flanking regions of BcMF15 gene.These preliminary results indicate that this DNA fragment upstream of the BcMF15 coding sequence could very possibly be a promoter with pollen specificity,the expresesion patterns may be influenced by environmental factors such as light,dehydration and other stress factors.（6）Construction of pBc15::GUS fusing vector was achieved.Recombinant plasmid was designated as pBI-pBc15.Plant expression vector was constructed by ligating this promoter and with gus gene then transferred into epidermis cell of onion by microprojectile bombardment.Histochemical GUS assay showed cell transformed with fused plasmid pBI=pBc15 of BcMF15 putative promoter may directed the expression of pBI-pBc15,and fused proteins were localized in the nuceil of transformed cells.It revealed the promoter region is functionally active.（7）The BcMF15 promoter-GUS fusion construct was stably transformed into wild type（Columbia） Arabidopsis plants by floral dip and homozygous transgenic plants were developed.Screening Kan^R plantlets and PCR analysis of T1 generation transgenic seedling were carried.Histochemical assays of gus activity and subsequent resin section analysis on promoter transformant plant showed that expression of pBclS::GUS was detected in anthers.更多还原