导　　师： 彭世清

学科专业： 0710

授予学位： 博士

作　　者： ;

机构地区： 海南大学

摘　　要： 巴西橡胶树以其胶乳产量高、质量好、易于获取而成为天然橡胶主要的商业来源,尽管天然橡胶生物合成途径等方面的研究已经取得了显著的进展,但天然橡胶生物合成的分子调节机制还不清楚。从橡胶合成的途径分析,参与橡胶生物合成的关键酶包括IPP异构酶和一系列的异戊烯基转移酶,如橡胶转移酶/(RuT/),法尼基焦磷酸合成酶/(FPS/),小橡胶粒子蛋白/(SRPP/)等,而SRPP在橡胶生物合成中起着类似于延长因子的作用。本研究以HbSRPP基因为研究切入点,分离和系统的研究了转录因子HbWRKY1的功能特性,旨在更深入的揭示小橡胶粒子蛋白在天然橡胶生物合成中的表达调控机制。 本研究通过Genome Walker的方法在巴西橡胶树基因组中克隆了HbSRPP启动子序列,该序列含有多个典型的真核生物启动子基本元件,如TATA-box、CAAT-box、 GATA-box等,且具有光响应、激素、组织特异性、非生物胁迫等应答元件。将HbSRPP启动子构建到诱饵载体上,通过酵母单杂交的方法在巴西橡胶树胶乳中分离到了HbWRKY1基因的全长cDNA序列,且HbWRKY1可以与HbSRPP启动子结合,具有转录激活能力。 生物信息学分析表明,HbWRKY1蛋白由479个氨基酸残基组成,且与其他植物WRKY类蛋白有高度同源性。HbWRKY1蛋白具有2个WRKY转录因子家族特有的结构域,且具有C2H2锌指结构。分子进化树分析表明,HbWRKY1属于WRKY基因家族第1组的成员。荧光定量PCR结果表明,HbWRKY1基因的表达具有组织特异性,胚状体中表达量较高,花、树皮、胶乳、叶片表达量较低。 亚细胞定位分析结果表明,在转化的洋葱表皮细胞中,HbWRKY1//GFP融合蛋白集中分布在细胞核内,而对照GFP蛋白在整个细胞中均有分布,证明了转录因子HbWRKY1定位在细胞核内。构建了HbWRKY1//GST原核表达载体,优化了HbWRKY1蛋白的诱导表达条件为：在37℃条件下经3mmol//L的IPTG诱导4h；且Western blot结果表明,诱导的蛋白即为HbWRKYl蛋白。 通过农杆菌介导法将HbSRPP启动子转化烟草植株,T1代GUS活性检测结果表明,HbSRPP启动子可以驱动GUS基因的表达。将HbWRKY1基因转入HbSRPP启动子转基因烟草T1代。对获得的pSRPP//HbWRKY1转基因烟草进行了GUS活性定量分析,结果表明,共表达的pSRPP//HbWRKY1转基因烟草GUS表达活性明显受到抑制,暗示了HbWRKY1转录因子对HbSRPP基因具有负调控作用,且HbWRKY1可能是天然橡胶生物合成途径中的负调控因子。 Hevea brasiliensis has been the only commercial source of natural rubber mainly because of its high yield, its quality, and the ease of harvesting. So far, researches on natural rubber biosynthesis pathway have made significant progress. Studies on the molecular regulation mechanism of natural rubber biosynthesis remain largely unknown. The key enzyme of participate in natural rubber biosynthesis, including a series of IPP isomerase and prenyl transferase, such as rubber transferase enzyme /(RT/), FPP synthase, Small rubber particle protein /(SRPP/) and so on. SRPP plays a direct role in elongating the rubber chain in rubber biosynthesis. In the study, based on HbSRPP gene, we isolated and analyzed systemically the characteristics and function of HbWRKYl, which will lay theoretical foundation for illuminating the regulation mechanism of SRPP in natural rubber biosynthesis The promoter region of HbSRPP was isolated from Hevea brasiliensis by Genome Walker method. TATA box, CAAT box, GATA-box and light-, hormone-, tissue-specific-, abiotic stress-responsive elements were found in the promoter region of HbSRPP. The promoter of HbSRPP was subcloned into the yeast bait vector. A full-length cDNA /(HbWRKYl/) was isolated from a cDNA library of the latex by yeast one-hybrid technology. Biochemical analysis demonstrated that the HbWRKYl protein was capable of binding to the HbSRPP promoter, and it demonstrated transactivation activity in yeast. HbWRKYl encodes a479amino acid protein containing two WRKY domains and a putative N-terminal Leu zipper; Bioinformatic analysis showed that the entire sequence of HbWRKYl has a high identity with those of other WRKY proteins; Analysis of the phylogenetic tree indicated that HbWRKYl protein belongs to Group I of WRKY transcription factor family. The Q-RT-PCR results showed that the expression of HbWRKYl gene was tissue-specific, with relative high transcription in somatic embryo, low transcription in flower, bark, latex and leaves. The results of subcellular localization indicated, in the transformed onion epidermal cells, the HbWRKY1-GFP fusion protein was localized in the nucleus, whereas the control GFP was distributed throughout the cell, demonstrating that HbWRKY1was targeted to nuclei. HbWRKYl was subcloned into pGEX-6p-1expression vector, the recombinant protein was induced4h with3mmol//L IPTG in the condition of37℃; Western-blot analysis revealed that the expression of protein was correct. The HbSRPP promoter was transformed into tobacco by Agrobacterium-mediated transformation. In the T/] plants of SRRPpro::GUS /(β-glucuronidase/), the GUS activity analysis revealed that the HbSRPP promoter drives expression of the GUS gene. HbWRKYl was transformed into the T1plants by Agrobacterium-mediated transformation, the GUS activity quantitative analysis revealed, co-expression of the effector construct35S::HbWRKY1with a reporter construct SRRPpro::GUS greatly suppressed expression of the GUS activities in stably transformed tobacco. These results strongly suggest that the HbWRKYl transcription factor does negatively regulate HbSRPP, HbWRKY1may be a negative regulator of natural rubber biosynthesis in Hevea brasiliensis.

关 键 词： 巴西橡胶树 觥基因 转录因子 酵母单杂交 转基因

分 类 号： [S794.1]

领　　域： [农业科学] [农业科学]