【作者】 鲁慧中；

【导师】 彭世清；

【作者基本信息】 海南大学 ， 植物分子遗传学， 2010， 硕士

【摘要】 巴西橡胶树(Hevea brasiliensis)是天然橡胶的主要来源。巴西橡胶树的幼态无性系具有生长速度快、产胶量高、抗逆性强的特点,是一种具有很大应用潜力的新型种植材料。对橡胶树幼态无性系高产的机制还不清楚,目前仅限于对幼态无性系和老态无性系之间差异的形态学观察、排胶生理参数等的研究,而缺乏直接的分子生物学、遗传学等方面的证据,在一定程度上限制了橡胶树幼态无性系在生产中的应用和大面积推广。因此开展橡胶树幼态无性系高产的分子基础研究将对我们进一步了解橡胶树幼态无性系增产的原因具有重要的理论意义；同时对推动橡胶树幼态无性系种植材料的推广和应用,提高天然橡胶的产量也将具有重要的实践意义。本课题组通过抑制缩减杂交(SSH)获得一个在幼态无性系和老态无性系之间差异表达的片段(HbSSH),经过BlastX分析,发现HbSSH所编码的氨基酸序列与蓖麻(Ricinus communis)硫氧还蛋白氨基酸序列的同源性很高。本研究根据HbSSH的序列信息设计引物,通过RT-PCR和RACE技术从巴西橡胶的胶乳中获得了目的基因(命名为HbTRX),该基因总长546bp,其中包括30bp的5’-UTR和144bp的3’-UTR,编码123个氨基酸,该氨基酸序列与蓖麻、毛果杨、葡萄、轮叶党参、拟南芥、大球果黄杉的TRX氨基酸序列同源性达到了79%、78%、77%、77%、72%、69%。生物信息学表明HbTRX具有TRX典型的WCXPC结构域。半定量RT-PCR结果显示,HbTRX基因的表达具有组织差异性,在愈伤中大量表达,胶乳中次之,在花、叶、芽、树皮中均微量表达；它在橡胶树733、海2、海1三种品系的老态无性系和幼态无性系胶乳中均有差异表达,均为老态无性系胶乳中较幼态胶乳中高表达；乙烯和茉莉酸对胶乳HbTRX基因的表达几乎没有影响。构建HbTRX的原核表达载体pET-30a-HbTRX,并在大肠杆菌BL21(DE3)中进行了表达,经过IPTG诱导4个小时之后,在SDS-PAGE中显示出特异性条带,产生包括His-tag(约5.4 kD)在内的约21.3 kD的重组HbTRX蛋白,和理论上蛋白的大小基本一致。超声波破碎细胞后,经SDS-PAGE检测发现重组蛋白在大肠杆菌BL21中是以可溶蛋白的形式存在的。为了进一步探究HbTRX的特性和功能,本研究采用Ni2+螯合亲和层析法分离出重组HbTRX蛋白,并构建了两组反应体系,第一组为25μg／ml重组HbTRX蛋白与牛胰岛素构成的反应体系,第二组为50μg／ml重组HbTRX蛋白与牛胰岛素构成的反应体系,这两组体系均有DTT的参与。结果显示,这两组反应均在650nm的光波下显示出逐渐上升的光吸收值,而50μg／ml重组HbTRX蛋白与牛胰岛素构成的反应体系的光吸收值更高,90min之后这两组反应均进入平台期。这个结果表明,重组HbTRX能降低牛胰岛素二硫化物的结合,具有氧化还原活性。更多还原

【Abstract】 Hevea brasiliensis is the main source of natural rubber. The juvenile clone of Hevea brasiliensis has great potential a pplication as a new type of planting material, with fast growth speed, high volume production of rubber, and the advantages of strong resistance. Now the research on morphology, physiology and cultivation of Hevea brasiliensis juvenile clone had made considerable progress, and the volume production of rubber reached a certain level. But there is no sufficient evidence of molecular biology to directly prove its high-yielding mechanism. The molecular biology research of rubber is still backward, thus limited the production applications and large-scale promotion of Hevea brasiliensis juvenile clone.So it has important theoretical and practical significance to do some work on the biological function of differentially expressed genes in rubber latex of old and juvenile clone of Hevea brasiliensis. A differentially expressed fragment of latex in old and juvenile clone of Hevea brasiliensis was obtained through the suppression subtractive hybridization by our reseach group.It encodes a thioredoxin-like amino acid analyzed by BlastX. Because thioredoxin is a class of multi-functional acidic protein existed in vivo and can regulate the activity of some key enzymes during the photosynthesis process in plant. The study of thioredoxin in rubber tree is shortage, and its function is not clear, therefore, by using the information about the acquired sequence (HbSSH), the thioredoxin was cloned by the technology of RT-PCR and RACE, named HbTRX. Sequence analysis revealed that the ORF of HbTRX encodes 123 amino acid. The deduced amino acid sequences of HbTRX showed high identities (79%,78%,77%,77%,72%,69%) to those of thioredoxin from Ricinus communis, Populus trichocarpa, Vitis vinifera, Codonopsis lanceolata, Arabidopsis thaliana and Pseudotsuga macrocarpa. Bioinformatics shows that HbTRX was a member of TRX-family, with a similar CXXC domain.Semi-quantitative RT-PCR analysis indicates that the HbTRX is most highly expressed in callus, and more highly expressed in latex than in flower, in enbryos, in leaf and bud. And it is highly expressed in old clone of every type of rubber tree. The transcription of HbTRX was not induced in the latex by jasmonic acid and ethylene. In order to further study the function of HbTRX, construct HbTRX prokaryotic expression vector pET-30a-HbTRX, specific protein band was detected by SDS-PAGE in the cell homogenate by induction with IPTG in the E.coli cells harboring pET-30a-HbTRX produced recombinant proteins with a total predicted molecular mass of 21.296kD. The recombinant proteins is a form of soluble protein. Ultrasonic broked cells with recombinant proteins. The fact was that recombinant protein was a soluble protein in E. coli (BL21) by SDS-PAGE.In order to understanding the features and functionality of HbTRX protein, the study separated recombinant proteins by Ni2+ chelate affinity chromatography, and constructed two reaction system with the presence of DTT. The first reaction system is composed of 25μg/ml recombinant protein and bovine insulin, the second is composed of 50μg/ml recombinant protein and bovine insulin. The results showed that, the two groups displayed that the light absorption value was gradually increased under in the 650nm light waves. The light absorption value of the second reaction was higher than the first reaction, and both of reaction groups went into plateau 90min later. This shows that, the recombinant protein in E. coli, can reduce bovine insulin disulfides bridges, with weak redox activity.更多还原