【作者】 黄德宝；

【导师】 唐朝荣；

【作者基本信息】 海南大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 在巴西橡胶树中,橡胶树的产胶功能是靠分布于树体的乳管系统实现的,胶乳是乳管细胞的细胞质。天然橡胶的生物合成是以蔗糖为原料,以乳管为加工场所进行的,乳管中的蔗糖供给能力与橡胶产量密切相关。因此,对橡胶树乳管中蔗糖供给问题的研究具有重要的意义。现已明确,在高等植物中,韧皮部介导的蔗糖运输是由一个中等规模的蔗糖转运蛋白(Sucrose transporter,SUT)基因家族参与完成的。研究蔗糖转运蛋白在橡胶树中的潜在生物学功能,具有极其重要的实践应用价值和理论意义。本研究首次对巴西橡胶树蔗糖转运蛋白基因进行了克隆,并对其表达特性进行了较为系统的分析,进一步探讨了这些基因可能的生物学功能。根据不同植物蔗糖转运蛋白氨基酸的保守序列设计简并引物,利用RT-PCR和RACE技术,从橡胶树中克隆了六个蔗糖转运蛋白基因的全长cDNA,分别命名为:HbSUT1、HbSUT2A、HbSUT2B、HbSUT3、HbSUT4和HbSUT5,相应序列已在GenBank中登录(DQ985466、DQ985467、DQ985465、EF067334、EF067335和EF067333)。HbSUT1、HbSUT2A、HbSUT2B、HbSUT3、HbSUT4和HbSUT5的cDNA分别编码531、611、611、535、498和498个氨基酸;生物信息学分析表明,这6个基因的编码蛋白均为跨膜结合蛋白,含有植物SUT蛋白典型的十二个跨膜结构域;利用酵母突变体所作的功能互补实验表明:这六个基因在酵母中的表达产物具有介导蔗糖转运的生理功能,能够使突变体酵母在以蔗糖为唯一碳源的培养基上生长,显示这些基因均编码正常蔗糖转运功能的SUT蛋白。利用半定量RT-PCR表达分析,研究了不同HbSUT基因的表达特性。在不同的橡胶树组织中,HbSUT基因表达模式具有明显的差异,呈现多样性。HbSUT1在枝皮和根中的表达量较高;HbSUT 2A除在枝皮,雌花,雄花中的表达量高外,还贯穿于叶片由叶芽,古铜期,淡绿期到稳定期的整个发育过程;HbSUT 2B在雄花中的表达量较高;HbSUT 3则在胶乳和雌花中的表达量最高;HbSUT 4在胶乳,枝皮中的表达量高于其它组织;HbSUT 5在枝皮和成熟的种子中表达量高。伤害和割胶均能诱导HbSUT3基因上调表达,而下调HbSUT5基因的表达,这表明在正常的丌割树中,HbSUT3可能在乳管蔗糖供给中起主要的生理作用;乙烯利刺激上调HbSUT2A、HbSUT2B和HbSUT3的表达,暗示这些基因与乙烯利刺激增强乳管代谢能力相关,可能共同参与乳管的蔗糖供给。此外,结合对不同产胶水平的橡胶树单株和不同橡胶树品系中不同HbSUT基因的表达分析,以及不同基因在死皮树和健康树中表达水平的比较,我们初步认为:HbSUT3基因可能在橡胶树的乳管蔗糖供给中起主要作用,而HbSUT5基因可能参与对蔗糖供给能力的调控。更多还原

【Abstract】 In Hevea brasiliensis, natural rubber biosynthesis depends on laticifer system. Latex is the cytoplasm of laticiferous cells. Natural rubber biosynthesis is accomplished with sucrose as the raw material, and latex vessels as the factory. Therefore, the capability of sucrose supply for latex vessel is vital for rubber production. It has been well known that in higher plants, a medium-sized gene family of sucrose transporter (SUT) is involved in the phloem-meditated sucrose translocation. However, in hevea, few are known about the SUT genes and their function. It has important academic meaning and applied value to study potential function of sucrose transporter genes in Hevea brasiliensis. In this study, we cloned six sucrose transporter genes, analyzed its expression patterns and did some forecast about its protein function.A pair of degenerate primers was designed according to the conserved regions which encode the sucrose transporter genes. Using the method of PCR and RACE , cloned six sucrose transporter genes from Hevea brasiliensis and named HbSUT1, HbSUT2A, HbSUT2B, HbSUT3, HbSUT4, HbSUT5. Six sucrose transporter genes from Hevea brasiliensis encode 531、611、611、535、498、498 amino acids. Biology informatics analysis indicated that six sucrose transporters from Hevea brasiliensis have twelve transmembrane domains. To demonstrate that the putative HbSUTs sequences identified above encode functional sucrose transporters, full-length HbSUTs cDNAs were constructed and then expressed in SEY6210.The results of complementation of yeast revealed that the HbSUT has physiological roles in yeast, which transformed with empty vector were unable to grow on the sucrose media while the HbSUTs construts enabled yeast cells to grow on sucrose alone.The expression of HbSUT genes was determined using RT-PCR analysis. The expression patterns in different tissues were variety, the mRNA levels of HbSUT1 are high in tress bark; the amount of mRNA of HbSUT2A are high in tress bark, female flower, male flower and through the ages of leaf development; the mRNA levels of HbSUT2B are high in male flower; the mRNA levels ofHbSUT3 are high in latex, female flower; the mRNA levels ofHbSUT4 are high in tress bark and latex; the mRNA levels of HbSUT5 are high in tress bark and mature seeds. Wounding and tapping could induce HbSUT3 up-regulated and HbSUT3 down-regulated. In tapping trees, HbSUT3 paly an important roles in sucrose supply to laticiferous cells. Ethylene could induce HbSUT2A, HbSUT2B, HbSUT3 up-regulated. These results suggest that the metabolize of laticiferous cells increase after ethylene treated. Three HbSUT genes have a cooperation roles in sugar supply. Furthmore, the results of expression patterns in different yield, different clones, between the healthy and TPD trees, it could be showed that HbSUT3 paly an important roles in sucrose supply to laticiferous cells while HbSUT5 has a potential regulatory role in Hevea brasiliensis.更多还原