杨树液泡膜糖转运蛋白基因功能和作用机制研究

Previous studies have shown that sugar transporters play a major role in the growth and development of woody plants. Our recent studies also showed that the sugar transporter proteins encoded by PtTST1.1, PtTST2.1 and PtTST3 were located on vacuole membrane, and the expression of them were regulated by phytohormones. Based on these findings, we hypothesize that overexpression or interference of tonoplast sugar transporter gene (TSTs) may promote the xylem formation and increase the biomass production of poplar. In this project, the effects of PtTST1.1, PtTST2.1 and PtTST3 on polar growth and xylem development will be investigated at gene, protein, cell and tissue levels. The relationship between these three genes and plant hormones will also be determined by measuring the contents of endogenous gibberellin and brassinolide. Finally, the transporting activity will be carried out by the complementation of the yeast defect mutant, and the measuring of sugar contents. The completion of this project will not only clarify the biological functions of PtTST1.1, PtTST2.1 and PtTST3, and the molecular mechanisms of them in regulation xylem development, but also provide research bases for the genetic breeding of trees with improved agronomic traits.

已有研究表明，糖转运蛋白在木本植物的生长发育过程中发挥着重大作用。我们近期的研究发现，杨树液泡膜糖转运蛋白定位在液泡膜上，并且PtTST1.1、PtTST2.1和PtTST3的表达受激素的调控。因此，推测杨树液泡膜糖转运蛋白基因（TSTs）的过表达或者反义表达能够促进杨树“库”--木质部的形成，从而增加杨树的生物量。本研究将以转基因杨树、拟南芥突变体和番茄为材料，通过一系列分子生物学方法，从基因、蛋白、组织和细胞水平研究目标基因在杨树生长和木质发育中的生物学功能；再通过测定内源赤霉素和油菜素内酯含量来确定这三个基因与激素的关系；最后通过酵母缺陷型突变体互补实验和糖含量的测定明确这三个基因的转运活性。本项目的实施不仅可以阐明PtTST1.1、PtTST2.1和PtTST3的生物学功能，同时可以揭示其调控木质部发育的分子机理，并为加速林木新品种的选育奠定遗传基础。

杨树是重要的生物质能源树种和环境保护用树，培育速生和生物量增加的杨树新品种具有巨大的经济价值和生态效益。本研究以转基因杨树和拟南芥突变体为材料，通过一系列的分子生物学方法从基因、组织和细胞水平研究了PtTST1.1和PtTST2.1在杨树生长和木质发育中的生物学功能。本研究从杨树测序品种毛果杨（Populous trichocarpa）中克隆到拟南芥TST（液泡膜糖转运蛋白基因）的同源基因PtTST1.1、PtTST2.1和PtTST3，同时将PtTST1.1、PtTST2.1和PtTST3的过表达载体通过农杆菌侵染导入山新杨（P. davidiana × P. bolleana）中，通过GUS组织化学染色、RT-PCR和PCR检测过表达植株。研究发现，过量表达PtTST1.1转基因株系在组培苗时期，温室苗时期，以及田间试验中的生长速度加快，直径变粗和株高变高；进一步分析发现过表达株系株高变高主要是由于杨树节间距和节间数目变多造成；直径变粗的原因是糖含量增加和木质部增多造成；过量表达PtTST1.1转基因株系能够提高拟南芥和杨树对盐胁迫的抗性。过表达PtTST2.1基因可以显著提高转基因植物的生长速度和生物量。本研究不仅阐明了PtTST1.1和PtTST2.1的生物学功能能，并为通过基因工程来改良林木木材产量及生长速度提供理论基础。