小叶杨TMM基因启动子克隆及表达模式分析

气孔作为植物与外界进行气体交换的通道,对植物自身的生长发育至关重要。研究表明植物TMM蛋白与植物气孔的形成发育密切相关。本研究利用PCR技术克隆了小叶杨(Populus simonii Carr.)TMM基因5’端上游的调控序列pPsTMM,长度为2336 bp。生物信息学分析结果表明:小叶杨启动子pPsTMM除了含有TATA-box、CAAT-box等核心启动子元件之外,还含有多种光响应元件、植物激素响应元件、抗逆胁迫相关元件以及生理代谢和生长发育相关元件等,说明转录活性还可能受到光、植物激素、逆境胁迫等因素诱导。利用双酶切法构建植物表达载体pBI121-pPsTMM-GUS,叶盘法稳定遗传转化烟草,GUS化学组织染色显示GUS主要集中在叶芽起始发育部位表达,说明pPsTMM启动活性具有组织特异性。对非生物胁迫的转基因烟草进行GUS化学染色以及进行荧光定量PCR,结果显示pPsTMM对不同因素的非生物胁迫响应程度存在差异性,干旱和盐胁迫下启动活性很低,高温和低温对其调控作用也不明显,但对外在施加的植物激素(GA,NAA,ABA)和水杨酸(SA)响应程度很高,说明pPsTMM启动子对物理因素的响应值要远低于化学因素的诱导。本研究为进一步阐明TMM蛋白表达模式与小叶杨气孔发育特性及抗旱性之间的分子机制提供理论基础。

Cloning and Expression Pattern Analysis of the Promoter of TMM Gene in Populus simonii

Stomata,as a channel for the exchange of plants with the outside world,is essential for plant growth and development.Recently researches showed that TMM protein was closely relative to the formation and development of plant stomata.The 5’-upstream regulatory sequence of the TMM gene in P.simonii Carr.was cloned by PCR with a length of 2336 bp,named pPsTMM.Bioinformatics analysis showed that pPsTMM contained not only TATA-box,CAAT-box and other core promoter elements,but also a variety of elements,such as light-responsive elements,plant hormone-responsive elements,stress-resistant elements,physiological metabolism and growth-related elements,which means that pPsTMM may can be induced by light,plant hormones and stress and so on.pPsTMM was inserted into a plant expression vector pBI121-pPsTMM-GUS by double enzyme digestion,and then was transformed in tobacco.GUS staining of PsTMM::GUS transgenic tobaccos showed that GUS were mainly expressed in the initiation and development parts of leaf buds,indicating that pPsTMM had promoter activity and tissue specificity.The results of GUS chemical staining and fluorescence quantitative PCR of PsTMM::GUS transgenic tobacco under different abiotic stress treatment showed that the response of pPsTMM was distinct.pPsTMM promoter activity was very low under drought and salt stress treatment,and the inducing efficiency under high and low temperature was also not obvious,but pPsTMM was highly responsive to exogenous application with phytohormones(GA,NAA,ABA)and salicylic acid(SA).We deduced that the response of pPsTMM to physical factors was much lower than to chemical factors.This research might provide base theory for the further study on the molecular mechanism among TMM expression pattern,stomata development,and drought resistance in P.simonii Carr.