烟草柠檬酸合成酶基因的克隆及其光诱导型植物表达载体的构建

Cloning of tobacco citrate synthase cDNA and construction of its light inducible plant expression vector

紫花苜蓿为多年生优质豆科牧草。我国南方地区酸性土壤分布比较广,铝害比较严重,限制了紫花苜蓿在南方地区的推广利用。提高有机酸合成酶基因的表达活性,增加有机酸的合成与分泌,有利于增强植物的耐铝性。本研究根据Genebank中已知的烟草柠檬酸合成酶(Citrate Synthase,cs)基因的序列,通过RT-PCR从烟草总RNA中扩增cs基因的cDNA,亚克隆于T载体得到重组载体pMD18-cs,对pMD18-cs中的插入片断进行核酸序列分析确认为cs基因的cDNA全长。用光诱导型启动子(Rubisco,小亚基的启动子)和双元载体pPZP211构建了cs基因的光诱导型植物表达载体pPZP211-PrbcS-cs,为利用基因工程手段提高紫花苜蓿耐铝毒能力,促进其在南方地区推广利用奠定了物质基础。

Medicago sativa is one of the most highly valued forage legumes. Aluminum toxicity is the main problem in the acid soils that are widely distributed in south China, and this restricts extending the use of M. sativa in this district. The aluminum-tolerance of plants can be enhanced by excretion of organic acid by increasing organic acid synthase gene activity. In this study, a eDNA for the cs gene （encodes citrate synthase） was amplified by RT-PCR from tobacco and subcloned into a T cloning vector to yield pMD18-cs. Sequence analyses confirmed that the insertion fragment in pMD18-cs contained the full length of cs eDNA. Using the promoter for the Rubisco small subunit gene （a light inducible promoter） and the binary vector pPZP211, a light inducible plant expression vector pPZP211-PrbcS-cs was constructed. It can be used for genetic engineering to enhance the aluminum-tolerance of M. sativa in order to extend the use of M. sativa in south China.