水稻乙酰乳酸合成酶基因的克隆和功能分析

Isolation and Functional Analysis of Rice Acetolactate-synthase (ALS)

乙酰乳酸合成酶(ALS)是植物和微生物中亮氨酸、异亮氨酸和缬氨酸合成途径的一个关键酶,该基因在植物抗除草剂基因工程中具有重要的应用价值。利用生物信息学分析,自水稻中分离了ALS基因的cDNA和基因组DNA序列。序列分析表明,ALS基因没有内含子,GC碱基含量高并不对称分布。软件预测ALS基因是单拷贝基因位于第4号染色体上,Southern杂交验证了此结果。构建ALS基因的植物表达载体并转化烟草,转基因烟草通过PCR-Southern验证。EXACT-RT-PCR结果表明,水稻ALS基因在转基因烟草的叶片和根部均有转录发生。利用甲嘧磺隆进行除草剂抗性测试,和对照烟草相比转基因烟草除草剂抗性高达50mg.L-1。

Acetolactate-synthase （ALS） is a key enzyme in biosynthetic pathway for valine, leucine, and isoleucine in plant and microorganism. It is important for plant insect-resistant genetic engineering. The cDNA sequence and genome DNA sequence of ALS gene were isolated. Sequence analysis indicated that ALS gene has no introns and its GC base content is asymmetrical. Software analysis result speculated that ALS has one copy locating on chromosome 4, which was confirrned by southern blotting. ALS plant expression vectors have been constructed in tobacco and transgenetic tobacco was confirmed by PCR southern blotting. EXACT-RT-PCR indicated rice ALS can be transcribed both tobacco leaves and roots. Data for bioassay of Sulfometuron-methyl showed that transgenetic tobacco displayed notably resistance to herbicide compared with wild plant, with highest content of 50 mg/L.