水稻β-石竹烯合成酶基因OsCAS的克隆鉴定、原核表达及其遗传转化

Cloning and prokaryotic expression of rice gene encoding β-caryophyllene synthase and its genetic transformation.

为阐明水稻β-石竹烯在调节水稻、害虫及其天敌相互关系中的作用,克隆鉴定了一个水稻β-石竹烯合成酶基因OsCAS,并对其原核表达与遗传转化进行研究.该基因的cDNA全长1731 bp,包含一个1728 bp的开放阅读框,编码一个由576个氨基酸组成的蛋白,预测分子量66.5 kDa.系统进化树分析表明,水稻OsCAS基因编码蛋白的氨基酸序列与同为单子叶植物玉米的β-石竹烯合成酶氨基酸序列同源性达99%,而与其他植物(拟南芥、青篙、黄瓜)的同源性仅51%.褐飞虱为害和茉莉酸处理能明显上调OsCAS基因的表达水平.同时原核表达了OsCAS基因,并利用农杆菌转化系统获得OsCAS基因过量表达和RNAi的水稻品系,为分析OsCAS基因的生化与生物学功能奠定了基础.

To elucidate the role of β-caryophyllene emitted from rice plants in mediating interactions among rice, herbivores and their natural enemies, a rice β-caryophyllene synthase gene OsCAS was cloned, expressed in E. coli and over-expressed or silenced in rice plants using an Agrobacterium-based transformation system. The gene OsCAS, a 1731 bp eDNA, contains an open reading frame of 1728 bp, which encodes a protein of 576 amino acids with a calculated molecular weight of 66.5 kDa. Phylogenetic tree analysis showed that the putative rice OsCAS had a high amino acid identity （99%） to maize （Zea mays） β-caryophyllene synthase, whereas a low amino acid identity （51%） to those in other plants including Arabodopsis （Arabidopsis thaliana）, Artemisia （Artemisia annua ） and cucumber （Cucurnis sativus ）. Infestation by rice brown planthopper Nilaparvata lugens （Stal） or jasmonic acid treatment enhanced significantly the expression levels of OsCAS gene. The recombinant OsCAS protein was expressed in E. coli and rice lines with over-expression or RNAi silencing of OsCAS gene were obtained by Agrobacterium-mediated transformation, which laid a foundation for elucidating the biochemical and ecological functions of OsCAS.