葡萄风信子MaFLS基因克隆与表达分析  

Isolation and Characterization of a Flavonol Synthase Gene from Muscari armeniacum

作　　者：杨慧萍[1] 刘雅莉[1] 娄倩[1] 李志根[1] 

机构地区：[1]西北农林科技大学旱区作物逆境生物学国家重点实验室,农业部西北地区园艺作物生物学与种质创制重点实验室,陕西杨陵712100

出　　处：《西北植物学报》2014年第8期1507-1513,共7页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家自然科学基金(31170652)

摘　　要：该研究利用PCR技术从葡萄风信子‘亚美尼亚’中克隆了1个黄酮醇合成酶(FLS)基因,命名为MaFLS。MaFLS的cDNA全长为1 076bp,开放阅读框为999bp,编码332个氨基酸,推测的蛋白质分子量38.51kD,理论等电点为5.09。同源比对和系统进化分析表明,MaFLS基因编码的氨基酸具有黄酮醇合成酶典型的2-ODD超家族保守结构域,与拟南芥和高粱FLS一致性分别为60%和83%。半定量PCR和荧光实时定量PCR表达分析表明,MaFLS在花器官中高表达,在根、茎、叶中微量表达,在完全未着色的花蕾期开始表达并于花完全开放未着色时期到达峰值,之后随花发育表达量逐渐降低。原核表达检测到1条大约38kD的外源蛋白,与预测的蛋白分子量相符。该研究结果为进一步探讨葡萄风信子MaFLS基因对黄酮类物质合成的调控作用奠定了基础。The gene MaFLS encoding the flavonol synthase（FLS）involved into flavonol synthesis was cloned from Muscari armeniacum.The full length cDNA of MaFLS was 1 076 bp,and the open reading frame length was 999 bp,which encoding aprotein polypeptide of 332 amino acids with a predicted molecular weight of 38.51 kD and pI of 5.09.The putative MaFLSdisplayed identities to the FLSs of Arabidopsis thaliana and Sorghum bicolor of 60%and 83%,respectively,and contained several typical conserved elements found in the 2-oxoglutarate-and Fe（Ⅱ）-dependent dioxygenases（2-ODD）.Quantitative real-time PCR analyzed the expression profiles of MaFLSin different tissues.The results demonstrated that MaFLS was constitutively expressed in roots,stems and leaves,with particularly high expression in flowers.The cDNA of MaFLS was then subcloned into pET-32 aand introduced into BL21（DE3）,and the recombinant plasmid was successfully expressed in the condition of 0.1mmol·L-1 IPTG at 25℃for 20 h.The molecular weight was found to be about 38 kD by checking with SDS-PAGE,nearly equal to the predicated.

关 键 词：葡萄风信子 黄酮醇合成酶 克隆 荧光实时定量 原核表达 

分 类 号：Q785[生物学—分子生物学] Q786