摘要
从贝达葡萄中提取总RNA,采用RT-PCR方法,克隆到了白藜芦醇合酶(resveratrol synthase,RS)基因的全长cDNA。将其与圆叶葡萄(Vitis rotundifolia)、华东葡萄(Vitis pseudoreticulata)、河南毛葡萄(Vitis ficifolia)、河岸葡萄(Vitis vulpine)、酿酒葡萄(Vitis virufera)、羊蹄甲(Bauhinia)、花生(Arachis hvpogaea)、虎杖(Polygonum cuspiaatum)、大黄(Rheum officinale)进行氨基酸同源性比对,结果表明:分离的RS基因与其他葡萄的RS基因编码的氨基酸同源性达到97%以上,与其他属的RS基因编码的氨基酸同源性达到64%以上。生物信息学分析表明,该蛋白分子量为43kD,等电点pI=6.2,包含392个氨基酸残基。将RS的全长cDNA插入到表达载体pET28a中构建重组表达质粒pET28a-RS,转化大肠杆菌BL21,SDS-PAGE电泳检测结果表明,以30℃、0.5mmol.L-1IPTG诱导4h时该基因表达效果最好,诱导产物为1个约43kD的蛋白,为进一步纯化和鉴定目的蛋白提供了基础。
The total RNA was extracted from leaves of Vitis ＂Beida＂,and the full-length cDNA sequence of resveratrol synthase（RS） was cloned from the total RNA.Blast analysis of the amino acid sequence from the cDNA sequence of resveratrol synthase（RS） in NCBI database comparing to other varieties and other generas showed that the amino acid sequence was over 97% and 66% identity,respectively.In this paper,the protein product was analyzed using bioinformation and molecular biology software.The result indicated that the protein molecular weight was 43kD,with isoelectric point（PI） 6.2,and 392 amino acid residues.RS cDNA fragment was inserted into the expression vector pET28a for constructing recombinant expression plasmid pET28a-RS,and then was transformed into E.coli BL21.SDS-PAGE electrophoresis analysis showed that expression effect was the best at the conditions of 30℃ and 0.5 mmol·L-1 ITPG introduction for 4 hours.The molecular weight of introduced product was about 43kD.This will provide a base for further purification and identification.
出处
《沈阳农业大学学报》
CAS
CSCD
北大核心
2011年第4期422-427,共6页
Journal of Shenyang Agricultural University
基金
哈尔滨市科技攻关计划项目(2003AA6CN088)
关键词
葡萄
白藜芦醇合酶
基因克隆
序列分析
原核表达
Vitis ＂Beida＂
resveratrol synthase
gene cloning
sequence analysis
prokaryotic expression
