【作者】 付洪冰； 张俊华； 崔崇士；

【Author】 FU Hong-bing,LIU Qi,ZHAO Xi,ZHANG Bi-xian,ZHANG Li-li,CUI Chong-shi (Horticulture College,Northeast Agricultural University,Harbin Heilongjiang 150030)

【机构】 东北农业大学园艺学院；

【摘要】 从东北山葡萄中提取总RNA,采用RT-PCR方法,克隆到了白藜芦醇合酶（Resveratrol Synthase,简称RS）基因的全长cDNA。将测序结果进行分析、拼接,用Primer Premier5.0软件将序列转化为氨基酸序列,与GenBank中己登录的圆叶葡萄、华东葡萄、河南毛葡萄、河岸葡萄、酿酒葡萄进行同属RS基因编码的氨基酸同源性比对,与GenBank中已登录的羊蹄甲、花生、虎杖、大黄进行不同属RS基因编码的氨基酸进行同源性比对,结果表明:东北山葡萄与其它葡萄的RS基因编码的氨基酸同源性达到97%以上;东北山葡萄与其它属的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 cDNA片段插入到表达载体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℃、0.5 mmol.L^-1IPTG诱导4小时时该基因表达效果最好,诱导产物为一个约43kD的蛋白,为进一步纯化和鉴定目的蛋白提供了基础。更多还原

【Abstract】 The total RNA was extracted from leaves of Vitis amurensis Rupr,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,isoelectric point(PI) was 6.2, and it included 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, molecular weight of introduced product was 43kD about.This will provide a base for further purification and identification.更多还原