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山葡萄PAL基因原核表达载体的构建及表达蛋白纯化
陈蒙,袁赫海,张宇,刘海峰
0
(延边大学农学院,延吉 133002)
摘要:
山葡萄苯丙胺酸解氨酶(PAL)是苯丙烷途径的关键酶,在果皮着色中能够调控花色苷合成途径。构建PAL基因原核表达重组质粒pET28a-PAL,并对重组蛋白表达条件进行优化,以山葡萄DNA扩增PAL并连接到pMD18-T,筛选得到阳性克隆后连接到pET28a原核表达载体上,重组质粒转化至E.coli BL21后经不同浓度的IPTG、不同诱导温度、不同诱导时间处理,通过DPS设计正交试验优化重组蛋白的表达条件,并将重组蛋白纯化。结果显示,正交试验不同处理下的蛋白表达差异显著(P<0.05),诱导因素的影响程度从强到弱分别为IPTG浓度、温度和时间。IPTG诱导浓度对其影响最显著(P=0.015<0.05),且IPTG浓度为0.6 mmol·L-1,诱导温度为22℃,诱导时间为5 h时,诱导重组蛋白PAL的效果最佳。成功构建其原核表达载体并使其在大肠杆菌中得到高效表达,为探究PAL蛋白的表达提供参考。
关键词:  山葡萄  PAL基因  正交设计  原核表达
DOI:10.13610/j.cnki.1672-352x.20191122.023
投稿时间:2019-01-21
基金项目:国家自然科学基金(31260067)和延边大学大学生创新创业训练资助项目共同资助。
Construction of prokaryotic expression vector of Vitis amurensis PAL gene and purification of the expressed protein
CHEN Meng,YUAN Hehai,ZHANG Yu,LIU Haifeng
(College of Agriculture, Yanbian University, Yanji 133002)
Abstract:
Vitis amurensis phenylalanine aminolysis enzyme (PAL) is a key enzyme in the phenylpropanoid pathway, which regulates the anthocyanin synthesis pathway in pericarp coloring. The prokaryotic expression recombinant plasmid pET28a-PAL was constructed, and the expression conditions of the recombinant protein were optimized. The PAL in the grape was amplified and ligated into pMD18-T. The positive clone was screened and ligated into the prokaryotic expression vector of pET28a. The recombinant plasmid was transformed into E. coli BL21 and further induced by different concentrations of IPTG, different induction temperature and induction time. The expression conditions of the recombinant protein were optimized by design orthogonal test of DPS, and the recombinant protein was purified. The results showed that the expression of the protein was significantly different under different treatments in orthogonal test (P<0.05), and the influences of induction factors on the protein expression from strong to weak were respectively IPTG concentration, temperature and time. The induced IPTG concentration had the most significant effect (P=0.015<0.05), and under the conditions of IPTG of 0.6 mmol·L-1, the induction temperature of 22°C, and the induction time of 5 h, the induced recombinant protein of PAL was the best. In conclusion, the prokaryotic expression vector was successfully constructed and highly expressed in E. coli, which would provide some references for exploring the expression of PAL protein.
Key words:  Vitis amurensis  PAL  orthogonal design  prokaryotic expression

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