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目的:克隆枸杞VDE基因的全长cDNA,通过对基因序列的生物信息学分析预测表达产物的结构特征和功能位点并验证其功能,为研究枸杞紫黄质循环的作用机理打下基础。方法:利用cDNA末端快速扩增和RT-PCR方法克隆枸杞VDE基因全长cDNA序列,生物软件分析VDE的生物学信息。构建VDE基因的原核表达载体pET-VDE,转化大肠后用IPTG诱导VDE过量表达;并构建体外反应体系对VDE表达蛋白酶功能进行验证。结果:LcVDE基因的ORF长1 413bp,编码的蛋白由470个氨基酸组成,分子量为53.61kDa,等电点为5.77。SDS-PAGE电泳结果表明,枸杞VDE基因在大肠杆菌中得到了过量表达。克隆基因表达蛋白进行紫黄质的脱环氧化反应,吸收光谱和HPLC的分析结果表明,表达蛋白催化了紫黄质的脱环氧化反应。结论:克隆得到的VDE基因编码的蛋白具有紫黄质脱环氧化酶的的功能与活性。
OBJECTIVE: To clone the full-length cDNA of VDE gene of Lycium barbarum and to predict its structure and function by bioinformatics analysis of gene sequence and to verify its function, which laid the foundation for studying the mechanism of violaxanthin cycle in Lycium barbarum. Methods: The complete cDNA sequence of VDE gene of Lycium barbarum was cloned by rapid amplification of cDNA ends and RT-PCR. Biological software was used to analyze the biological information of VDE gene. The prokaryotic expression vector pET-VDE of VDE gene was constructed and overexpression of VDE was induced by IPTG after transformation into the large intestine. The in vitro reaction system was constructed to verify the function of VDE-expressing protease. Results: The ORF of LcVDE gene was 1 413bp in length. The encoded protein consisted of 470 amino acids with a molecular weight of 53.61 kDa and an isoelectric point of 5.77. SDS-PAGE electrophoresis results show that the wolfberry VDE gene was overexpressed in E. coli. The epoxidation of violaxanthin by the cloned gene was carried out. The results of absorption spectroscopy and HPLC showed that the expressed protein catalyzed the epoxidation of violaxanthin. Conclusion: The protein encoded by the cloned VDE gene has the function and activity of violaxanthin deacylases.