枸杞紫黄质脱环氧化酶(LcVDE)基因的克隆和分析

Cloning and Analysis of a Novel Gene Codifying for Violaxanthin De-epoxidase from Lycium chinense

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： Violaxanthinde-epoxidase play an important role in the violaxanthin cycle. The aim of the study is toclone the full-length eDNA of LeVDE from Lycium chinense, to predict the architectural feature, functional sites and the secondary structures of LcVDE through bioinformatics analysis and to confirm its functions. Method：The full-length eDNA of LcVDE was cloned from Lycium chinense using rapid-amplification of eDNA ends essay and RT-PCR. Bioinformatics software were used to analyse the amino acid sequences of LcVDE protein, and constructed the prokaryotie expression vectorp ET-VDE and transformed into E. coli BL21. Overexpression of LcVDE in the E. coli BL21 was induced by IPTG, and in the vitro system the LcVDE function was confirmed. Results： Bioinformatics analysis showed that the open reading frame of LcVDE gene is 1 413bp, encodes a putative polypeptide of 470aa with molecular mass of 53.61 kDa and isoeleetric point of 5.77. According to the analysis of SDS-PAGE, the LcVDE over-expressed in E. coli. The protein was added to thevitro reaction system, absorption spectrometry and HPLC indicated that the expressed protein catalyzed violaxanthin de-epoxidationreaction. Conclusion： In this study, the protein encoded by the LcVDE gene has the same function toviolaxanthin de-epoxidase.