栀子类胡萝卜素剪切双加氧酶基因GjCCD4的克隆与原核表达

发布时间：2018-02-28 16:02

本文关键词： 栀子类胡萝卜素剪切双加氧酶基因克隆　出处：《生物技术》2016年01期 　论文类型：期刊论文

【摘要】：[目的]类胡萝卜素剪切双加氧酶(Carotenoid cleavage oxygenase,CCD)是西红花总苷生物合成途径的关键酶,本研究拟从栀子果实中克隆CCD基因。[方法]在NCBI数据库中搜索CCD蛋白,获得种子序列后,构建系统进化树,按照亲缘关系分组。再利用CODEHOP法,针对每个组设计简并引物,在栀子果皮和果肉c DNA中扩增,获得保守区段。然后通过RACE获得基因全长。构建pet28a原核表达载体,转化BL21(DE3)菌株后在30℃培养4 h,转化菌株Aritic Expression(DE3)后在15℃培养过夜,诱导蛋白表达。[结果]克隆了一个GjCCD4基因,长1 863 bp,编码的蛋白质N端序列变动较大,C端保守性强,N-末端含有一个叶绿体定位的信号肽。系统发育分析表明,GjCCD4蛋白与拟南芥和西红花的同源蛋白聚在同一个簇。在BL21(DE3)和AE(DE3)菌株的沉淀中表达出大量包涵体形式的融合蛋白,在BL21(DE3)菌株的上清中表达出少量可溶蛋白。[结论]获得了一个类胡萝卜素剪切双加氧酶基因GjCCD4,通过原核表达获得了少量可溶蛋白。
[Abstract]:[Objective] carotenoid shear dioxygenase (Carotenoid cleavage, oxygenase, CCD) is a key enzyme in the biosynthesis of crocin from gardenia fruit, this study intends to clone CCD gene. Methods search CCD protein in the NCBI database, seed sequences, phylogenetic tree, grouped according to their relationships. Using the CODEHOP method, for each group of degenerate primers and amplified in Gardenia peel and pulp C DNA, obtain the conserved segment. Then obtain the full-length genes by RACE. Prokaryotic expression vector of pET28a was constructed, and transformed into BL21 (DE3) strain at 30 C 4 h culture, strain Aritic Expression (DE3) to cultivate. At 15 DEG C, induced protein expression. The cloning of a GjCCD4 gene, 1863 BP in length, encoding protein N terminal sequence changes, C terminal conservative, N- terminal signal peptide containing a chloroplast localized system development. Analysis showed that GjCCD4 protein homology with Arabidopsis and saffron together in the same cluster. In BL21 (DE3) and AE (DE3) expression of a large number of inclusion bodies in the form of fusion protein strains in the precipitation of BL21 (DE3) on the expression of a small amount of soluble protein. Conclusion] to obtain a carotenoid shear dioxygenase gene of strain GjCCD4 was obtained, a small amount of soluble protein by prokaryotic expression.

【作者单位】：江西民族传统药现代科技与产业发展协同创新中心/江西中医药大学;江西省中药种质资源工程技术研究中心/江西中医药大学;桂林医学院;
【基金】：江西省自然科学基金项目(“栀子玉米黄素剪切加双氧酶基因的克隆与功能分析”,No.20122BAB205070)资助国家自然科学基金项目(“栀子果实中西红花总苷合成途径CCD基因的筛选与功能分析”,No.31360362)资助
【分类号】：Q943.2

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