《植物生理学报》 2014, 50(10): 1574-1584

油茶3羟酰CoA脱水酶基因的克隆与表达分析

王建勇¹, 谭晓风^1,*, 曾艳玲¹, 龙洪旭¹, 陈鸿鹏², 刘凯³

¹中南林业科技大学, 经济林培育与保护教育部重点实验室, 经济林育种与栽培国家林业局重点实验室, 长沙410004; ²国家林业局桉树研究开发中心, 广东湛江524022; ³广西林业科学研究院, 南宁530001

通信作者：谭晓风；E-mail: tanxiaofengcn@126.com；Tel: 0731-85623416

摘　要：

3羟酰CoA脱水酶是一类催化3羟酰CoA脱水转化成为烯酰CoA形式的酶。本研究以国审油茶品种‘华硕’种子为材料, 在已构建的转录组和表达谱数据库的基础之上, 采用RACE技术, 克隆到一个油茶3羟酰CoA脱水酶基因的cDNA全长, 命名为CoHCD (GenBank登录号KJ910336)。该基因cDNA全长为1 145 bp, 含有666 bp的开放读码框, 编码221个氨基酸, 分子量为25.2 kDa, 理论等电点pI为9.4, 疏水残基占整个氨基酸残基的48.9%, 是亲水性蛋白, 具有4个比较明显的跨膜区和蛋白质酪氨酸磷酸酶基序“HGXXGXXRS”。在基因cDNA全长序列的基础上, 分别成功地构建了原核表达载体、超表达载体和RNA干扰载体, 其中, 原核表达载体在宿主细胞BL21(DE3)上成功诱导表达, 获得表观分子量约为25 kDa的相应目的蛋白。实时荧光定量PCR分析表明, 在5个不同发育时期的油茶种子中CoHCD高效表达主要集中在8~10月, 转录最高峰发生在9月, 其表达量在种子发育过程中呈增加趋势。

关键词：油茶; 3羟酰CoA脱水酶; 克隆; 表达分析

收稿：2014-06-13   修定：2014-08-22

资助：国家自然科学基金(31070603)、湖南省自然科学基金(14JJ2104)和中南林业科技大学青年基金重点项目(QJ2011008A)。

Cloning and Expression Analysis of a 3-Hydroxyacyl-CoA Dehydratase Gene from Camellia oleifera

WANG Jian-Yong¹, TAN Xiao-Feng^1,*, ZENG Yan-Ling¹, LONG Hong-Xu¹, CHEN Hong-Peng², LIU Kai³

¹Key Laboratory of Cultivation and Protection for Non-Wood Forest Trees, Key Lab of Non-Wood Forest Product of State Forestry Administration, Central South University of Forestry and Technology, Changsha 410004, China; ²China Eucalypt Research Centre, Zhanjiang, Guangdong 524022, China; ³Guangxi Academy of Forestry, Nanning 530001, China

Corresponding author: TAN Xiao-Feng; E-mail: tanxiaofengcn@126.com; Tel: 0731-85623416

Abstract:

3-Hydroxyacyl-CoA dehydratase (HCD) is a kind of protein that is the dehydrateion of the 3-hydroxyacyl- CoA to an enoyl-CoA. In this paper, a full-length cDNA of 3-hydroxyacyl-CoA dehydratase in Camellia oleifera ‘Huashuo’ seeds was cloned using reverse transcription PCR (RT-PCR), RACE technique, and basing’ transcriptome and expression profiling database of C. oleifera seed. The full length cDNA was 1 145 bp, and the gene was named CoHCD (GenBank No. KJ910336). The sequence had an open reading frame of 666 bp, encoding 221 amino acids rich in hydrophobic residues (48.9%), showing a hydrophilic protein. Sequence analysis showed that molecular mass of the CoHCD protein was 25.2 kDa with a theoretical pI of 9.4. CoHCD had four transmembrane domains and contained a protein tyrosine phosphatase motif “HGXXGXXRS” that was common to the PTPLA gene families. The expression vectors of CoHCD were constructed successfully, and the BL21(DE3) bacteria harboring the pET30a-CoHCD was induced to express the about 25 kDa target protein. The relative expression abundance of CoHCD at 5 different developmental stages of seed were analyzed using RT-PCR. The CoHCD gene expression was up-regulated during the seed developmental stages (with the highest point occurring in middle September and high-efficiency expression in August to October), which was consistent with the accumulation of the lipid synthesis of C. oleifera seeds.

Key words: Camellia oleifera; 3-hydroxyacyl-CoA dehydratase; clone; expression analysis