《植物生理学报》 2014, 50(12): 1845-1856

油茶脂酰辅酶A硫酯酶基因的克隆与表达分析

谭晓风^1,*, 王建勇¹, 龙洪旭¹, 曾艳玲¹, 梅芳芳², 刘凯³, 陈鸿鹏⁴

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华中师范大学生命科学学院, 武汉430079; ³广西林业科学研究院, 南宁530001; ⁴国家林业局桉树研究开发中心, 广东湛江524022

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

摘　要：

脂酰辅酶A硫酯酶是催化脂酰CoA水解成自由脂肪酸(FFA)和辅酶A (CoASH)的一类酶, 它通过维持胞内脂酰CoA、FFA和CoASH等的水平来参与生命体内的生理过程。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, 分离和克隆到一个油茶脂酰CoA硫酯酶基因的全长cDNA序列, 命名为CoACOT (GenBank登录号KJ910339)。该基因cDNA全长为1 588 bp, 含有1 164 bp 的开放读码框, 编码387个氨基酸; 基因编码的蛋白CoACOT具有cNMP结合域保守序列“VVREGEAGDGVYFIWDG”和C端的保守三联肽“SKL”, 属于过氧化物酶体蛋白。成功构建了表达载体, 其中, 原核表达载体在宿主细胞BL21 (DE3)上成功诱导表达, 获得分子量约为44 kDa的目的蛋白。荧光定量PCR分析表明, CoACOT基因在种子不同的发育期均有表达, 种子膨大期最低, 经油脂合成期上调表达之后, CoACOT基因的相对表达量维持在一个相对稳定的较高水平。结果显示CoACOT基因可能调控着油茶种子的油脂合成。

关键词：油茶; 脂酰辅酶A硫酯酶; 克隆; 表达分析; 载体构建

收稿：2014-09-05   修定：2014-11-04

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 Acyl-CoA Thioesterase Gene from Camellia oleifera

TAN Xiao-Feng^1,*, WANG Jian-Yong¹, LONG Hong-Xu¹, ZENG Yan-Ling¹, Mei Fang-Fang², LIU Kai³, CHEN Hong-Peng⁴

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; ²Hubei Key Laboratory of Integrative Biology, College of Life Sciences, Central China Normal University, Wuhan 430079, China; ³Guangxi Academy of Forestry, Nanning 530001, China; ⁴China Eucalypt Research Centre, Zhanjiang, Guangdong 524022, China

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

Abstract:

Acyl-CoA thioesterases are a group of enzymes that catalyze the hydrolysis of acyl-CoAs to the free fatty acid (FFA) and coenzyme A (CoASH), providing the potential to regulate intracellular levels of acyl-CoAs, FFAs and CoASH. In this paper, state trial oil-tea variety ‘Huashuo’ as material, a full-length cDNA of acyl-CoA thioesterase genes from Camellia oleifera was isolated and cloned by RACE technology and reverse transcription PCR (RT-PCR). This gene was named CoACOT (GenBank accession number KJ910339) and the full-length cDNA was found to be 1 588 bp. CoACOT had an open reading frame of 1 164 bp, encoding 387 amino acids. CoACOT contained a conserved sequences of “VVREGEAGDGVYFIWDG” in cNMP binding domain and a highly conserved triple peptides of “SKL” in the C-terminal, belonging to a peroxysome protein. The expression vectors of CoACOT were constructed successfully, and the recombinant vector of pET30a-Co-ACOT was induced to expressed the about 44 kDa target product in the BL21 (DE3) bacteria. The real-time quantitive PCR showed the expression level of CoACOT was the lowest in seed expanding stage, and then remained almost a relatively stable higher level after up-regulation in the lipid synthesis. The results showed CoACOT, as a auxiliary gene, may regulate and maintain the physiological balance in lipid synthesis of C. oleifera seeds.

Key words: Camellia oleifera; acyl-CoA thioesterase; clone; expression analysis; vector construction