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首页> 中文期刊> 《林业科学》 >油茶丙二酰单酰CoA：ACP转酰基酶基因的克隆与原核表达

油茶丙二酰单酰CoA：ACP转酰基酶基因的克隆与原核表达

         
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摘要

Objective] Camellia oleifera is one of the four famous woody plants for edible oil in China. Camellia oil is a high quality and edible oil,mostly composed of unsaturated fatty acids (UFA),such as oleic acid and linoleic acid, and the average UFA content in Camellia oil is above 90%. But oil content of oil-tea seeds is low and vulnerable to adverse environmental conditions,restricting rapid development of the Camellia oil industry. Cloned pivotal genes from C. oleifera can be used as a biotechnological alternative for improving the molecular breeding of C. oleifera. Malonyl-CoA:ACP transacylase is one part of the fatty acid synthase ( FAS Ⅱ) complex,which is responsible for transferring the malonyl group from malonyl-CoA to the holo-acyl carrier protein (ACP). Malonyl-ACP,the product of this enzymatic reaction,is the key building block for de novo fatty acid biosynthesis.[Method]Seeds of the variety C. oleifera‘Huashuo’were used for DNA extraction,and a full-length cDNA of malonyl-CoA:ACP transacylase gene in seeds was cloned using RACE technique. Transcriptome data and expression profiles of C. oleifera seeds were used for bioinformatics analysis of the gene. Normal techniques of digestion and connection was used to construct prokaryotic expression vector, and the α-ketoglutarate dehydrogenase ( KDH )-coupled assay system was used to detect the enzymatic activity of the expressed product.[Result]The cDNA of CoMCAT was 1 867 bp with an open reading frame of 1 131 bp encoding 376 amino acids. It was named as CoMCAT ( GenBank accession number: KJ910337 ) . Sequence analysis showed that the protein encoded by CoMCAT had a chloroplast transit peptide of 58 amino acids and contained two highly conserved motifs of‘GQGXQ’and‘GXSXG’which were respectively located on malonyl-CoA binding site and ACP binding site. The BL21 (DE3) bacteria harboring pET30a-CoMCAT was induced to express the recombinant protein which was about 40 kDa. The enzymatic activity of the recombinant CoMCAT was 2. 384 U·μg -1 under the optimum condition at 30 ℃ with pH 7. 0.[Conclusion]The molecular cloning of the CoMCAT gene and its bioinformatics analysis,and expression characteristics in prokaryotic cells are first reported study of C. oleifera seeds. The results not only enriches the C. oleifera gene bank of MCATs,but lays an essential foundation for further study of the gene function and molecular genetic breeding in C. oleifera.%【目的】油茶种仁含油率较低且易遭逆性环境等的影响，这些都严重影响和制约了油茶产业的快速发展。丙二酰单酰 CoA：ACP转酰酶是脂肪酸合成酶( FASⅡ)复合体的组成酶之一，在脂肪酸合成中起着装载作用，它的酶促反应产物丙二酰单酰 ACP是脂肪酸合成关键组成部分。开展该基因的功能研究，能为油茶的分子遗传育种奠定基础。【方法】以油茶品种‘华硕’种子为材料，借助其种子转录组和表达谱数据库，通过 RACE 技术，克隆丙二酰单酰 CoA：ACP转酰酶基因并对该基因进行生物信息学分析。利用常规的酶切连接技术构建基因的原核表达载体，并利用α酮戊二酸脱氢酶( KDH)偶联系统测定表达产物的酶活性。【结果】丙二酰单酰 CoA：ACP转酰酶基因的全长 cDNA 1867 bp，含有1131 bp 的开放读码框，编码376个氨基酸，具有58个氨基酸长度的叶绿体转运肽，该基因被命名为 CoMCAT (GenBank 登录号 KJ910337)。在 malonyl-CoA结合位点和 ACP结合位点上分别存在高度保守的基序“GQGXQ”和“GXSXG”。原核表达载体 pET30a-CoMCAT 在 BL21(DE3)细胞中被成功地诱导表达，目的重组蛋白分子量约为40 kDa。酶活性分析表明，重组蛋白 CoMCAT 的最佳催化温度为30℃，最佳 pH 为7.0，比酶活性约为2.384 U·μg －1。【结论】以本实验室构建的油茶转录组数据库为基础，首次克隆获得油茶MCAT基因的全长 cDNA序列，构建其原核表达载体在宿主细胞 BL21(DE3)中成功诱导表达目的蛋白，并对重组蛋白进行初步的酶活性分析。研究结果为今后进一步深入开展和揭示油茶 MCAT 基因在油脂合成的代谢过程中的作用奠定基础。

著录项

  • 来源
    《林业科学》 |2015年第3期|148-154|共7页
  • 作者

    王建勇; 谭晓风; 陈鸿鹏; 曾艳玲; 龙洪旭; 梅芳芳; 刘凯;

  • 作者单位

    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;

    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;

    国家林业局桉树研究开发中心 湛江 524022;

    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;

    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;

    华中师范大学生命科学学院 武汉 430079;

    广西林业科学研究院 南宁 530001;

  • 原文格式 PDF
  • 正文语种 chi
  • 中图分类 树木遗传学;
  • 关键词

    油茶; 丙二酰单酰CoA:ACP转酰酶; 基因克隆; 原核表达; 酶活性;

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