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Scientia Silvae Sinicae ›› 2015, Vol. 51 ›› Issue (3): 148-154.doi: 10.11707/j.1001-7488.20150319

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Cloning and Prokaryotic Expression of a Malonyl-CoA:ACP Transacylase Gene from Camellia oleifera

Wang Jianyong1, Tan Xiaofeng1, Chen Hongpeng2, Zeng Yanling1, Long Hongxu1, Mei Fangfang3, Liu Kai4   

  1. 1. Key Laboratory of Cultivation and Protection for Non-Wood Forest Trees of Ministry of Education Key Laboratory of Non-Wood Forest Product of State Forestry Administration Central South University of Forestry and Technology Changsha 410004;
    2. China Eucalypt Research Centre Zhanjiang 524022;
    3. College of Life Sciences, Central China Normal University Wuhan 430079;
    4. Guangxi Academy of Forestry Nanning 530001
  • Received:2014-06-10 Revised:2014-10-05 Published:2015-04-10

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Abstract:

【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 RNA 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.

Key words: Camellia oleifera, malonyl-CoA:ACP transacylase, gene cloning, prokaryotic expression, enzymatic activity

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