莱茵衣藻酰基辅酶A合成酶cDNA克隆及其酵母表达

doi:10.13560/j.cnki.biotech.bull.1985.2015.09.016

生物技术通报 ›› 2015, Vol. 31 ›› Issue (9): 119-124.doi: 10.13560/j.cnki.biotech.bull.1985.2015.09.016

莱茵衣藻酰基辅酶A合成酶cDNA克隆及其酵母表达

宋燕子, 贾彬, 林柏成, 胡章立, 黄瑛

(深圳市海洋生物资源与生态环境重点实验室深圳市海洋藻类开发与应用工程实验室深圳大学生命科学学院，深圳 518060)

收稿日期:2015-01-21 出版日期:2015-09-15 发布日期:2015-09-16
作者简介:宋燕子，女，硕士研究生，研究方向：海洋生物学；E-mail：swallowsong@163.com
基金资助:
国家自然科学基金项目(31100582，31470431)，中国博士后科学基金项目(2014M562199)，深圳市科技计划项目(JCYJ20120613112512654)

cDNA Cloning and Yeast Expression of Acyl-CoA Synthetase of Chlamydomonas reinhardtii

Song Yanzi, Jia Bin, Lin Baicheng, Hu Zhangli, Huang Ying

(Shenzhen Key Laboratory of Marine Bioresource and Eco-environmental Science，Shenzhen Engineering Laboratory of Marine Algae Biotechnology，College of Life Sciences，Shenzhen University，Shenzhen 518060)

Received:2015-01-21 Published:2015-09-15 Online:2015-09-16

摘要/Abstract

摘要： 旨在预测并克隆莱茵衣藻酰基辅酶A合成酶cDNA(cracs)，分析其在酵母中的功能。RT-PCR克隆cracs序列，ClustalW和MEGA6.0软件分别分析其编码蛋白保守序列和进化树，表达并分析其在酵母YB525中的底物偏好性。结果表明，首次在莱茵衣藻中克隆获得一个cracs，测序表明其序列大小为2 004 bp，编码667个氨基酸，编码蛋白crACS的预测分子量为72.3 kD，包含酰基辅酶A合成酶的两个保守区：AMP-binding区和FACS区。进化树比对显示，crACS与拟南芥的长链酰基辅酶A合成酶LACSs具有较高的同源性。酵母表达显示cracs编码蛋白能互补酵母YB525 LACS的缺陷表型，活化并优先利用C16:n>1和C14:n>0。莱茵衣藻cracs编码蛋白可活化外源脂肪酸，属于酰基辅酶A合成酶家族。

关键词: 莱茵衣藻, 酰基辅酶A合成酶, 酵母YB525, 进化树, 脂肪酸

Abstract: This work aims to predict and clone cDNA of Chlamydomonas reinhardtii acyl-CoA synthetase(gene cracs), and analyze its function in yeast Saccharomyces cerevisiae YB525. The cracs sequence was cloned by RT-PCR, its conserved sequence of encoded protein and phylogenetic tree were analyzed with ClustalW and MEGA6.0, then the substrate specificity in YB525 of expressed gene was analyzed. As the results, a cracs was cloned for the first time with sequence of 2 004 bp and encoded a 72.3 kD protein crACS of 667 amino acids containing two conserved regions of including acyl-CoA synthetase：the AMP-binding domain and the FACS motif. The phylogenetic tree analysis indicated that crACS shared high homology with LACs of Arabidopsis thaliana. Yeast expression experiments showed that crACS restored acyl-CoA synthetase deficient phenotype of YB525 and assimilated foreign palmitoleic acid and myristic acid. Conclusively, cracs of C. reinhardtii can activate exogenous fatty acid and belongs to acyl-CoA synthetase family.

Key words: Chlamydomonas reinhardtii, acyl-CoA synthetase, yeast YB525, phylogenetic tree, fatty acid

宋燕子, 贾彬, 林柏成, 胡章立, 黄瑛. 莱茵衣藻酰基辅酶A合成酶cDNA克隆及其酵母表达[J]. 生物技术通报, 2015, 31(9): 119-124.

Song Yanzi, Jia Bin, Lin Baicheng, Hu Zhangli, Huang Ying. cDNA Cloning and Yeast Expression of Acyl-CoA Synthetase of Chlamydomonas reinhardtii[J]. Biotechnology Bulletin, 2015, 31(9): 119-124.

参考文献

[1]Steinberg SJ, Morgenthaler J, Heinzer AK, et al. Very long-chain acyl-CoA synthetases：Human “bubblegum” represents a new family of proteins capable of activating very long-chain fatty acids[J]. Biological Chemistry, 2000, 275(45)：35162-35169.
[2]Fulda M, Shockey J, Werber M, et al. Two long-chain acyl-CoA synthetases from Arabidopsis thaliana involved in peroxisomal fatty acid β-oxidation[J]. Plant, 2002, 32(1)：93-103.
[3]Black PN, Dirusso CC, Sherin D, et al. Affinity labeling fatty acyl-CoA synthetase with 9-p-azidophenoxy nonanoic acid and the identification of the fatty acid-binding site[J]. Biological Chemistry, 2000, 275(49)：38547-38553.
[4]Babbit PC, Kenyon GL. Ancestry of the 4-chlorobenzoate dehalogenase：analysis of amino acid sequence identities among families of acy1：adenyl ligases, enoyl-CoA hydratases/isomerases, and acyl-CoA thioesterasest[J]. Biochemistry, 1992, 31(24)：5594-5604.
[5]Pei Z, Oey NA, Zuidervaart MM, et al. The acyl-CoA Synthetase "Bubblegum"(Lipidosin)：Furture characterization and role in neuronal fatty acid β-oxdation[J]. Biological Chemistry, 2003, 278(47)：47070-47078.
[6]Sato A, Matstmura R, Hoshino N, et al. Responsibility of regulatory gene expression and repressed protein synthesis for triacylglycerol accumulation on sulfur-starvation in Chlamydomonas reinhardtii[J]. Frontiers in Plant Science, 2014, 5(444)：1-12.
[7]Ibá?ez-Salazar A, Rosales-Mendoza S, Rocha-U-ribe A, et al. Over-expression of Dof-type transcription factor increases lipid production in Chlamydomonas reinhardtii[J]. Journal of Biotechnology, 2014, 184：27-38.
[8]Deng X, Cai J, Li Y, et al. Expression and knockdown of the PEPC1 gene affect carbon flux in the biosynthesis of triacylglycerols by the green alga Chlamydomonas reinhardtii[J]. Biotechnology Letters, 2014, 36(11)：2199-2208.
[9]Zhang L, Ma X, Yang G, et al. Isolation and characterization of a long-chain acyl-coenzyme A synthetase encoding gene from the marine microalga Nannochloropsis oculata[J]. Applied Phycology, 2012, 24(4)：873-880.
[10]Xu Z, Zhang Y, Wang Z, et al. A highly efficient protocol transformation of Saccharomyces cerevisiae and Pichia pastoris by electroporation[J]. Acta Scientiarum Naturealum Univeritatis Sunyatseni, 2010, 49(3)：98-101.
[11]Shockey JM, Fulda MS, Browse JA. Arabidopsis contains nine long-chain acyl-Coenzyme A synthetase genes that participate in fatty acid and glycerolipid metabolism[J]. Plant Physiology, 2002, 129(4)：1710-1722.
[12]Watkins PA. Fatty acid activation[J]. Progress in Lipid Researh, 1997, 36(1)：55-83.
[13]于莉莉, 谭小力, 侯文胜. 大豆长链脂酰辅酶A合成酶基因GmLACS在酵母中的表达[J]. 大豆科学, 2011, 30(5)：719-722.
[14]朱福各, 谭小力, 崇保强, 等. 油菜脂酰CoA合成酶基因pXT166的鉴定和功能分析[J]. 中国油料作物学报, 2009, 31(3)：274-278.
[15]王建林, 邵新峰, 梁振普, 等. 铜绿假单胞菌长链脂酰辅酶A合成酶的克隆及活性鉴定[J]. 河南科学, 2014, 32(3)：355-361.
[16]Tonon T, Qing R, Harvey D, et al. Identification of a long-chain polyunsaturated fatty acid acyl-Coenzyme A synthetase from the Diatom Thalassiosira pseudonana[J]. Plant Physiology, 2005, 138(1)：402-408.
[17]Lü S, Song T, Kosma DK, et al. Arabidopsis CER8 encodes long-chain acyl-coA synthase 1(LACS1)that has overlapping functons with LACS2 in plant wax and cuitin synthesis[J]. Plant, 2009, 59(4)：553-564.
[18]Fulda M, Shockey J, Werber M, et al. Two long-chain acyl-CoA synthetases from Arabidopsis thaliana involved in peroxisomal fatty acid beta-oxidation[J]. Plant, 2002, 32(1)：93-103.
[19]Guo X, Jiang M, Wan X, et al. Identification and biochemical characterization of five long-chain acyl-coenzyme A synthetases from the diatom Phaeodactylum tricornutum[J]. Plant Physiology and Biochemistry, 2014, 74：33-41.

莱茵衣藻酰基辅酶A合成酶cDNA克隆及其酵母表达

cDNA Cloning and Yeast Expression of Acyl-CoA Synthetase of Chlamydomonas reinhardtii

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