Cloning and Prokaryotic Expression of Wax Synthase Gene of the Chinese White Wax Scale

LIU Bo-wen; WANG Xue-qing; SUN Tao; QI Qian; YU Shu-hui; YANG Pu; CHEN Xiao-ming

Volume 29 Issue 4

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Cloning and Prokaryotic Expression of Wax Synthase Gene of the Chinese White Wax Scale

1.
Research Institute of Resources Insects, Chinese Academy of Forestry, Key laboratory of Cultivating and Utilization of Resources Insects of State Forestry Administration, Kunming 650224, Yunnan, China

Received Date: 2016-02-15

Abstract

[Objective] To obtain the full length cDNA sequence of the wax synthase (ws) gene of the white wax insect, Ericerus pela Chavannes, and heterologously express the enzyme in Escherichia coli. [Method] The 3' and 5' ends of the ws gene were obtained separately by using RACE, and the prokaryotic expression vector was constructed after the analysis of the full length cDNA sequence of the ws gene. Finally, the expression of WS in Ericerus coli BL21 was induced by IPTG. [Result] Sequence analysis showed that, the full-length cDNA of the ws gene was 1 518 bp, which included the 5'-UTR (untranslated region) with 94 bp, 3'-UTR with 68 bp, and the open reading frame with 1 356 bp. The gene was deduced to encode 452 amino acid residues with the putative protein molecular weight of 51.8 kDa, and the theoretical isoelectric point of 6.35. Western Blot result showed that, the WS enzyme was expressed successfully in Ericerus coli. [Conclusion] In this study, the full length cDNA of the ws gene of Ericerus pela was obtained, and this enzyme was successfully expressed in Ericerus coli, which provides references for the research on wax ester synthesis in other insects.
- Ericerus pela,
- wax synthase,
- full-length cDNA sequence,
- prokaryotic expression

References

[1]	陈晓鸣, 冯颖. 资源昆虫学概论[M]. 北京:北京科学出版社, 2009: 54-78.
[2]	何晨柳, 孔任秋, 胡晗华. 产蜡酯聚球藻PCC 7002的构建[J]. 水生生物学报, 2010, 34(6):1202-1205.
[3]	谢映平, 薛皎亮, 张艳峰, 等. 蚧虫蜡泌物的化学研究进展[J]. 昆虫知识, 2004, 41(6): 512-518.
[4]	杨璞, 徐冬丽, 陈晓鸣, 等. 蜡酯合成途径及关键酶的研究发展[J]. 中国细胞生物学报, 2012, 34(7): 695-703.
[5]	Turkish A R, Sturley S L. The genetics of neutral lipid biosynthesis: an evolutionary perspective[J]. Am J Physiol Endocrinol Metab, 2009, 297(1): E19-27.
[6]	Yang P, Zhu J Y, Gong Z J, et al. Transcriptome analysis of the Chinese white wax scale Ericerus pela with focus on genes involved in wax biosynthesis[J]. PLoS ONE, 2012, 7(4): e35719.
[7]	J 萨姆布鲁克, DW拉塞尔. 分子克隆实验指南[M]. 北京:科学出版社, 2002: 418-423; 1256-1259; 1570-1576.
[8]	Gasteiger E, Gattiker A, Hoogland C, et al. ExPASy: The proteomics server for in-depth protein knowledge and analysis[J]. Appl Environ Microbiol, 2003, 31(13): 3784-3788.
[9]	Krogh A, Larsson B, von Heijine G, et al. Predicting transmembrane protein topology with a hidden Markov model: application to complete genome[J]. J Mol Biol, 2001, 305(3): 567-580.
[10]	Petersen T N, Brunak S, Von Heijne G, et al. SignalP 4.0: discriminating signal peptides from transmembrane regions[J]. Nat Methods, 2011, 8(10): 785-786.
[11]	Rottig A, Steinbuchel A. Acyltransferase in bacteria[J]. Microbiol Mol Bio Rev, 2013, 77(2): 277.
[12]	Biester E M, Hellenbrand J, Gruber J, et al. Identification of avian wax synthases[J]. BMC Biochem, 2012, 13(4): 4.
[13]	Biester E M, Hellenbrand J, Gruber J. Multifunctional acyltransferases from Tetrahymena thermophila[J]. Lipids, 2012, 47(4): 371-381.
[14]	Teerawanichpan P, Qiu X. Fatty acyl-CoA reductase and wax synthase from Euglena gracilis in the biosynthesis of mediumchain wax esters[J]. Lipids, 2010, 45(3): 263-273.
[15]	Klypina N, Hanson S F. Arabidopsis thaliana wax synthase gene homologues show diverse expression patterns that suggest a specialized role for these genes in reproductive organs[J]. Plant Science, 2008, 175: 312-320.
[16]	Li F L, Wu X M, Lam P, et al. Identification of the wax ester synthase/acyl-coenzyme A: Diacylglycerol acyltransferase WSD I required for stem wax ester biosynthesis in Arabidopsis[J]. Plant Physiol, 2008, 148(1): 97-107.
[17]	King A, Nam J W, Han J, et al. Cuticular wax biosynthesis in petunia petals: cloning and characterization of an alcohol-acyltransferase that synthesizes[J]. Planta, 2007, 226(2): 381-394.
[18]	Stoveken T, Kalscheuer R, Malkus U, et al. The wax ester synthase/acyl coenzyme A: diacylglycerol acyl-tansferase from Acinetobacter sp. Strain ADP1: characterization of a novel type of acyltransferase[J]. J Bacteriol, 2005, 187(4): 1369-1376.
[19]	Cheng J B, Russell D W. Mammalian wax biosynthesis. II. Expression cloning of wax synthase cDNAs encoding a member of the acyltransferase enzyme family[J]. J Biol Chem, 2004, 279(36): 37798-37807.
[20]	Kalscheuer R, Steinbuchel A. A novel bifunctional wax ester synthase/acyl-CoA: diacylglycerol acyltransferase mediates wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus ADP1[J]. J Biol Chem, 2003, 278(10): 8075-8082.
[21]	Lardizabal K D, Metz J G, Sakamoto T, et al. Purification of a jojoba embryo wax synthase, cloning of its cDNA, and production of high levels of wax in seeds of trangenic Arabidopsis[J]. Plant Physiol, 2000, 122(3): 645-655.
[22]	Uthoff S, Stoveken T, Weber N, et al. Thio wax ester synthesis utilizing the unspecific bifunctional wax ester synthase/acyl coenzyme A:diacylglycerol acyltransferase of Acinetobacter sp. strain ADP1[J]. Appl Environ Microbiol, 2005, 71(2): 790-796.

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

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Cloning and Prokaryotic Expression of Wax Synthase Gene of the Chinese White Wax Scale

1. Research Institute of Resources Insects, Chinese Academy of Forestry, Key laboratory of Cultivating and Utilization of Resources Insects of State Forestry Administration, Kunming 650224, Yunnan, China

Received Date: 2016-02-15

Abstract: [Objective] To obtain the full length cDNA sequence of the wax synthase (ws) gene of the white wax insect, Ericerus pela Chavannes, and heterologously express the enzyme in Escherichia coli. [Method] The 3' and 5' ends of the ws gene were obtained separately by using RACE, and the prokaryotic expression vector was constructed after the analysis of the full length cDNA sequence of the ws gene. Finally, the expression of WS in Ericerus coli BL21 was induced by IPTG. [Result] Sequence analysis showed that, the full-length cDNA of the ws gene was 1 518 bp, which included the 5'-UTR (untranslated region) with 94 bp, 3'-UTR with 68 bp, and the open reading frame with 1 356 bp. The gene was deduced to encode 452 amino acid residues with the putative protein molecular weight of 51.8 kDa, and the theoretical isoelectric point of 6.35. Western Blot result showed that, the WS enzyme was expressed successfully in Ericerus coli. [Conclusion] In this study, the full length cDNA of the ws gene of Ericerus pela was obtained, and this enzyme was successfully expressed in Ericerus coli, which provides references for the research on wax ester synthesis in other insects.

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Reference (22)

[1]	陈晓鸣, 冯颖. 资源昆虫学概论[M]. 北京:北京科学出版社, 2009: 54-78.
[2]	何晨柳, 孔任秋, 胡晗华. 产蜡酯聚球藻PCC 7002的构建[J]. 水生生物学报, 2010, 34(6):1202-1205.
[3]	谢映平, 薛皎亮, 张艳峰, 等. 蚧虫蜡泌物的化学研究进展[J]. 昆虫知识, 2004, 41(6): 512-518.
[4]	杨璞, 徐冬丽, 陈晓鸣, 等. 蜡酯合成途径及关键酶的研究发展[J]. 中国细胞生物学报, 2012, 34(7): 695-703.
[5]	Turkish A R, Sturley S L. The genetics of neutral lipid biosynthesis: an evolutionary perspective[J]. Am J Physiol Endocrinol Metab, 2009, 297(1): E19-27.
[6]	Yang P, Zhu J Y, Gong Z J, et al. Transcriptome analysis of the Chinese white wax scale Ericerus pela with focus on genes involved in wax biosynthesis[J]. PLoS ONE, 2012, 7(4): e35719.
[7]	J 萨姆布鲁克, DW拉塞尔. 分子克隆实验指南[M]. 北京:科学出版社, 2002: 418-423; 1256-1259; 1570-1576.
[8]	Gasteiger E, Gattiker A, Hoogland C, et al. ExPASy: The proteomics server for in-depth protein knowledge and analysis[J]. Appl Environ Microbiol, 2003, 31(13): 3784-3788.
[9]	Krogh A, Larsson B, von Heijine G, et al. Predicting transmembrane protein topology with a hidden Markov model: application to complete genome[J]. J Mol Biol, 2001, 305(3): 567-580.
[10]	Petersen T N, Brunak S, Von Heijne G, et al. SignalP 4.0: discriminating signal peptides from transmembrane regions[J]. Nat Methods, 2011, 8(10): 785-786.
[11]	Rottig A, Steinbuchel A. Acyltransferase in bacteria[J]. Microbiol Mol Bio Rev, 2013, 77(2): 277.
[12]	Biester E M, Hellenbrand J, Gruber J, et al. Identification of avian wax synthases[J]. BMC Biochem, 2012, 13(4): 4.
[13]	Biester E M, Hellenbrand J, Gruber J. Multifunctional acyltransferases from Tetrahymena thermophila[J]. Lipids, 2012, 47(4): 371-381.
[14]	Teerawanichpan P, Qiu X. Fatty acyl-CoA reductase and wax synthase from Euglena gracilis in the biosynthesis of mediumchain wax esters[J]. Lipids, 2010, 45(3): 263-273.
[15]	Klypina N, Hanson S F. Arabidopsis thaliana wax synthase gene homologues show diverse expression patterns that suggest a specialized role for these genes in reproductive organs[J]. Plant Science, 2008, 175: 312-320.
[16]	Li F L, Wu X M, Lam P, et al. Identification of the wax ester synthase/acyl-coenzyme A: Diacylglycerol acyltransferase WSD I required for stem wax ester biosynthesis in Arabidopsis[J]. Plant Physiol, 2008, 148(1): 97-107.
[17]	King A, Nam J W, Han J, et al. Cuticular wax biosynthesis in petunia petals: cloning and characterization of an alcohol-acyltransferase that synthesizes[J]. Planta, 2007, 226(2): 381-394.
[18]	Stoveken T, Kalscheuer R, Malkus U, et al. The wax ester synthase/acyl coenzyme A: diacylglycerol acyl-tansferase from Acinetobacter sp. Strain ADP1: characterization of a novel type of acyltransferase[J]. J Bacteriol, 2005, 187(4): 1369-1376.
[19]	Cheng J B, Russell D W. Mammalian wax biosynthesis. II. Expression cloning of wax synthase cDNAs encoding a member of the acyltransferase enzyme family[J]. J Biol Chem, 2004, 279(36): 37798-37807.
[20]	Kalscheuer R, Steinbuchel A. A novel bifunctional wax ester synthase/acyl-CoA: diacylglycerol acyltransferase mediates wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus ADP1[J]. J Biol Chem, 2003, 278(10): 8075-8082.
[21]	Lardizabal K D, Metz J G, Sakamoto T, et al. Purification of a jojoba embryo wax synthase, cloning of its cDNA, and production of high levels of wax in seeds of trangenic Arabidopsis[J]. Plant Physiol, 2000, 122(3): 645-655.
[22]	Uthoff S, Stoveken T, Weber N, et al. Thio wax ester synthesis utilizing the unspecific bifunctional wax ester synthase/acyl coenzyme A:diacylglycerol acyltransferase of Acinetobacter sp. strain ADP1[J]. Appl Environ Microbiol, 2005, 71(2): 790-796.

Cloning and Prokaryotic Expression of Wax Synthase Gene of the Chinese White Wax Scale

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通讯作者: 陈斌, bchen63@163.com

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Cloning and Prokaryotic Expression of Wax Synthase Gene of the Chinese White Wax Scale

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