Construction of a High-efficient Expression Vector of Δ12 Fatty Acid Desaturase in Peanut and Its Prokaryotical Expression

Dongmei Yin; Dangqun Cui; Bin Jia

doi:10.1016/S1673-8527(07)60009-5

Volume 34 Issue 1

Jan. 2007

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Article Navigation > Journal of Genetics and Genomics > 2007 > 34(1): 81-88

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Construction of a High-efficient Expression Vector of Δ12 Fatty Acid Desaturase in Peanut and Its Prokaryotical Expression

doi: 10.1016/S1673-8527(07)60009-5

a
Department of Agronomy, Henan Agricultural University, Zhengzhou 450002, China
b
The Center of Science Laboratory, Henan Academy of Agricultural Science, Zhengzhou 450002, China

Received Date: 2006-02-17
Accepted Date: 2006-06-15

Available Online: 2007-04-18

Publish Date: 2007-01-20

Abstract

Abstract

A full-length sequence coding for Δ¹² fatty acid desaturase gene from peanut (Arachis hypogaea L.) was cloned into the expression vector, pRSETB, to generate recombinant plasmid pRSET/HO-A, which was subsequently transformed into expression Escherichia. coli BL21(DE3)pLysS. The Δ¹² fatty acid desaturase was highly expressed in E. coli BL21(DE3)pLysS in the presence of isopropyl-D-thiogalactopyranoside (IPTG). The fusion protein was purified and used to form a reaction system in vitro by adding oleic acid as substrate and incubating it at 20°C for 6 h. Total fatty acids was extracted and methlesterified and then analyzed with gas chromatography. A novel peak corresponding to linoleic acid methyl ester standards was detected with the same retention time. GC-MS (gas chromatogram and gas chromatogram-mass spectrometry) analysis showed that the novel peak was linoleic acid methyl ester. These results exhibited Δ¹² fatty acid desaturase activity, which could convert oleic acid to linoleic acid specifically.
- peanut,
- prokaryotical expression,
- function identification

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References(34)

References

[1]	Page, RA, Okada, et al. Acetyl-CoA carboxylase exerts strong flux control over lipid synthesis in plants Biochem Biophys Acta (1994),pp. 369-372
[2]	Schmidt, H, Dresselhaus, et al. Purification and PCR-based cDNA cloning of a plastidial n-6 desaturase Plant Mol Biol, 26 (1994),pp. 631-642
[3]	Alonso, DL, Maroto, et al. Evolution of the membranebound fatty acid desaturases Biochemical System Ecol, 31 (2003),pp. 1111-1124
[4]	Cho, HP, Nakamura, et al. Cloning, expression, and nutritional regulation of the mammalian Delta-6 desaturase Biol Chem, 274 (1999),pp. 471-477
[5]	Heppard, EP, Kinney, et al. Developmental and growth temperature regulation of two different microsomal omega-6 desaturase genes in soybeans Plant Physiol, 110 (1996),pp. 311-319
[6]	Los, DA, Murata, et al. Structure and expression of fatty acid desaturases Biochim Biophys Acta, 1394 (1998),pp. 3-15
[7]	Ishizaki-Nishizawa, O, Fujii, et al. Low-temperature resistance of higher plants is significantly enhanced by a nonspecific cyanobacterial desaturase Nat Biotechnol., 14 (1996),pp. 1003-1006
[8]	Zhou, YH, Chen, et al. Genetic manipulation and gene engineering of fatty acid metabolism in plant seeds Chinese Bulletin of Botany, 15 (1998),pp. 16-23
[9]	Browse, J, McConn, et al. Mutants of Arabidopsis deficient in the synthesis of alpha-linolenate. Biochemical and genetic characterization of the endoplasmic reticulum linoleoyl desaturase Biol Chem, 268 (1993),pp. 16345-16351
[10]	Sayanova, O, Smith, et al. Expression of a borage desaturase cDNA containing an N-terminal cytochrome b5 domain results in the accumulation of high levels of delta6-desaturated fatty acids in transgenic tobacco Proc Nat Acad Sci USA, 94 (1997),pp. 4211-4216
[11]	Pirtle, IL, Kongcharoensuntorn, et al. Molecular cloning and functional expression of the gene for a cotton Delta-12 fatty acid desaturase (FAD2) Biochim Biophys Acta, 1522 (2001),pp. 122-129
[12]	Bell, E, Creelman, et al. A chloroplast lipoxygenase is required for wound-induced jasmonic acid accumulation in Arabidopsis Proc Natl Acad Sci USA, 92 (1995),pp. 8675-8679
[13]	Sambrook, J, Fritsch, et al.
[14]	Kou, XY, Yu, et al. Research on the method of fatty and fatty acid methylation Research and Development of Food, 26 (2005),pp. 46-47
[15]	Ramos, CR, Abreu, et al. Braz J Med Biol Res, 37 (2004),pp. 1103-1109
[16]	Choi, JW, Ra, et al. Enhancement of bovine growth hormone gene expression by increasing the plasmid copy number Biotechnol Lett, 21 (1999),pp. 1-5
[17]	Ribas, AV, Ho, et al. High-level expression of tetanus toxin fragment C-thioredoxin fusion protein in Escherichia coli Biotechnol Appl Biochem (2000),pp. 91-94
[18]	Areas, AP, Oliveira, et al. Protein Expr Purif, 25 (2002),pp. 481-487
[19]	Ye, Q, Sui, et al. Construction, prokaryotic expression and identification of mutant SEA(D227A) Journal of Chinese Immunology (2004),pp. 521-524
[20]	Qiu, LN, Ma, et al. Expression and purification of recombinant rhTNF2 b in expression vector pRSET C Cell Mol Immunol, 15 (1999),pp. 167-170
[21]	Chen, S, Rao, et al. Construction and expression of single chain variable fragments (ScFv) against human CD19 antigen Chinese Journal of Biotechnology, 21 (2005),pp. 686-691
[22]	Xu, WT, Huang, et al. Journal of Agricultural Biotechnology, 12 (2004),pp. 583-585
[23]	Berberich, T, Harada, et al. Two maize genes encoding omega-3 fatty acid desaturase and their differential expression to temperature Plant Mol Biol, 36 (1998),pp. 297-306
[24]	Slocombe, SP, Piffanelli, et al. Temporal and tissue-specific regulation of a Brassica napus stearoylacyl carrier protein desaturase gene Plant Physiol, 104 (1994),pp. 1167-1176
[25]	Nishiuchi, T, Hamada, et al. Wounding changes the spatial expression pattern of the Arabidopsis plastid omega-3 fatty acid desaturase gene (FAD7) through different signal transduction pathways Plant Cell, 9 (1997),pp. 1701-1712
[26]	He, JM, Wang, et al. Southwest China Journal of Agricultural Sciences, 16 (2003),pp. 34-36
[27]	Shi, DQ, Zhou, et al. Journal of Agricultural Biotechnology, 9 (2001),pp. 359-362
[28]	Xiong, XH, Guan, et al. Journal of Hunan Agricultural University, 29 (2003),pp. 188-191
[29]	Dong, N, Lin, et al. Mol Plant Breed, 2 (2004),pp. 655-699
[30]	Li, MC, Liu, et al. Acta Agronomica Sinica, 30 (2004),pp. 618-621
[31]	Bu, YP, Wang, et al. Biotechnology (2003),pp. 6-8
[32]	Nabloussi, A, Fernandez-Martinez, et al. Crop Sci, 45 (2005),pp. 202-208
[33]	Knutzon, DS, Thompson, et al. Modification of Brassica seed oil by anti-sense expression of a stearoyl-acyl carrier protein desaturase gene Proc Natl Acad Sci USA, 89 (1992),pp. 2624-2628
[34]	Jadhav, A, Katavic, et al. Metab Eng, 7 (2005),pp. 215-220