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[1]杨晓霞,何仕武,魏云林,等.粘红酵母?12-脂肪酸脱氢酶基因的克隆和表达[J].应用与环境生物学报,2015,21(03):421-426.[doi:10.3724/SP.J.1145.2014.11024]
 YANG Xiaoxia,HE Shiwu,WEI Yunlin,et al.Cloning and heterologous expression of a Δ12-desaturase gene from Rhodotorula glutinis[J].Chinese Journal of Applied & Environmental Biology,2015,21(03):421-426.[doi:10.3724/SP.J.1145.2014.11024]
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粘红酵母?12-脂肪酸脱氢酶基因的克隆和表达()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
21卷
期数:
2015年03期
页码:
421-426
栏目:
研究论文
出版日期:
2015-06-25

文章信息/Info

Title:
Cloning and heterologous expression of a Δ12-desaturase gene from Rhodotorula glutinis
作者:
杨晓霞何仕武魏云林林连兵季秀玲张琦
昆明理工大学生命科学与技术学院 昆明 650500
Author(s):
YANG Xiaoxia HE Shiwu WEI Yunlin LIN Lianbing JI Xiuling ZHANG Qi
Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China
关键词:
粘红酵母Δ12-脂肪酸脱氢酶亚油酸克隆和表达
Keywords:
Rhodotorula glutinis Δ12-fatty acid desaurase gene linoleic acid cloning and expression
分类号:
Q78 : Q936
DOI:
10.3724/SP.J.1145.2014.11024
文献标志码:
A
摘要:
Δ12-脂肪酸脱氢酶一般特异性催化在油酸的Δ12位引入双键转变成亚油酸. 为了从粘红酵母YM25079中克隆全长Δ12-脂肪酸脱氢酶基因序列,参考已知的Δ12-脂肪酸脱氢酶基因序列设计基因特异性引物,通过PCR扩增获得到全长为1 353 bp的cDNA序列,序列分析结果表明该序列具有一个编码450个氨基酸的完整开放阅读框,所编码蛋白质的大小为50.9 × 103. 与报道的Δ12-脂肪酸脱氢酶一样,推测的氨基酸序列具有膜整合脂肪酸脱氢酶特异性的3个组氨酸保守区,表明该序列为一个新的编码Δ12-脂肪酸脱氢酶的基因. 为了验证其功能,把开放阅读框序列亚克隆到表达载体pYES3/CT,构建重组表达载体pYRGD12,并转化到酿酒酵母的缺陷型菌株INVScl进行表达. 脂肪酸气相色谱(GC)分析表明,该序列所编码的蛋白质具有Δ12-脂肪酸脱氢酶活性,能将油酸转化为亚油酸,亚油酸的含量占酵母总脂肪酸的4.31%. 以上结果表明,PCR所获得序列是新的Δ12-脂肪酸脱氢酶基因.
Abstract:
Δ12- fatty acid desaturase generally introduces a new double bond specifically at Δ12 position of 18C oleic acid to generate linoleic acid. This study aimed to clone the full-length ?12- fatty acid desaturase gene from Rhodotorula glutinis strain YM25079. Based on the sequence information of the known yeast Δ12-fatty acid desaturase genes, a pair of specific primers was designed and used for the amplification of YM25079 Δ12- fatty acid desaturase gene. The obtained sequence was further transformed into Saccharomyces cerevisiae strain INVScl for functional analysis. A full-length cDNA sequence of 1 353 bp was amplified from R. glutinis YM25079. Sequence analysis showed this sequence comprised an open reading frame encoding 458 amino acids of 50.9 × 103. The deduced amino-acid sequence showed similarity to the known Δ12-fatty acid desaturases which comprise three characteristic conserved histidine-rich regions for the membrane-bound desaturases, indicating this cDNA sequence is a novel Δ12-fatty acid desaturase gene. The sequence was further cloned into the expression vector pYES3/CT to generate a recombinant plasmid pY3RGD12, which was subsequently transformed into S. cerevisiae strain INVScl for heterologous expression. Total fatty acids analysis of the transformed yeast cells by gas chromatography (GC) showed a novel peak corresponding to the standards of linoleic acid methyl ester with the same retention time, which was absent in the cell transformed with empty vector. The ratio of this new fatty acid to total fatty acids was 4.31%. The results indicated that the PCR-amplified sequence is a novel ?12-fatty acid desaturase gene.

参考文献/References:

1 张琦, 王志, 何仕武, 季秀玲, 林连兵, 魏云林. 多不饱和脂肪酸对微生物低温适应性的影响[J]. 生命科学, 2012, 24 (1): 58-63 [Zhang Q, Wang Z, He SW, Ji XL, Lin LB, Wei YL. Effects of polyunsaturated fatty acids on the cold adaptation of microoganisms [J]. Chin Bull Life Sci, 2012, 24 (1): 58-63]
2 Gill I, Valivety R. Polyunsaturated fatty acids, part 1: occurrence, biological activities and applications [J]. Trends Biotechnol, 1997, 15 (10): 401-409
3 Bureau DP, Hua K, Harris AM. The effect of dietary lipid and long-chain n-3 PUFA levels on growth, energy utilization, carcass quality, and immune function of rainbow trout, Oncorhynchus mykiss [J]. J World Aquacul Soc, 2008, 39 (1): 1-21
4 Sakuradani E, Kobayashi M, Ashikari T, Shimizu S. Identification of Δ12-fatty acid desaturase from arachidonic acid-producing Mortierella fungus by heterologous expression in the yeast Saccharomyces cerevisiae and the fungus Aspergillus oryzae [J]. Eur J Biochem, 1999, 261 (3): 812-820
5 Meesapyodsuk D, Qiu X. The front-end desaturase: structure, function, evolution and biotechnological use [J]. Lipids, 2012, 47 (3): 227-237
6 Wada H, Combos Z, Murata N. Enhancement of chilling tolerance of a cyanobacterium by genetic manipulation of fatty acid desaturation [J]. Nature, 1990, 347 (6289): 200-203
7 Takeno S, Sakuradani E, Tomi A, Inohara-Ochiai M, Kawashima H, Ashikari T, Shimizu S. Improvement of the fatty acid composition of an oil-producing filamentous fungus, Mortierella alpina 1S-4, through RNA interference with Δ12-desaturase gene expression [J]. Appl Environ Microbiol, 2005, 71 (9): 5124-5128
8 Bu?ek A, Matou?ková P, Sychrová H, Pichová I, Hru?ková-Heidingsfeldová O. Δ12-fatty acid eesaturase from Candida parapsilosis is a multifunctional desaturase producing a range of polyunsaturated and hydroxylated fatty acids [J]. PloS ONE, 2014, 9 (3): e93322
9 Zhou XR, Green AG, Singh SP. Caenorhabditis elegans Δ12-desaturase FAT-2 is a bifunctional desaturase able to desaturate a diverse range of fatty acid substrates at the Δ12 and Δ15 positions [J]. J Biol Chem, 2011, 286 (51): 43644-43650
10 Kikukawa H, Sakuradani E, Kishino S, Park SB, Ando A, Shima J, Ochiai M, Shimizu S, Ogawa J. Characterization of a trifunctional fatty acid desaturase from oleaginous filamentous fungus Mortierella alpina 1S-4 using a yeast expression system [J]. J Biosci Bioeng, 2013, 116 (6): 672-676
11 杨昭杰, 李凌彦, 胡彬彬, 林连兵, 魏云林, 季秀玲, 张琦. 多不饱和脂肪酸与粘红酵母低温适应性的关系[J]. 应用与环境生物学报, 2014, 20 (2): 233-237 [Yang ZJ, Li LY, Hu BB, Lin LB, Wei YL, Ji XL. Correlation of polyunsaturated fatty acids and cold adaptation of Rhodotorula glutinis [J]. Chin J Appl Environ Biol, 2014, 20 (2): 233-237]
12 张琦, 李明春, 孙颖, 马海庭, 任勇, 邢来君. 少根根霉?6-脂肪酸脱氢酶基因的克隆和表达[J]. 遗传学报, 2004, 31 (7): 740-749 [Zhang Q, Li MC, Sun Y, Ma HT, Ren Y, Xing LJ. Cloning and heterologous expression of a novel Δ6-desaturase gene from Rhizopus arrhizus NK030037 [J]. J Gen Genomics, 2004, 31 (7): 740-749]
13 Los DA, Murata N. Structure and expression of fatty acid desaturases [J]. Biochim Biophys Acta, 1998, 1394 (1): 3-15
14 张琦, 李明春, 孙红妍, 孙颖, 马海庭, 邢来君. ?6-脂肪酸脱氢酶的分子生物学研究进展[J]. 生物工程学报, 2004, 20 (3): 319-324 [Zhang Q, Li MC, Sun HY, Sun Y, Ma HT, Xing LJ. Progress on molecular biology of ?6-fatty acid desaturase. Chin J Biotechnol, 2004, 20 (3): 319-324]
15 Uemura H. Synthesis and production of unsaturated and polyunsaturated fatty acids in yeast: current state and perspectives [J]. Appl Microbiol Biotechnol, 2012, 95 (1): 1-12
16 Ageitos JM, Vallejo JA, Veiga-Crespo P, Villa TG. Oily yeasts as oleaginous cell factories [J]. Appl Microbiol Biotechnol, 2011, 90 (4): 1219-1227
17 Beopoulos A, Cescut J, Haddouche R, Uribelarrea JL, Molina-Jouve C, Nicaud JM. Yarrowia lipolytica as a model for bio-oil production [J]. Prog Lipid Res, 2009, 48 (6): 375-387
18 Paul D1, Magbanua Z, Arick M 2nd, French T, Bridges SM, Burgess SC, Lawrence ML. Genome sequence of the oleaginous yeast Rhodotorula glutinis ATCC 204091 [J]. Genome Announc, 2014, 2 (1): e00046-14

相似文献/References:

[1]杨昭杰,李凌彦,胡彬彬,等.多不饱和脂肪酸与粘红酵母低温适应性的关系[J].应用与环境生物学报,2014,20(02):233.[doi:10.3724/SP.J.1145.2014.00233]
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备注/Memo

备注/Memo:
国家自然科学基金项目(31160016,31260034)、云南省应用基础研究基金资助项目(KKSA201126005)和教育部回国人员科研启动基金(KKQA201226003)资助 Supported by the National Natural Science Foundation of China (31160016, 31260034), the Application and Basic Research Fund of Yunnan Province (KKSA201126005), and the Scientific Research Foundation for Returned Overseas Chinese Scholars, State Education Ministry of China (KKQA201226003)
更新日期/Last Update: 2015-06-23