油葵脂肪酸去饱和酶基因HaFAD2-1的克隆与功能鉴定
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摘要
为研究油葵FAD2-1基因在脂肪酸合成中的催化功能,利用RT-PCR技术,从油葵品种新葵杂4号未成熟种子中克隆HaFAD2-1基因的全长cDNA序列。将该基因序列构建穿梭表达载体pYES2-HaFAD2-1,并转化到营养缺陷型酿酒酵母菌株INVSc1中。将重组酵母诱导培养后对其总脂肪酸进行GC-MS分析。结果显示,HaFAD2-1基因编码一个长378个氨基酸的脂肪酸去饱和酶蛋白,分子质量43 719,等电点(pI)为8.38,具有3个高度保守的组氨酸簇。脂肪酸甲酯气相色谱分析结果表明HaFAD2-1基因在酿酒酵母中获得表达,能将油酸转化为亚油酸,证明克隆得到的HaFAD2-1具有完整的催化功能。
To study the catalytic function of oil sunflower FAD2-1 gene in fatty acid synthesis,a full length cDNA of FAD2 named HaFAD2-1 was isolated from developing seeds of Helianthus annuus L.Xinza 4 by RT-PCR.The HaFAD2-1gene encodes a-378 aa oleic acid desaturase with predicted molecular weight of 43 719,isoelectric point(pI) value of 8.38 and three his-boxes.HaFAD2-1 gene was sub-cloned into the shuttle vector pYES2 to generate the recombinant vector pYES2-HaFAD2-1 which was transformed into a defective mutant INVSc1 strain of Saccharomyces cerevisiae for expression.The total fatty acids of recombinant yeast were analyzed by GC-MS after induced culture.The result showed that transgenic yeast with the vector pYES2-HaFAD2-1 could catalyze oleic acid into linoleic acid,indicating that HaFAD2-1 is a functional enzyme of FAD2.
To study the catalytic function of oil sunflower FAD2-1 gene in fatty acid synthesis,a full length cDNA of FAD2 named HaFAD2-1 was isolated from developing seeds of Helianthus annuus L.Xinza 4 by RT-PCR.The HaFAD2-1gene encodes a-378 aa oleic acid desaturase with predicted molecular weight of 43 719,isoelectric point(pI) value of 8.38 and three his-boxes.HaFAD2-1 gene was sub-cloned into the shuttle vector pYES2 to generate the recombinant vector pYES2-HaFAD2-1 which was transformed into a defective mutant INVSc1 strain of Saccharomyces cerevisiae for expression.The total fatty acids of recombinant yeast were analyzed by GC-MS after induced culture.The result showed that transgenic yeast with the vector pYES2-HaFAD2-1 could catalyze oleic acid into linoleic acid,indicating that HaFAD2-1 is a functional enzyme of FAD2.
引文
[1]LOS D A,MURATA N.Structure and expression of fatty acid desaturases[J].Biochimica et Biophysica Acta,1998,1394(1):3-15.
[2]CAO S J,ZHOU X R,CRAIG C W et al.A large and functionally diverse family of FAD2 genes in safflower(Carthamus tinctorius L.)[J].Plant Biology,2013,13(5):1-18.
[3]SIMOPOULOS A P.Genetic variants in the metabolism of omega-6and omega-3 fatty acids:their role in the determination of nutritional requirements and chronic disease risk[J].Experimental Biology and Medicine,2010,235(7):785-795.
[4]LEE K R,SOO I S,JUNG J H,et al.Functional analysis and tissue-differential expression of four FAD2 genes in amphidiploid Brassica napus derived from Brassica rapa and Brassica oleracea[J].Gene,2013,531(2):253-262.
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[6]LIU Q,SINGH S P,GREEN A G.High-stearic and high-oleic cottonseed oils produced by hp RNA-mediated posttranscriptional gene silencing[J].Plant Physiology,2002,129:1732-1743.
[7]WAGNER N,MROCZKA A,ROBERTS P D,et al.RNAi trigger fragment truncation attenuates soybean FAD2-1 transcript suppression and yields intermediate oil phenotypes[J].Plant Biotechnology Journal,2011,9(7):723-728.
[8]孙黎,寇尚龙,欧阳超,等.油葵含油量相关基因在烟草中的表达[J].西北植物学报,2011,31(5):861-867.
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[10]HUNTER K,ROSE A H.Lipid composition of Saccharomyces cerevisiae as influenced by growth temperature[J].Biochimica et Biophysica Acta,1972,260(4):639-653.
[11]NIU B,GUO L,ZHAO M M,et al.Molecular cloning,characterization,and expression of anω-3 fatty acid desaturase gene from Sapium sebiferum[J].Journal of Bioscience and Bioengineering,2008,106(4):375-380.
[12]ISKANDAROV U,KHOZIN-GOLDBERG I,COHEN Z.Identification and characterization of△12,△6,and△5 desaturases from the green microalga Parietochloris incisa[J].Lipids,2010,45(6):519-530.
[13]房志家,陈婷,郝贺龙,等.酿酒酵母转化方法的新探索[J].实验室研究与探索,2012,31(4):5-8.
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[15]KARGIOTIDOU A,DELI D,GALANOPOULOU D,et al.Low temperature and light regulate delta 12 fatty acid desaturases(FAD2)at a transcriptional level in cotton(Gossypium hirsutum)[J].Journal of Experimental Botany,2008,59(8):2043-2056.
[16]HE X,TURNER C,CHEN G Q,et al.Cloning and characterization of a c DNA encoding diacylglycerl acyltransferase from castor bean[J].Lipids,2004,39(4):311-318.
[17]JOHN M D,DORSELYN C C,JUI-CHANG W K,et al.Molecular analysis of a biofunctional fatty acid conjugase/desaturase from tung.Implications for the evolution of plant fatty acid diversity[J].Plant Physiology,2002,130(4):2027-2038.
[18]LOS D A,MURATA N.Structure and expression of fatty acid desaturases[J].Biochimica et Biophysica Acta,1998,1394(1):3-15.
[19]SHANKLIN J,CAHOON E B.Desaturation and related modifications of fatty acids[J].Annual Review of Plant Physiology and Plant Molecular Biology,1998,49(6):611-641.
[20]MOCHE M,SHANKLIN J,GHOSHAL A,et al.Azide and acetate complexes plus two iron-depleted crystalsetures of the di-iron enzyme delta9 stearoyl-acyl carrier protein desaturase implications for oxygen activation and catalytic intermediates[J].Journal of Biological Chemistry,2003,278(27):25072-25080.
[21]SCHEFFLER J A,SHARPE A G,SCHMIDT H,et al.Desaturase multigene families of Brassica napus arose through genome duplication[J].Theoretical and Applied Genetics,1997,94(5),583-591.
[22]KIM M J,KIM H,SHIN J S,et al.Seed-specific expression of sesame microsomal oleic acid desaturase is controlled by combinatorial properties between negative cis-regulatory elements in the SeFAD2 promoter and enhancers in the 5'-UTR intron[J].Molecular Genetics and Genomics,2006,276(4):351-368.
[23]BELO A,ZHENG P,LUCK S,et al.Whole genome scan detects an allelic variant of fad2 associated with increased oleic acid levels in maize[J].Molecular Genetics and Genomics,2008,279(1):1-10.
[24]ZHANG D,PIRTLE I L,PARK S J,et al.Identification and expression of a new delta-12 fatty acid desaturase(FAD2-4)gene in upland cotton and its functional expression in yeast and Arabidopsis thaliana plants[J].Plant Physiology and Biochemistry,2009,47(6):462-471.
[25]MARTINEZ-RIVAS J M,SPERLING P,LUHS W,et al.Spatial and temporal regulation of three different microsomal oleate desaturase genes(FAD2)from normal-type and high-oleic varieties of sunflower(Helianthus annuus L.)[J].Molecular Breeding,2001,8(2):159-168.
[26]CHI X Y,YANG Q L,PAN L J et al.Isolation and characterization of fatty acid desaturase genes from peanut(Arachis hypogaea L.)[J].Plant Cell Report,2011,30(8):1393-1404.
[27]LU Y,CHI X,YANG Q,et al.Molecular cloning and stress-dependent expression of a gene encoding D12-fatty acid desaturase in the Antarctic microalga Chlorella vulgaris NJ-7[J].Extremophiles,2009,13(6):875-884.
[28]LU Y,CHI X,LI Z,et al.Isolation and characterization of a stress-dependent plastidial△12fatty acid desaturase from the Antarctic microalga Chlorella vulgaris NJ-7[J].Lipids,2010,45(2):179-187.
[29]CUI J J,HE S W.Identification and characterization of a novel biofunctional△12/△15-fatty acid desaturase gene from Rhodosporidium kratochvilovae[J].Biotechnology Letters,2016,38(7):1155-1164.
[2]CAO S J,ZHOU X R,CRAIG C W et al.A large and functionally diverse family of FAD2 genes in safflower(Carthamus tinctorius L.)[J].Plant Biology,2013,13(5):1-18.
[3]SIMOPOULOS A P.Genetic variants in the metabolism of omega-6and omega-3 fatty acids:their role in the determination of nutritional requirements and chronic disease risk[J].Experimental Biology and Medicine,2010,235(7):785-795.
[4]LEE K R,SOO I S,JUNG J H,et al.Functional analysis and tissue-differential expression of four FAD2 genes in amphidiploid Brassica napus derived from Brassica rapa and Brassica oleracea[J].Gene,2013,531(2):253-262.
[5]STOUTJESDIJK P A,SINGH S P,LIU Q,et al.Hp RNA-mediated targeting of the Arabidopsis thaliana FAD2 gene gives highly efficient and stable silencing[J].Plant Physiology,2002,129:1723-1731.
[6]LIU Q,SINGH S P,GREEN A G.High-stearic and high-oleic cottonseed oils produced by hp RNA-mediated posttranscriptional gene silencing[J].Plant Physiology,2002,129:1732-1743.
[7]WAGNER N,MROCZKA A,ROBERTS P D,et al.RNAi trigger fragment truncation attenuates soybean FAD2-1 transcript suppression and yields intermediate oil phenotypes[J].Plant Biotechnology Journal,2011,9(7):723-728.
[8]孙黎,寇尚龙,欧阳超,等.油葵含油量相关基因在烟草中的表达[J].西北植物学报,2011,31(5):861-867.
[9]刘公社,ALAIN B,彭克敬.向日葵研究与开发[M].北京:中国科学技术出版社,1994.
[10]HUNTER K,ROSE A H.Lipid composition of Saccharomyces cerevisiae as influenced by growth temperature[J].Biochimica et Biophysica Acta,1972,260(4):639-653.
[11]NIU B,GUO L,ZHAO M M,et al.Molecular cloning,characterization,and expression of anω-3 fatty acid desaturase gene from Sapium sebiferum[J].Journal of Bioscience and Bioengineering,2008,106(4):375-380.
[12]ISKANDAROV U,KHOZIN-GOLDBERG I,COHEN Z.Identification and characterization of△12,△6,and△5 desaturases from the green microalga Parietochloris incisa[J].Lipids,2010,45(6):519-530.
[13]房志家,陈婷,郝贺龙,等.酿酒酵母转化方法的新探索[J].实验室研究与探索,2012,31(4):5-8.
[14]ADAMS A,COTTSCHLING D E,KAISER C A,et al.Methods in yeast genetics:a cold spring harbor laboratory course manual[M].New York:Cold Spring Harbor Laboratory Press,1997:81-88.
[15]KARGIOTIDOU A,DELI D,GALANOPOULOU D,et al.Low temperature and light regulate delta 12 fatty acid desaturases(FAD2)at a transcriptional level in cotton(Gossypium hirsutum)[J].Journal of Experimental Botany,2008,59(8):2043-2056.
[16]HE X,TURNER C,CHEN G Q,et al.Cloning and characterization of a c DNA encoding diacylglycerl acyltransferase from castor bean[J].Lipids,2004,39(4):311-318.
[17]JOHN M D,DORSELYN C C,JUI-CHANG W K,et al.Molecular analysis of a biofunctional fatty acid conjugase/desaturase from tung.Implications for the evolution of plant fatty acid diversity[J].Plant Physiology,2002,130(4):2027-2038.
[18]LOS D A,MURATA N.Structure and expression of fatty acid desaturases[J].Biochimica et Biophysica Acta,1998,1394(1):3-15.
[19]SHANKLIN J,CAHOON E B.Desaturation and related modifications of fatty acids[J].Annual Review of Plant Physiology and Plant Molecular Biology,1998,49(6):611-641.
[20]MOCHE M,SHANKLIN J,GHOSHAL A,et al.Azide and acetate complexes plus two iron-depleted crystalsetures of the di-iron enzyme delta9 stearoyl-acyl carrier protein desaturase implications for oxygen activation and catalytic intermediates[J].Journal of Biological Chemistry,2003,278(27):25072-25080.
[21]SCHEFFLER J A,SHARPE A G,SCHMIDT H,et al.Desaturase multigene families of Brassica napus arose through genome duplication[J].Theoretical and Applied Genetics,1997,94(5),583-591.
[22]KIM M J,KIM H,SHIN J S,et al.Seed-specific expression of sesame microsomal oleic acid desaturase is controlled by combinatorial properties between negative cis-regulatory elements in the SeFAD2 promoter and enhancers in the 5'-UTR intron[J].Molecular Genetics and Genomics,2006,276(4):351-368.
[23]BELO A,ZHENG P,LUCK S,et al.Whole genome scan detects an allelic variant of fad2 associated with increased oleic acid levels in maize[J].Molecular Genetics and Genomics,2008,279(1):1-10.
[24]ZHANG D,PIRTLE I L,PARK S J,et al.Identification and expression of a new delta-12 fatty acid desaturase(FAD2-4)gene in upland cotton and its functional expression in yeast and Arabidopsis thaliana plants[J].Plant Physiology and Biochemistry,2009,47(6):462-471.
[25]MARTINEZ-RIVAS J M,SPERLING P,LUHS W,et al.Spatial and temporal regulation of three different microsomal oleate desaturase genes(FAD2)from normal-type and high-oleic varieties of sunflower(Helianthus annuus L.)[J].Molecular Breeding,2001,8(2):159-168.
[26]CHI X Y,YANG Q L,PAN L J et al.Isolation and characterization of fatty acid desaturase genes from peanut(Arachis hypogaea L.)[J].Plant Cell Report,2011,30(8):1393-1404.
[27]LU Y,CHI X,YANG Q,et al.Molecular cloning and stress-dependent expression of a gene encoding D12-fatty acid desaturase in the Antarctic microalga Chlorella vulgaris NJ-7[J].Extremophiles,2009,13(6):875-884.
[28]LU Y,CHI X,LI Z,et al.Isolation and characterization of a stress-dependent plastidial△12fatty acid desaturase from the Antarctic microalga Chlorella vulgaris NJ-7[J].Lipids,2010,45(2):179-187.
[29]CUI J J,HE S W.Identification and characterization of a novel biofunctional△12/△15-fatty acid desaturase gene from Rhodosporidium kratochvilovae[J].Biotechnology Letters,2016,38(7):1155-1164.