一个凤丹PoFAD2基因家族新成员PoFAD2-2的克隆及表达模式分析
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摘要
脂肪酸脱氢酶2(FAD2)能将油酸催化成亚油酸,是单不饱和脂肪酸转化为多不饱和脂肪酸的关键酶。本研究根据筛选到的PoFAD2-2基因序列,克隆到凤丹PoFAD2-2基因全长的c DNA,命名为PoFAD2-2。其开放阅读框为1 155 bp,编码384个氨基酸,蛋白质分子量为44 000,等电点为8. 51。生物信息学分析结果显示,该基因编码的蛋白质偏亲水性,包含5个跨膜结构域,属于膜结合型的FAD超家族。多序列比对与系统进化树分析结果发现,PoFAD2-2基因与PoFAD2-1基因亲缘关系较远,对于PoFAD2-2基因,凤丹在进化上与油橄榄[Olea europaea(common olive)(KY652929.1)]、靛木[Wrightia tinctoria (GU190864.1)]的相似性较高。荧光定量分析结果表明,PoFAD2-2在根和子房中没有表达,在花中的相对表达量最高,随着种子逐渐成熟,PoFAD2-2在种子中的表达量先逐渐降低再升高然后再降低,表明此基因表达具有组织特异性。PoFAD2-2基因在种子发育进程中的表达趋势与PoFAD2-1相同。
Fatty acid desaturase 2( FAD2) catalyzes the conversion of oleic acid to linoleic acid,and it is a key enzyme which converts monounsaturated fatty acids to polyunsaturated fatty acids. In this study,the full-length c DNA of PoFAD2-2 gene was cloned from PoFAD2-2 gene and named PoFAD2-2. Its open reading frame was 1 155 bp,384 amino acids were encoded,the molecular weight of protein was 44 000,the isoelectric point was 8. 51. Bioinformatics analysis results showed that the protein encoded by the gene was hydrophilic,contained five transmembrane domains,and belonged to the membrane-bound FAD superfamily. Multiple sequence alignment and phylogenetic analysis found that,PoFAD2-2 gene was more distantly related to PoFAD2-1 gene. PoFAD2-2 was evolutionarily similar to Olea europaea and Wrightia tinctoria. Quantitative real-time PCR results showed that PoFAD2-2 was not expressed in roots and ovaries,and the expression level of PoFAD2-2 was highest in petals. With the gradual maturation of seeds,the expression of PoFAD2-2 in the seeds was gradually decreased, then increased and then decreased. This gene expression was tissue-specific. The gene expression trend of PoFAD2-2 in seed development was the same as that of PoFAD2-1.
Fatty acid desaturase 2( FAD2) catalyzes the conversion of oleic acid to linoleic acid,and it is a key enzyme which converts monounsaturated fatty acids to polyunsaturated fatty acids. In this study,the full-length c DNA of PoFAD2-2 gene was cloned from PoFAD2-2 gene and named PoFAD2-2. Its open reading frame was 1 155 bp,384 amino acids were encoded,the molecular weight of protein was 44 000,the isoelectric point was 8. 51. Bioinformatics analysis results showed that the protein encoded by the gene was hydrophilic,contained five transmembrane domains,and belonged to the membrane-bound FAD superfamily. Multiple sequence alignment and phylogenetic analysis found that,PoFAD2-2 gene was more distantly related to PoFAD2-1 gene. PoFAD2-2 was evolutionarily similar to Olea europaea and Wrightia tinctoria. Quantitative real-time PCR results showed that PoFAD2-2 was not expressed in roots and ovaries,and the expression level of PoFAD2-2 was highest in petals. With the gradual maturation of seeds,the expression of PoFAD2-2 in the seeds was gradually decreased, then increased and then decreased. This gene expression was tissue-specific. The gene expression trend of PoFAD2-2 in seed development was the same as that of PoFAD2-1.
引文
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[8] YANG M,ZHENG G,ZHANG F,et al. FAD2-silencing has pleiotropic effect on polar lipids of leaves and varied effect in different organs of transgenic tobacco[J]. Plant Science,2006,170(1):170-177.
[9] YUAN. Abiotic stresses and phytohormones regulate expression of FAD2 gene in Arabidopsis thaliana[J]. Journal of Integrative Agriculture,2012,11(1):62-72.
[10]官玲亮,侯凯,陈郡雯,等.ω-6和ω-3脂肪酸脱氢酶家族系统进化与功能分化[J].遗传,2013,35(5):643-654.
[11] HEPPARD E P,KINNEY A J,STECCA K L,et al. Developmental and growth temperature regulation of two different microsomalω-6 desaturase genes in soybeans[J]. Plant Physiology,1996,110(1):311-319.
[12] TANG G Q,NOVITZKY W P,CAROL G H,et al. Oleate desaturase enzymes of soybean:evidence of regulation through differential stability and phosphorylation[J]. Plant Journal,2005,44(3):433-446.
[13] KISHORE K,SINHA S K,KUMAR R,et al. Isolation and characterization of microsomal omega-6-desaturase gene(FAD2-1)from soybean[J]. Indian Journal of Experimental Biology,2007,45(4):390-397.
[14] LI L,WANG X,GAI J,et al. Molecular cloning and characterization of a novel microsomal oleate desaturase gene from soybean[J].Journal of Plant Physiology,2007,164(11):1516-1526.
[15] LI L,WANG X,GAI J,et al. Isolation and characterization of a seed-specific isoform of microsomal omega-6 fatty acid desaturase gene(FAD2-1B)from soybean[J]. DNA Sequence,2008,19(1):28-36.
[16] HERNNDEZ M L,MANCHA M,MARTNEZ-RIVAS J M. Molecular cloning and characterization of genes encoding two microsomal oleate desaturases(FAD2)from olive[J]. Phytochemistry,2005,66(12):1417-1426.
[17] TAO F,ZHU S W,FAN J,et al. Cloning and sequence analysis of maize FAD2 gene[J]. Journal of Plant Physiology and Molecular Biology,2006,32(6):649-656.
[18]何早柯,周茜萍,王梦瑶,等.油葵脂肪酸去饱和酶基因Ha FAD2-1的克隆与功能鉴定[J].江苏农业学报,2017,33(2):273-279.
[19]宋淑香.凤丹脂肪酸去饱和酶基因PoFAD2的克隆及其表达分析[D].泰安:山东农业大学,2016.
[20] LIVAK K J,SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△Ctmethod[J]. Methods,2001,25(4):402-408.
[21] DONG C J,CAO N,ZHANG Z G,et al. Characterization of the fatty acid desaturase genes in cucumber:structure,phylogeny,and expression patterns[J]. PLoS ONE, 2016, 11(3):e0149917.
[22] WANG Y,ZHANG X,ZHAO Y,et al. Insights into the novel members of the FAD2 gene family involved in high-oleate fluxes in peanut[J]. Genome,2015,58(8):375-383.
[23] JUNG J H,KIM H,GO Y S,et al. Identification of functional BrFAD2-1 gene encoding microsomal delta-12 fatty acid desaturase from Brassica rapa and development of Brassica napus containing high oleic acid contents[J]. Plant Cell Reports,2011,30(10):1881-1892.
[24] LEE K R,SOHN S I,JIN H J,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.
[25]王仲伟,温强,汤诗杰,等.一个油茶FAD2基因家族新成员的克隆及分析[J].分子植物育种,2017,15(1):84-90.
[26] LIU W,LI W,HE Q,et al. Characterization of 19 genes encoding membrane-bound fatty acid desaturases and their expression profiles in Gossypium raimondii under low temperature[J]. PLoS ONE,2015,10(4):e0123281.
[27] SURESHA G S,RAI R D,SANTHA I M. Molecular cloning,expression analysis and growth temperature dependent regulation of a novel oleate desaturase gene(FAD2)homologue from Brassica juncea[J]. Australian Journal of Crop Science,2012,6(2):296-308.
[28] KIM H,GO Y S,KIM A Y,et al. Isolation and functional analysis of three microsomal delta-12 fatty acid desaturase genes from Camelina sativa(L.)cv. CAME[J]. Journal of Plant Biotechnology,2014,41(3):146-158.
[29] KONGCHAROENSUNTORN W. Isolation and analysis of cotton genomic clones encompassing a fatty acid desaturase(FAD2)gene[D]. Denton:University of North Texas,2001.
[30] LIU Q,BRUBAKER C L,GREEN A G,et al. Evolution of the FAD2-1 fatty acid desaturase 5'UTR intron and the molecular systematics of Gossypium(Malvaceae)[J]. American Journal of Botany,2001,88(1):92-102.
[31] HEPPARD E P,KINNEY A J,STECCA K L,et al. Development and growth temperature regulation of two different microsomalω-6desaturase genes in soybeans[J]. Plant Physiology,1996,110(1):311-319.
[32] HERNANDEZ M L,PADILLA M N,MANCHA M,et al. Expression analysis identifies FAD2-2 as the olive oleate desaturase gene mainly responsible for the linoleic acid content in virgin olive oil[J]. Journal of Agricultural&Food Chemistry,2009,57(14):6199-6206.
[33] 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 Biochemistry,2009,47(6):462-471.
[2]曾硕士,江黎明,元冬娟.脂肪酸去饱和酶的研究进展[J].生物技术通报,1998,20(4):816-820.
[3] SABETTA W,BLANCO A,ZELASCO S,et al. FAD7 gene identification and fatty acids phenotypic variation in an olive collection by EcoTILLING and sequencing approaches[J]. Plant Physiology&Biochemistry,2013,69(8):1-8.
[4] RAJWADE A V,KADOO N Y,BORIKAR S P,et al. Differential transcriptional activity of SAD, FAD2, and FAD3, desaturase genes in developing seeds of linseed contributes to varietal variation inα-linolenic acid content[J]. Phytochemistry,2014,98:41-53.
[5]王萍,张银波,江木兰.多不饱和脂肪酸的研究进展[J].中国油脂,2008,33(12):42-46.
[6]夏晗,王兴军,李孟军,等.利用基因工程改良植物脂肪酸和提高植物含油量的研究进展[J].生物工程学报,2010,26(6):735-743.
[7]叶静,胡海涛,王长春,等.植物脂肪酸去饱和酶与抗冷性研究进展[J].广东农业科学,2011,38(12):131-134.
[8] YANG M,ZHENG G,ZHANG F,et al. FAD2-silencing has pleiotropic effect on polar lipids of leaves and varied effect in different organs of transgenic tobacco[J]. Plant Science,2006,170(1):170-177.
[9] YUAN. Abiotic stresses and phytohormones regulate expression of FAD2 gene in Arabidopsis thaliana[J]. Journal of Integrative Agriculture,2012,11(1):62-72.
[10]官玲亮,侯凯,陈郡雯,等.ω-6和ω-3脂肪酸脱氢酶家族系统进化与功能分化[J].遗传,2013,35(5):643-654.
[11] HEPPARD E P,KINNEY A J,STECCA K L,et al. Developmental and growth temperature regulation of two different microsomalω-6 desaturase genes in soybeans[J]. Plant Physiology,1996,110(1):311-319.
[12] TANG G Q,NOVITZKY W P,CAROL G H,et al. Oleate desaturase enzymes of soybean:evidence of regulation through differential stability and phosphorylation[J]. Plant Journal,2005,44(3):433-446.
[13] KISHORE K,SINHA S K,KUMAR R,et al. Isolation and characterization of microsomal omega-6-desaturase gene(FAD2-1)from soybean[J]. Indian Journal of Experimental Biology,2007,45(4):390-397.
[14] LI L,WANG X,GAI J,et al. Molecular cloning and characterization of a novel microsomal oleate desaturase gene from soybean[J].Journal of Plant Physiology,2007,164(11):1516-1526.
[15] LI L,WANG X,GAI J,et al. Isolation and characterization of a seed-specific isoform of microsomal omega-6 fatty acid desaturase gene(FAD2-1B)from soybean[J]. DNA Sequence,2008,19(1):28-36.
[16] HERNNDEZ M L,MANCHA M,MARTNEZ-RIVAS J M. Molecular cloning and characterization of genes encoding two microsomal oleate desaturases(FAD2)from olive[J]. Phytochemistry,2005,66(12):1417-1426.
[17] TAO F,ZHU S W,FAN J,et al. Cloning and sequence analysis of maize FAD2 gene[J]. Journal of Plant Physiology and Molecular Biology,2006,32(6):649-656.
[18]何早柯,周茜萍,王梦瑶,等.油葵脂肪酸去饱和酶基因Ha FAD2-1的克隆与功能鉴定[J].江苏农业学报,2017,33(2):273-279.
[19]宋淑香.凤丹脂肪酸去饱和酶基因PoFAD2的克隆及其表达分析[D].泰安:山东农业大学,2016.
[20] LIVAK K J,SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△Ctmethod[J]. Methods,2001,25(4):402-408.
[21] DONG C J,CAO N,ZHANG Z G,et al. Characterization of the fatty acid desaturase genes in cucumber:structure,phylogeny,and expression patterns[J]. PLoS ONE, 2016, 11(3):e0149917.
[22] WANG Y,ZHANG X,ZHAO Y,et al. Insights into the novel members of the FAD2 gene family involved in high-oleate fluxes in peanut[J]. Genome,2015,58(8):375-383.
[23] JUNG J H,KIM H,GO Y S,et al. Identification of functional BrFAD2-1 gene encoding microsomal delta-12 fatty acid desaturase from Brassica rapa and development of Brassica napus containing high oleic acid contents[J]. Plant Cell Reports,2011,30(10):1881-1892.
[24] LEE K R,SOHN S I,JIN H J,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.
[25]王仲伟,温强,汤诗杰,等.一个油茶FAD2基因家族新成员的克隆及分析[J].分子植物育种,2017,15(1):84-90.
[26] LIU W,LI W,HE Q,et al. Characterization of 19 genes encoding membrane-bound fatty acid desaturases and their expression profiles in Gossypium raimondii under low temperature[J]. PLoS ONE,2015,10(4):e0123281.
[27] SURESHA G S,RAI R D,SANTHA I M. Molecular cloning,expression analysis and growth temperature dependent regulation of a novel oleate desaturase gene(FAD2)homologue from Brassica juncea[J]. Australian Journal of Crop Science,2012,6(2):296-308.
[28] KIM H,GO Y S,KIM A Y,et al. Isolation and functional analysis of three microsomal delta-12 fatty acid desaturase genes from Camelina sativa(L.)cv. CAME[J]. Journal of Plant Biotechnology,2014,41(3):146-158.
[29] KONGCHAROENSUNTORN W. Isolation and analysis of cotton genomic clones encompassing a fatty acid desaturase(FAD2)gene[D]. Denton:University of North Texas,2001.
[30] LIU Q,BRUBAKER C L,GREEN A G,et al. Evolution of the FAD2-1 fatty acid desaturase 5'UTR intron and the molecular systematics of Gossypium(Malvaceae)[J]. American Journal of Botany,2001,88(1):92-102.
[31] HEPPARD E P,KINNEY A J,STECCA K L,et al. Development and growth temperature regulation of two different microsomalω-6desaturase genes in soybeans[J]. Plant Physiology,1996,110(1):311-319.
[32] HERNANDEZ M L,PADILLA M N,MANCHA M,et al. Expression analysis identifies FAD2-2 as the olive oleate desaturase gene mainly responsible for the linoleic acid content in virgin olive oil[J]. Journal of Agricultural&Food Chemistry,2009,57(14):6199-6206.
[33] 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 Biochemistry,2009,47(6):462-471.