续随子ElDGAT1基因的克隆、序列分析及表达特性
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摘要
二酰甘油酰基转移酶(Diacylglycerol acyltransferase,DGAT)是植物三酰甘油(TAG)合成最后一步反应的关键酶和限速酶,属于酰基转移酶超家族。为探究续随子(Euphorbia lathyris L.)中二酰甘油酰基转移酶1(DGAT1)对于调控种子油脂合成的作用机理,依据续随子转录组数据,采用RT-PCR技术分离和鉴定出一个 DGAT1基因(命名为 ElDGAT1),运用生物信息学方法对获得的序列进行分析,通过qPCR技术研究 ElDGAT1基因在续随子不同器官中的表达特性。结果表明:从续随子中克隆获得 ElDGAT1基因的cDNA序列,编码区长为1 530 bp,共编码509个氨基酸;预测ElDGAT1蛋白定位于内质网上,且为疏水性膜结合蛋白;其二级结构以α-螺旋和无规则卷曲为主,编码的蛋白含有9个典型的跨膜螺旋区。基于DGAT1蛋白的系统发育分析表明:续随子与乌桕的亲缘关系最近,与橡胶树和木薯的同源性次之。qPCR检测结果发现, ElDGAT1基因在续随子根中表达量最低,种子中的表达量相对较高,在花后15 d、30 d、45 d的表达量呈递增趋势。研究结果为进一步分析续随子种子油脂合成积累的调控机理及 ElDGAT1 基因的生物学功能提供依据。
Diacylglycerol acyltransferase(DGAT) belonging to the acyltransferase superfamily, is a key and rate-limiting enzyme in the final step of triacylglycerol(TAG) synthesis. In order to investigate its regulatory mechanism underlying seed oil synthesis in Euphorbia lathyris, we isolated and identified a DGAT1 gene(named ElDGAT1) by RT-PCR based on transcriptome data, using bioinformatics methods analyzed its cDNA sequence, and detected its expression in different organs by qPCR. Our results showed that the length of ElDGAT1 is 1 530 bp, encoding 509 amino acids. ElDGAT1 protein is a hydrophobic membrance binding protein may and localized in the endoplasmic reticulum;its secondary structure is mainly composed of alpha helix and irregular curl, and contains 9 typical transmembrane regions. Phylogenetic analysis based on DGAT1 protein showed that Euphorbia lathyris has a closer relationship with Triadica sebifera, and the homology with the Hevea brasiliensis and Manihot esculenta is second. qPCR results showed that the expression of ElDGAT1 Gene was the lowest in the roots, relatively high in the seeds, and constantly increased at 15, 30 and 45 days after flowering. Our results provided a basis for further study on the regulation mechanism and biological function of the oil accumulation in the seed of Euphorbia lathyris.
Diacylglycerol acyltransferase(DGAT) belonging to the acyltransferase superfamily, is a key and rate-limiting enzyme in the final step of triacylglycerol(TAG) synthesis. In order to investigate its regulatory mechanism underlying seed oil synthesis in Euphorbia lathyris, we isolated and identified a DGAT1 gene(named ElDGAT1) by RT-PCR based on transcriptome data, using bioinformatics methods analyzed its cDNA sequence, and detected its expression in different organs by qPCR. Our results showed that the length of ElDGAT1 is 1 530 bp, encoding 509 amino acids. ElDGAT1 protein is a hydrophobic membrance binding protein may and localized in the endoplasmic reticulum;its secondary structure is mainly composed of alpha helix and irregular curl, and contains 9 typical transmembrane regions. Phylogenetic analysis based on DGAT1 protein showed that Euphorbia lathyris has a closer relationship with Triadica sebifera, and the homology with the Hevea brasiliensis and Manihot esculenta is second. qPCR results showed that the expression of ElDGAT1 Gene was the lowest in the roots, relatively high in the seeds, and constantly increased at 15, 30 and 45 days after flowering. Our results provided a basis for further study on the regulation mechanism and biological function of the oil accumulation in the seed of Euphorbia lathyris.
引文
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[18] 李春霞.续随子三个候选内参基因的克隆及液泡膜Na+/H+逆向转运蛋白的表达分析[D].南京:南京师范大学,2013.LI CH X.Cloning and expression analysis of tonoplast Na+/H+ antiporter of three candidate internal reference genes of euphorbia lathyris[D].Nanjing:Nanjing Normal University,2013.
[19] 杨利民,韩梅.一种多用途植物:续随子的利用价值[J].生物学通报,1994,29(8):46-47.YANG L M,HAN M.A multi-purpose plant:the use value of Euphorbia lathyris L.[J].Bulletin of Biology,1994,29(8):46-47.
[20] 金梦阳,马冲,危文亮,等.新型能源植物续随子的核型分析[J].中国油料作物学报,2007,29(2):213-215.JIN M Y,MA CH,WEI W L,et al.Karyotype studies of new energy plant Euphorbia lathyris L.[J].Chinese Journal of Oil Crop Sciences,2007,29(2):213-215.
[21] WANG R,HANNA M A,ZHOU W W,et al.Production and selected fuel properties of biodiesel from promising non-edible oils:Euphorbia lathyris L.Sapium sebiferum L.and Jatropha curcas L.[J].Bioresource Technology,2011,102(2):1194-1199.
[22] WU Y,LI R,HILDEBRAND D F.Biosynthesis and metabolic engineering of palmitoleate production,an important contributor to human health and sustainable industry[J].Progress in Lipid Research,2012,51(4):340-349.
[23] 刘贵芹,邵群,黄荣峰,等.大豆DGAT基因家族的鉴定和表达分析[J].中国农学通报,2013,29(12):55-61.LIU G Q,SHAO Q,HUANG R F,et al.Characterization and expression analysis of DGAT gene family in soybean[J].Chinese Agricultural Science Bulletin,2013,29(12):55-61.
[24] 元博.续随子生长发育特性及其种子脂肪酸积累动态的研究[D].山西太谷:山西农业大学,2018.YUAN B.The research on the growth and development characteristics of Euphorbia lathyris and dynamic change[D].Taigu Shanxi:Shanxi Agricultural University,2018.
[25] CHEN G Q,TURNER C,HE X,et al.Expression profiles of genes involved in fatty acid and triacylglycerol synthesis in castor bean (Ricinus communis L.) [J].Lipids,2007,42(3):263-274.
[26] HOBBS D H,LU C,HILLS M J.Cloning of a cDNA encoding diacylglycerol acyltransferase from Arabidopsis thaliana and its functional expression [J].Febs Letters,1999,452(3):145-149.
[27] WANG H,ZHANG J,GAI J,et al.Cloning and comparative analysis of the gene encoding diacylglycerol acyltransferase from wild type and cultivated soybean[J].Theoretical and Applied Genetics,2006,112(6):1086-1097.
[28] SAHA S,ENUGUTTI B,RAJAKUMARI S,et al.Cytosolic triacylglycerol biosynthetic pathway in oilseeds molecular cloning and expression of peanut cytosolic diacylglycerol acyltransferase[J].Plant Physiology,2006,141(4):1533-1543.
[2] 中国科学院中国植物志编辑委员会.中国植物志第43卷第3分册[M].北京:科学出版社,1997.Chinese Flora Editorial Board of the Chinese Academy of Sciences.Chinese Flora 44th Volume Ⅲ[M].Beijing:Science Press,1997.
[3] 贾良智,周俊.中国油脂植物[M].北京:科学出版社,1987:193-196.JIA L ZH,ZHOU J.Chinese Oil Plant[M].Beijing:Science Press,1987:193-196.
[4] 秦燕,彭凯文,宁正祥.植物油脂保健作用研究进展[J].现代食品科技,1996(1):12-16.QIN Y,PENG K W,NING ZH X.Research progress on the health effects of plant oils[J].Modern Food Science and Technology,1996(1):12-16.
[5] 张庆会,徐步东.植物油脂的保健与药用价值[J].生物学通报,2002,37(4):27.ZHANG Q H,XU B D.Health and medicinal value of vegetable oils[J].Bulletin of Biology,2002,37(4):27.
[6] 危文亮,金梦阳,马冲,等.续随子油脂肪酸组成分析[J].中国油脂,2007,32(5):70-71.WEI W L,JIN M Y,MA CH,et al.Fatty acid composition analysis of Euphorbia lathyris L.seed oil[J].China Oils and Fats,2007,32(5):70-71.
[7] 李昌珠,蒋丽娟,程树棋.生物柴油—绿色能源[M].北京:化学工业出版社,2004:66-67.LI CH ZH,JIANG L J,CHENG SH Q.Biodiesel - Green Energy[M].Beijing:Chemical Industry Press,2004:66-67.
[8] 刘艳,周燕,高述民.植物油脂的生物合成及生物质能源产业化[J].中国农学通报,2007,23(3):491-493.LIU Y,ZHOU Y,GAO SH M.Biosynthesis of plant oils and biomass energy industrialization[J].Chinese Agricultural Science Bulletin,2007,23(3):491-493.
[9] COLEMAN R A,LEE D P.Enzymes of triacylglycerol synthesis and their regulation [J].Progress in Lipid Research,2004,43(2):134-176.
[10] SETTLAGE S B,KWAMYUEN P,WILSON R F.Relation between diacylglycerol acyltransferase activity and oil concentration in soybean [J]Journal of the American Oil Chemists Society,1998,75(7):775-781.
[11] STYMNE S,STOBART K.Triacylglycerol Biosynthesis[M].Lipids:Structure & Function,1987:175-214.
[12] XU J,FRANCIS T,MIETKIEWSKA E,et al.Cloning and characterization of an acyl-CoA-dependent diacylglycerol acyltransferase 1 ( DGAT1) gene from Tropaeolum majus and a study of the functional motifs of the DGAT protein using site-directed mutagenesis to modify enzyme activity and oil content [J].Plant Biotechnology Journal,2010,6(8):799-818.
[13] WESELAKE R J,SHAH S,TANG M G,et al.Metabolic control analysis is helpful for informed genetic manipulation of oilseed rape (Brassica napus) to increase seed oil content [J].Journal of Experimental Botany,2008,59(13):3543-3549.
[14] ZHENG P,ALLEN W B,ROESLER K,et al.A phenylalanine in DGAT is a key determinant of oil content and composition in maize [J].Nature Genetics,2008,40(3):367-372.
[15] BOUVIER-NAVE P,BENVENISTE P,OELKERS P,et al.Expression in yeast and tobacco of plant cDNAs encoding acyl-CoA:diacylglycerol acyltransferase [J].European journal of biochemistry,2010,267(1):85-96.
[16] HE X H,GRACE Q C,LIN J T,et al.Regulation of diacylglycerolacyl transferase in developing seeds of castor [J].Lipids,2004,39(9):865-871.
[17] 刘志勇,王孝宣,高建昌,等.番茄S-腺苷蛋氨酸脱羧酶基因slsamdc1的克隆与序列分析[J].园艺学报,2008,35(8):1137-1146.LIU ZH Y,WANG X X,GAO J CH,et al.Cloning and sequence analysisofa S-adenosylmethionine decarboxylase gene(SlSAMDC 1) in tomato[J].Acta Horticulturae Sinica,2008,35(8):1137-1146.
[18] 李春霞.续随子三个候选内参基因的克隆及液泡膜Na+/H+逆向转运蛋白的表达分析[D].南京:南京师范大学,2013.LI CH X.Cloning and expression analysis of tonoplast Na+/H+ antiporter of three candidate internal reference genes of euphorbia lathyris[D].Nanjing:Nanjing Normal University,2013.
[19] 杨利民,韩梅.一种多用途植物:续随子的利用价值[J].生物学通报,1994,29(8):46-47.YANG L M,HAN M.A multi-purpose plant:the use value of Euphorbia lathyris L.[J].Bulletin of Biology,1994,29(8):46-47.
[20] 金梦阳,马冲,危文亮,等.新型能源植物续随子的核型分析[J].中国油料作物学报,2007,29(2):213-215.JIN M Y,MA CH,WEI W L,et al.Karyotype studies of new energy plant Euphorbia lathyris L.[J].Chinese Journal of Oil Crop Sciences,2007,29(2):213-215.
[21] WANG R,HANNA M A,ZHOU W W,et al.Production and selected fuel properties of biodiesel from promising non-edible oils:Euphorbia lathyris L.Sapium sebiferum L.and Jatropha curcas L.[J].Bioresource Technology,2011,102(2):1194-1199.
[22] WU Y,LI R,HILDEBRAND D F.Biosynthesis and metabolic engineering of palmitoleate production,an important contributor to human health and sustainable industry[J].Progress in Lipid Research,2012,51(4):340-349.
[23] 刘贵芹,邵群,黄荣峰,等.大豆DGAT基因家族的鉴定和表达分析[J].中国农学通报,2013,29(12):55-61.LIU G Q,SHAO Q,HUANG R F,et al.Characterization and expression analysis of DGAT gene family in soybean[J].Chinese Agricultural Science Bulletin,2013,29(12):55-61.
[24] 元博.续随子生长发育特性及其种子脂肪酸积累动态的研究[D].山西太谷:山西农业大学,2018.YUAN B.The research on the growth and development characteristics of Euphorbia lathyris and dynamic change[D].Taigu Shanxi:Shanxi Agricultural University,2018.
[25] CHEN G Q,TURNER C,HE X,et al.Expression profiles of genes involved in fatty acid and triacylglycerol synthesis in castor bean (Ricinus communis L.) [J].Lipids,2007,42(3):263-274.
[26] HOBBS D H,LU C,HILLS M J.Cloning of a cDNA encoding diacylglycerol acyltransferase from Arabidopsis thaliana and its functional expression [J].Febs Letters,1999,452(3):145-149.
[27] WANG H,ZHANG J,GAI J,et al.Cloning and comparative analysis of the gene encoding diacylglycerol acyltransferase from wild type and cultivated soybean[J].Theoretical and Applied Genetics,2006,112(6):1086-1097.
[28] SAHA S,ENUGUTTI B,RAJAKUMARI S,et al.Cytosolic triacylglycerol biosynthetic pathway in oilseeds molecular cloning and expression of peanut cytosolic diacylglycerol acyltransferase[J].Plant Physiology,2006,141(4):1533-1543.