紫苏PfDGAT1酵母功能互补分析
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摘要
[目的]本试验将已克隆的紫苏种子中高表达二酰甘油酰基转移酶(DGAT)基因PfDGAT1转化至酿酒酵母突变型菌株H1246进行酵母互补功能验证,研究二酰甘油酰基转移酶是否具有催化油脂合成的作用。[方法]通过构建紫苏DGAT基因的酵母表达载体pYES2.0-PfDGAT1并转化至酿酒酵母突变型菌株H1246,用尼罗红对转基因酵母细胞染色,检测转基因酵母中是否能合成油体;利用薄层层析(TLC)试验检测转基因酵母中是否存在三酰甘油(TAG)。[结果]转pYES2.0-PfDGAT1载体的突变型酵母菌株H1246能够检测到有油体存在,而转pYES2.0空载体的突变型酵母菌株H1246中没有检测到油体,说明紫苏PfDGAT1基因的表达恢复了酿酒酵母突变型菌株H1246油体缺陷的表型,可以催化TAG合成。[结论]PfDGAT1基因在紫苏种子油脂合成积累过程中发挥重要作用,该研究结果为深入解析PfDGAT1基因在紫苏油脂合成途径中的功能奠定了基础。
[Objective]In the previous study,the diacylglycerolacyltransferase(DGAT)gene PfDGAT1 has been cloned from perilla seeds and highly expressed.In this study,yeast Saccharomyces cerevisiae mutant strain H1246 was used to test the function of DGAT for catalyzing oil synthesis by yeast functional complementation experiments.[Methods]The yeast expression vector pYES2.0-PfDGAT1 of PfDGAT1 gene was successfully constructed by double digestion,and the recombinant plasmid was transformed into the yeast mutant strain H1246.The transgenic yeast cells were stained with Nile Red to detect whether oil body could be synthesized in the transgenic yeast.TLC test was used to detect TAG presented in thransgenic yeast.[Results]Oil bodies were able to be detected in the mutant strain H1246 transfected with pYES2.0-PfDGAT1,but not in the mutant strain H1246 transfected with pYES2.0 empty vector,whichindicated that the oil body defectingin Saccharomyces cerevisiae mutant strain H1246 was restored by the expression of PfDGAT1 gene.The diacylglycerolacyltransferase could catalyze TAG synthesis.[Conclusion]The PfPDAT1 gene plays an important role in the synthesis and accumulation of perilla seed oil.These results lay the foundation for the further study of the function of PfDGAT1 gene in the fatty acid metabolism pathway of perilla.
[Objective]In the previous study,the diacylglycerolacyltransferase(DGAT)gene PfDGAT1 has been cloned from perilla seeds and highly expressed.In this study,yeast Saccharomyces cerevisiae mutant strain H1246 was used to test the function of DGAT for catalyzing oil synthesis by yeast functional complementation experiments.[Methods]The yeast expression vector pYES2.0-PfDGAT1 of PfDGAT1 gene was successfully constructed by double digestion,and the recombinant plasmid was transformed into the yeast mutant strain H1246.The transgenic yeast cells were stained with Nile Red to detect whether oil body could be synthesized in the transgenic yeast.TLC test was used to detect TAG presented in thransgenic yeast.[Results]Oil bodies were able to be detected in the mutant strain H1246 transfected with pYES2.0-PfDGAT1,but not in the mutant strain H1246 transfected with pYES2.0 empty vector,whichindicated that the oil body defectingin Saccharomyces cerevisiae mutant strain H1246 was restored by the expression of PfDGAT1 gene.The diacylglycerolacyltransferase could catalyze TAG synthesis.[Conclusion]The PfPDAT1 gene plays an important role in the synthesis and accumulation of perilla seed oil.These results lay the foundation for the further study of the function of PfDGAT1 gene in the fatty acid metabolism pathway of perilla.
引文
[1]王计平,张玲慧,赵静,等.紫苏种子脂肪酸代谢及关键酶基因调控油脂合成规律的研究[J].中国粮油学报,2016,31(3):91-95.
[2]Settlage S B,Kwanyuen P,Wilson R F.Relation between diacylglycerol acyltransferase activity and oil concentration in soybean[J].The Journal of the American Oil Chemists’Society,1998,75:775-781.
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[9]Kalscheuer R,Stoveken T,Malkus U,et al.Analysis of storage lipid accumulation in Alcanivorax borkumensis:evidence for alternative triacylglycerol biosynthesis routes in bacteria[J].Journal of Bacteriology,2007,189:918-928.
[10]乔晓芳,王春艳,顾宝华,等.无糖基化修饰GLP-1-Fc(N126A)在酵母中的表达、纯化[J].郑州大学学报(理学版),2016,48(4):86-89.
[11]Meesapyodsuk D,Reed D W,Savile C K,et al.Characterization of the regiochemistry and cryptoregiochemistry of a Caenorhabditis elegans fatty acid desaturase(FAT-1)expressed in Saccharomyces cerevisiae[J].Biochemistry,2000,39(39):11948-11954.
[12]刘辉,邓治,杨洪,等.橡胶树HbMC2在酵母中的表达和抗逆性分析[J].生物技术通报,2018,34(9):202-208.
[13]朱红,王春,王芳,等.人源SIRT4蛋白在毕赤酵母中的表达及纯化[J].郑州大学学报(医学版),2018,53(4):443-447.
[14]冯德明,温佩颖,赵畅,等.刚毛柽柳ThDREB基因在酵母中的表达及抗逆能力分析[J].植物研究,2017,37(1):63-68.
[15]夏凌峰,史雪,杨昊虹,等.小麦β-酮脂酰CoA合成酶基因KCS的克隆与酵母表达[J].麦类作物学报,2016,36(9):1121-1129.
[16]徐荣华,邱丽俊,阳天泉,等.小桐子磷脂二酰甘油酰基转移酶(JcPDAT1)cDNA的克隆与功能鉴定[J].中国油料作物学报,2013,35(2):123-130.
[17]谭太龙,冯韬,罗海燕,等.甘蓝型油菜磷脂二酰甘油酰基转移酶(BnPDAT1)cDNA的克隆和功能鉴定[J].作物学报,2016,42(5):658-666.
[18]徐荣华,崔涛,王建才,等.蒺藜苜蓿DGAT1基因的克隆和功能鉴定[J].西北植物学报,2016(10):1941-1947.
[2]Settlage S B,Kwanyuen P,Wilson R F.Relation between diacylglycerol acyltransferase activity and oil concentration in soybean[J].The Journal of the American Oil Chemists’Society,1998,75:775-781.
[3]Maisonneuve S,Bessoule J J,Lessire R,et al.Expression of rapeseed microsomal lysophosphatidic acid acyltransferase isozymes enhances seed oil content in Arabidopsis[J].Plant physiology,2010,152(2):670-684.
[4]Murphy D J,Mukherjee K D.Acyltransferases in subcellular fractions of developing seeds of rape(Brassica napus L.)[J].Lipids,1987,22(5):293-298.
[5]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.
[6]Srensen B M,Furukawa-Stoffer T L,Marshall K S,et al.Storage lipid accumulation and acyltransferase action in developing flaxseed[J].Lipids,2005,40(10):1043-1049.
[7]Jako C,Kumar A,Wei Y D,et al.Seed-specific over-expression of an Arabidopsis cDNA encoding a diacylg-lycerol acyltransferase enhances seed oil content and seed weight[J].Plant Physiology,2001,126:861-874.
[8]Siloto R M P,Truksa M,He X H,et al.Simple methods to detect triacylglycerol biosynthesis in a yeast based recombinant system[J].Lipids,2009,44:963-973.
[9]Kalscheuer R,Stoveken T,Malkus U,et al.Analysis of storage lipid accumulation in Alcanivorax borkumensis:evidence for alternative triacylglycerol biosynthesis routes in bacteria[J].Journal of Bacteriology,2007,189:918-928.
[10]乔晓芳,王春艳,顾宝华,等.无糖基化修饰GLP-1-Fc(N126A)在酵母中的表达、纯化[J].郑州大学学报(理学版),2016,48(4):86-89.
[11]Meesapyodsuk D,Reed D W,Savile C K,et al.Characterization of the regiochemistry and cryptoregiochemistry of a Caenorhabditis elegans fatty acid desaturase(FAT-1)expressed in Saccharomyces cerevisiae[J].Biochemistry,2000,39(39):11948-11954.
[12]刘辉,邓治,杨洪,等.橡胶树HbMC2在酵母中的表达和抗逆性分析[J].生物技术通报,2018,34(9):202-208.
[13]朱红,王春,王芳,等.人源SIRT4蛋白在毕赤酵母中的表达及纯化[J].郑州大学学报(医学版),2018,53(4):443-447.
[14]冯德明,温佩颖,赵畅,等.刚毛柽柳ThDREB基因在酵母中的表达及抗逆能力分析[J].植物研究,2017,37(1):63-68.
[15]夏凌峰,史雪,杨昊虹,等.小麦β-酮脂酰CoA合成酶基因KCS的克隆与酵母表达[J].麦类作物学报,2016,36(9):1121-1129.
[16]徐荣华,邱丽俊,阳天泉,等.小桐子磷脂二酰甘油酰基转移酶(JcPDAT1)cDNA的克隆与功能鉴定[J].中国油料作物学报,2013,35(2):123-130.
[17]谭太龙,冯韬,罗海燕,等.甘蓝型油菜磷脂二酰甘油酰基转移酶(BnPDAT1)cDNA的克隆和功能鉴定[J].作物学报,2016,42(5):658-666.
[18]徐荣华,崔涛,王建才,等.蒺藜苜蓿DGAT1基因的克隆和功能鉴定[J].西北植物学报,2016(10):1941-1947.