沙棘油脂合成关键基因GPD1和DGAT的克隆及功能验证  

Cloning and functional validation of key genes GPD1 and DGAT involving in seed oil biosynthesis in sea buckthorn

作　　者：赵思阳 阮成江 丁健 卢顺光[2] 温秀凤[2] 胡建忠[2] ZHAO Siyang;RUAN Chengjiang;DING Jian;LU Shunguang;WEN Xiufeng;HU Jianzhong(Institute of Plant Resources,Dalian Minzu University,Dalian 116600,Liaoning,China;Seabuckthorn Development Management Center,Ministry of Water Resources,Beijing 100038,China)

机构地区：[1]大连民族大学资源植物研究所,辽宁大连116600 [2]水利部沙棘开发管理中心,北京100038

出　　处：《中南林业科技大学学报》2023年第8期149-158,168,共11页Journal of Central South University of Forestry & Technology

基　　金：国家自然科学基金项目(32071799,32111540255,32261133521);西藏中央引导地方项目(XZ202201YD0025C);水利部沙棘开发管理中心项目(2023-zg-kj-018);大连民族大学-西藏农牧学院联合基金项目(DLMZ-NMXY2021001)。

摘　　要：【目的】沙棘是水土保持和防沙治沙的重要木本油料树种,种子油富含生物活性成分,种子含油率低是制约沙棘开发和利用的主要瓶颈。本研究通过克隆沙棘种子油脂合成关键基因GPD1(glycerol-3-phosphate dehydrogenase)、DGAT1(diacylglycerol acyltransferase 1)和DGAT2(diacylglycerol acyltransferase 2),并进行了拟南芥异源过表达,以期为高油沙棘培育提供科学依据。【方法】基于沙棘转录组测序结果,以沙棘种子cDNA为模板克隆HrGPD1、HrDGAT1和HrDGAT2基因,通过In-fusion连接技术构建pCAMBIA1300-mCherry表达载体,通过农杆菌介导法将表达载体转入农杆菌GV3101,采用农杆菌蘸花法获取过表达油脂合成关键基因的拟南芥植株。利用实时荧光定量qRT-PCR分析HrGPD1、HrDGAT1和HrDGAT2在转基因拟南芥中的表达情况,利用氯仿甲醇法对T2代转基因和野生型的拟南芥进行含油量检测。【结果】通过PCR技术扩增得到了正确的HrGPD1、HrDGAT1和HrDGAT2基因序列,HrGPD1全长975 bp,编码氨基酸324个,分子量为35.55 kDa,等电点为5.36;HrDGAT1全长1608 bp,编码氨基酸535个,分子量为61.24 kDa,等电点为8.84;HrDGAT2全长993 bp,编码氨基酸330个,分子量为37.21 kDa,等电点为9.72。GPD1蛋白序列中含有1个保守结构域,属于甘油-3-磷酸脱氢酶家族成员,与枣和桑树亲缘关系较近;DGAT1、DGAT2蛋白序列中各含有1个保守结构域,属于膜结合O-酰基转移酶家族成员,DGAT1与枣亲缘关系较近,DGAT2与苹果和梨亲缘关系较近。通过农杆菌转化技术得到了稳定过表达HrGPD1、HrDGAT1和HrDGAT2的拟南芥株系。与野生型相比,过表达HrGPD1、HrDGAT1和HrDGAT2基因拟南芥T2代种子的含油量分别提高了5.09%、4.73%和2.51%。【结论】克隆获得沙棘HrGPD1、HrDGAT1和HrDGAT2的全长cDNA序列并鉴定功能,发现HrGPD1、HrDGAT1和HrDGAT2可以提高转基因拟南芥的种子含油量,为沙棘高油品种的培育提供了基因材料,对沙棘�【Objective】Sea buckthorn was one of important woody oil A low for soil and water conservation and sand prevention and control,and its seed oil contained rich bioactive components.A low seed oil content was the main bottleneck restricting the development and utilization of sea buckthorn.The objective of this study was to clone key genes of GPD1(glycerol-3-phosphate dehydrogenase),DGAT1(diacylglycerol acyltransferase 1)and DGAT2(diacylglycerol acyltransferase 2),which were related to seed oil biosynthesis in sea buckthorn;and test their function by heterologous over expression.This will provide a scientific basis for breeding sea buckthorn cultivars with high seed oil.【Method】According to the transcriptome sequencing results of sea buckthorn,HrGPD1,HrDGAT1 and HrDGAT2 genes were cloned using the cDNA template of seabuckthorn seeds.Pcambia1300-mCherry expression vector was constructed by in fusion ligation technology.The expression vector was transferred into GV3101 by the Agrobacterium-mediated method,and the Arabidopsis thaliana strains with an overexpressed of the three key genes were obtained by Agrobacterium impregnation.The expressions of three target genes in transgenic A.thaliana were determined by qRT-PCR.The oil contents of T2 transgenic and wild-type A.thaliana seeds were detected by chloroform methanol method.【Result】The correct gene sequences of HrGPD1,HrDGAT1 and HrDGAT2 were amplified by PCR,HrGPD1 consisted of 975 nucleotides encoding a protein of 324 amino acids with a calculated molecular mass of 35.55 kDa and a predicted pI of 5.36.HrDGAT1 consisted of 1608 nucleotides encoding a protein of 535 amino acids with a calculated molecular mass of 61.24 kDa and a predicted pI of 8.84.HrDGAT2 consisted of 993 nucleotides encoding a protein of 330 amino acids with a calculated molecular mass of 37.21 kDa and a predicted pI of 9.72.GPD1 protein contained one conserved function domain,which had been identified as a glycerol-3-phosphate dehydrogenase family members.HrGPD1,ZiGPD1 and MnGPD1 had be

关 键 词：沙棘 油脂合成 关键基因 过表达 含油量 

分 类 号：S793.6[农业科学—林木遗传育种]