马齿苋PoFAD6基因克隆、原核表达及对低温胁迫的响应  

Cloning and Prokaryotic Expression of PoFAD6 Gene in Portulaca oleracea L.and Its Response to Low Temperature Stress

作　　者：周麟笔 韩洪强 王彪 赵大芹[1] 马关鹏 刘峰 文林宏[1] ZHOU Linbi;HAN Hongqiang;WANG Biao;ZHAO Daqin;MA Guanpeng;LIU Feng;WEN Linhong(Institute of Horticulture,Guizhou Academy of Agricultural Sciences/Guizhou Engineering Technology Research Center of Horticulture,Guiyang 550006,China;School of Ethnic-Minority Medicine,Guizhou Minzu University,Guiyang 550025,China;College of Landscape and Ecology,Ningbo City College of Vocational Technology,Ningbo Zhejiang 315100,China)

机构地区：[1]贵州省农业科学院园艺研究所,贵阳550006 [2]贵州民族大学民族医药学院,贵阳550025 [3]宁波城市职业技术学院景观生态学院,浙江宁波315100

出　　处：《种子》2023年第10期1-7,15,共8页Seed

基　　金：贵州省科技计划项目(黔科合支撑[2022]重点019号、黔科合服企[2022]005);贵阳市科技计划项目(筑科合同[2021]5-1号);宁波市公益类科技计划项目(2021S026);贵州省高等学校绿色化学与资源环境创新团队(黔教技[2022]13号)。

摘　　要：本研究以马齿苋为材料,采用同源克隆获得马齿苋ω-6脂肪酸脱氢酶PoFAD6基因,并对其进行了生物信息学分析、原核表达及在低温胁迫下的表达研究。结果表明,PoFAD6基因CDS全长为1380 bp,推导编码459个氨基酸,其氨基酸序列具有Membrane-FADS-like超家族共同的保守结构域。PoFAD6蛋白N端无信号肽,具有4个跨膜结构,为不稳定亲水蛋白,亚细胞预测定位于叶绿体上,二级结构主要包括α-螺旋和不规则卷曲。氨基酸同源比对和系统发育树分析结果表明,PoFAD6与藜麦FAD6氨基酸序列一致性最高,为77.54%,亲缘关系最近。PoFAD6融合蛋白可在大肠杆菌BL21表达宿主中成功表达,分子量约为53 kD。RT-qPCR结果表明,5℃低温处理0~24 h,PoFAD6基因的表达模式呈先升后降的趋势,说明PoFAD6基因能响应低温胁迫。In the study,using Portulaca oleracea L.as materials,theω-6 fatty acid desaturase PoFAD6 gene from Portulaca oleracea L.was obtained by homologous cloning,and bioinformatics analysis,prokaryotic expression and expression under low temperature stress were carried out.The results showed that the CDS of PoFAD6 gene was 1380 bp in length,encoding 459 amino acids.The amino acid sequence had the conserved domain of the Membrane-FADS-like superfamily.PoFAD6 protein had no signal peptide at the N-terminus and had four transmembrane structures,which was an unstable hydrophilic protein.Subcellular prediction was located on the chloroplast.The secondary structure mainly includedα-helix and random coil.The results of amino acid homology comparison and phylogenetic tree analysis showed that PoFAD6 had the highest amino acid sequence identity with FAD6 of Chenopodium quinoa,which was 77.54%,and the genetic relationship was the closest.The PoFAD6 fusion protein was successfully expressed in Escherichia coli BL21 strain,with a molecular weight of about 53 kD.The results of RT-qPCR showed that the expression pattern of PoFAD6 gene increased first and then decreased during 0-24 hours of low temperature treatment at 5℃,indicating that PoFAD6 gene could respond to low temperature stress.

关 键 词：马齿苋 ω-6脂肪酸脱氢酶 原核表达 低温胁迫 

分 类 号：S567.219[农业科学—中草药栽培]