油莎豆5-烯醇式丙酮酰莽草酸-3-磷酸合酶基因CeEPSPS的克隆与分析  

Cloning and Characterization of CeEPSPS,a Gene Encoding5-Enolpyruvylshikimate-3-Phosphate Synthase from Tigernut(Cyperus esculentus L.)

作　　者：邹智 肖艳华 张丽 赵永国 ZOU Zhi;XIAO Yanhua;ZHANG Li;ZHAO Yongguo(Sanya Research Institute,Chinese Academy of Tropical Agricultural Sciences/Institute of Tropical Bioscience and Biotechnology,Chinese Academy of Tropical Agricultural Sciences/Hainan Key Laboratory for Biosafety Monitoring and Molecular Breeding in Off-Season Reproduction Regions,Haikou,Hainan 571101,China;College of Life Science,South-Central University for Nationalities/Hubei Provincial Key Laboratory for Protection and Application of Special Plants in Wuling Area of China,Wuhan,Hubei 430074,China;Guangdong University of Petrochemical Technology,Maoming,Guangdong 525000,China)

机构地区：[1]中国热带农业科学院三亚研究院/中国热带农业科学院热带生物技术研究所/海南省南繁生物安全与分子育种重点实验室,海南海口571101 [2]中南民族大学生命科学学院/武陵山区特色资源植物种质保护与利用湖北省重点实验室,湖北武汉430074 [3]广东石油化工学院生物与食品工程学院,广东茂名525000

出　　处：《热带作物学报》2023年第1期26-34,共9页Chinese Journal of Tropical Crops

基　　金：海南省自然科学基金项目(No.320RC705);国家自然科学基金项目(No.31971688);中国热带农业科学院基本科研业务费专项资金(No.1630052017011)。

摘　　要：起源于非洲和地中海沿岸的油莎豆以适应性广、产油量高而成为我国的新型油料作物。为促进该物种的开发与利用,研究基于基因组和转录组数据对油莎豆及代表性单子叶植物的EPSPS基因进行系统鉴定。结果表明:与大多数植物类似,油莎豆仅含有1个EPSPS基因,其包含7个内含子,命名为CeEPSPS;RT-PCR分离到的CeEPSPS编码区序列为1584 bp,预测编码514个氨基酸,其N端的前70个残基为叶绿体信号肽,77~508位残基为高度保守的EPSP合酶结构域(PF00275);与EPSP合酶结构域相比,信号肽在不同植物中变异较大;CeEPSPS成熟蛋白的理论分子量为47.32 kDa,等电点为5.49,总平均疏水指数为0.069,脂肪族指数为93.76,不稳定系数为31.73,与其他物种相近,均属于稳定的疏水型酸性蛋白;进化分析支持油莎豆划归为禾本目莎草科。序列比对和基因组测序分析结果显示,56份油莎豆种质不存在已报道的草甘膦抗性变异。qRT-PCR分析结果显示,CeEPSPS主要在成熟叶片和块茎中表达,其表达水平显著高于芽、幼嫩叶片、衰老叶片和芽茎。此外,本研究还构建了CeEPSPS的植物过表达载体,这为下一步的草甘膦抗性育种奠定了坚实的基础。Tigernut(Cyperus esculentus L.)is a perennial herb that is native to Africa and the Mediterranean coast.Due to the shortage of edible vegetable oil and biodiesel in China,tigernut has been emerging as a promising oil crop for its wide adaptability and high oil production per unit.To promote the development and utilization of this special crop,EPSPS(5-enolpyruvylshikimate-3-phosphate synthase)genes,which encode a key enzyme targeted by herbicide glyphosate in the shikimate pathway for the synthesis of aromatic amino acids,were identified from tigernut as well as several representative monocots based on genome and transcriptome data available.Comparative analyses revealed that(a)tigernut harbors a single EPSPS gene(denoted CeEPSPS)with seven introns as observed in most plant species;(b)its complete coding sequence of 1584 bp was further isolated using RT-PCR;(c)among 514 amino acids encoded by CeEPSPS,the first 70 residues from the N-terminal were characterized as the chloroplast signal peptide,whereas the peptide from sites 77 to 508 belongs to the highly conserved EPSP_synthase domain(under the Pfam accession number of PF00275);(d)compared with the EPSP_synthase domain,the chloroplast signal peptide is relatively more variable;and(e)the maturation protein of CeEPSPS was predicted to possess the theoretical molecular weight(Mw)of 47.32kDa,the isolectric point(pI)of 5.49,the grand average of hydropathicity(GRAVY)of 0.069,the aliphatic index(AI)of93.76,and the instability index(Ⅱ)of 31.73,which is similar to other reported EPSPSs and could be categorized as a type of hydrophilic,acidic,and stable proteins;(f)phylogenetic analysis using deduced EPSPS proteins supports that tigernut is a Cyperaceae plant within Poales.Further sequence alignment and genome resequencing analyses revealed that no target site resistance mutations to glyphosate were found in all 56 germplasms investigated in this study.Gene expression analysis using qRT-PCR showed that CeEPSPS was dominantly expressed in mature leaves and tubers,which we

关 键 词：油莎豆 莎草科 油料作物 5-烯醇式丙酮酰莽草酸-3-磷酸合酶 草甘膦 

分 类 号：S565.9[农业科学—作物学]