坛紫菜PhGME基因克隆及差异表达分析  

Cloning and Differential Expression Analysis of GDP-Mannose-3,5-Epimerase(PhGME)Gene from Pyropia haitanensis

作　　者：魏鹏华 王文磊 许凯[1,2,3] 徐燕 纪德华[1,2,3] 谢潮添 陈昌生[1,2,3] WEI Penghua;WANG Wenlei;XU Kai;XU Yan;JI Dehua;XIE Chaotian;CHEN Changsheng(Fisheries College,Jimei University,Xiamen 361021,China;Fujian Engineering Research Center of Aquatic Breeding and Healthy Aquaculture,Xiamen 361021,China;Key Laboratory of Healthy Mariculture for the East China Sea,Ministry of Agriculture andRural Affairs,Xiamen 361021,China)

机构地区：[1]集美大学水产学院,福建厦门361021 [2]福建省水产生物育种与健康养殖工程研究中心,福建厦门361021 [3]农村农业部东海健康养殖重点实验室,福建厦门361021

出　　处：《集美大学学报（自然科学版）》2022年第2期97-106,共10页Journal of Jimei University：Natural Science

基　　金：国家重点研发项目(2018YFD0901500);国家自然科学基金项目(41806185);福建省重大科技专项(2019N20803);现代农业产业技术体系专项(CARS-50)。

摘　　要：GDP-甘露糖-3,5-表异构酶(GME)是抗坏血酸合成过程中的关键限速酶。以坛紫菜(Pyropia haitanensis)转录组测序获得的unigene序列为基础,采用普通PCR技术克隆获得了坛紫菜编码GME的基因序列PhGME。序列分析结果表明,PhGME基因序列全长1411 bp,包含一个1260 bp的开放阅读框,所编码的多肽包含419个氨基酸,相对分子质量为46.46 ku,理论等电点为6.76,具有GME特有的底物结合位点。多序列比对和系统进化树分析结果显示,PhGME与角叉菜同源且关系较近。差异表达分析结果表明,随着高盐胁迫时间的增加,TK品系处理组坛紫菜PhGME基因的表达水平均显著高于对照组,同时TK品系的AsA含量在胁迫处理6 h后明显增加。由此推测该基因的上调表达可能有助于抗坏血酸的合成,进而调控藻体抵抗盐胁迫。以上结果说明,PhGME在坛紫菜应对盐胁迫过程中发挥着重要作用,为坛紫菜抗逆功能的研究提供了理论参考。GDP-mannose-3,5-epimerase(GME)is a key rate limiting enzyme in ascorbic acid synthesis.In this study,based on the unigene sequences obtained via whole transcriptome sequencing of Pyropia haitanensis,the full-length of GME gene was obtained by PCR,and named PhGME.The full-length cDNA of PhGME gene comprised of 1411 bp nucleotides and contained an open reading frame of 1260 bp,encoding a protein of 419 amino acid residued with the predicted molecular weight of 46.46 ku and theoretical isoelectric point of 6.76.The sequence of PhGME contained specific active sites of GME.Multiple sequence alignment and phylogenetic analysis indicated that the PhGME had a close homology relationship with Chondrus crispus.The expressions of the PhGME gene,as measured by real-time quantitative PCR,under different treatment time points of the same strain,with the increase of hypersaline stress time,the expression level of PhGME gene of P.haitanensis in the TK strain treatment group was significantly higher than that in the control group,and the AsA content of the TK strain was significantly increased after 6 h of stress treatment.It was speculated that the up-regulated expression of this gene may contribute to the synthesis of ascorbic acid,thereby regulating the algae to resist salt stress.These results suggested that the PhGME plays an important role in the response of P.haitanensis to hypersaline stress,which provided a theoretical reference for the study of stress resistance of P.haitanensis.

关 键 词：坛紫菜 GDP-甘露糖-3 5-表异构酶 基因克隆 抗坏血酸 盐胁迫 

分 类 号：S968.431[农业科学—水产养殖]