甜菜M14品系BvM14-Tpx基因克隆及原核表达体系下应答氧化胁迫功能的初步研究  

Cloning of Thioredoxin Peroxidase Gene in Sugar Beet M14 Line and Its Function Responsive to Oxidative Stress in Prokaryotic Expression System

作　　者：张庭跃 赵晨曦 陈思学[1,2] 李洪丽 Zhang Tingyue;Zhao Chenxi;Chen Sixue;Li Hongli(Engineering Research Center of Agricultural Microbiology Technology,Ministry of Education,Heilongjiang University,Harbin 150500;Key Laboratory of Molecular Biology,College of Heilongjiang Province,School of Life Sciences,Heilongjiang University,Harbin 150080)

机构地区：[1]黑龙江大学农业微生物技术教育部工程研究中心,哈尔滨150500 [2]黑龙江大学生命科学学院黑龙江省普通高校分子生物学重点实验室,哈尔滨150080

出　　处：《中国农学通报》2020年第32期30-38,共9页Chinese Agricultural Science Bulletin

基　　金：黑龙江省省属高等学校基本科研业务费基础研究项目“甜菜再生体系的建立及优化”(KJCXZD201718);国家自然科学基金面上项目“甜菜M14品系乙二醛酶Ⅰ基因抗盐能力的转录因子功能研究”(31671751)。

摘　　要：为了研究甜菜M14品系BvM14-Tpx基因的抗氧化功能,本试验以带有野生白花甜菜第9号染色体的单体附加系M14品系为试验材料,利用RACE技术获得甜菜M14品系硫氧还蛋白过氧化物酶基因(BvM14-Tpx) cDNA全长,对其进行生物信息学分析,利用Real-time PCR和半定量RT-PCR技术对该基因进行组织特异性表达分析,在原核表达体系下进行BvM14-Tpx基因应答氧化胁迫研究。生物信息学分析结果表明,BvM14-Tpx基因cDNA全长为1044 bp,包含最大的ORF为489 bp,编码162个氨基酸;含有过氧化物酶Ⅱ(PrxⅡ)型的保守结构域;BvM14-Tpx蛋白与豌豆(Pisum sativum L.)和苜蓿(Medicago truncatula L.)中Tpx蛋白的亲缘性较高。组织特异性表达分析结果表明,BvM14-Tpx基因在甜菜M14品系各组织表达量从高到低的顺序是根、茎、叶、花。通过原核表达体系下BvM14-Tpx基因应答氧化胁迫的研究,表明BvM14-Tpx基因能够提高大肠杆菌对于环境中氧化胁迫的适应能力,减轻H2O2对细菌生长的抑制。本研究对挖掘甜菜M14品系优质基因,提高甜菜对于非生物胁迫的抗性以及开展甜菜遗传改良工作具有重要意义。The purpose is to study the oxidative-resistance function of BvM14-Tpx gene. In this study, the monosomic addition line M14 with the alien No.9 chromosome from the wild white flower sugar beet species was used as the experimental material. The cDNA full length of thioredoxin peroxidase gene(BvM14-Tpx) in sugar beet M14 line was obtained by RACE technique, and the bioinformatics analysis was carried out. The tissue-specific expression of BvM14-Tpx gene was analyzed by Real-time PCR and semi-quantitative RTPCR. The BvM14-Tpx gene in response to oxidative stress was studied in prokaryotic expression system. The results of bioinformatics analysis showed that the total cDNA length of BvM14-Tpx gene was 1044 bp including the largest ORF 489 bp, which encoded 162 amino acids and contained the conserved domain of peroxidase Ⅱ(Prx Ⅱ). BvM14-Tpx protein had close genetic relationship with the Tpx protein from Pisum sativum L. and Medicago truncatula L.. The results of tissue specific expression analysis showed that the expression level of BvM14-Tpx gene from high to low was root, stem, leaf and flower. The research on BvM14-Tpx gene in response to oxidative stress in prokaryotic expression system showed that BvM14-Tpx gene could improve the adaptability of Escherichia coli to oxidative stress and reduce the inhibition of H2O2 on bacterial growth. This research is of certain significance for mining the high quality genes of sugar beet M14 line,improving the resistance of sugar beet to abiotic stress and carrying out genetic improvement of sugar beet.

关 键 词：甜菜M14品系 硫氧还蛋白过氧化物酶基因 生物信息学 原核表达 氧化胁迫 

分 类 号：S566.3[农业科学—作物学]