番茄泛素结合酶变体基因鉴定及表达分析

Identification and expression analysis of ubiquitin E2 enzyme variant gene family in tomato

【目的】开展番茄泛素结合酶变体(UEV)家族基因鉴定、组织特异性和非生物胁迫表达模式分析,为阐明UEV基因在番茄生长和逆境响应中的功能研究奠定基础。【方法】利用已知拟南芥UEV家族基因为探针,在番茄基因组数据库中查找并下载番茄UEV基因序列,同时查找其CDS、氨基酸长度及染色体位置等信息。利用生物信息学方法,通过在线工具GSDS 2.0、Expasy-protparam、Plant-PLoc、MEME对获得序列的基因结构、蛋白质分子量、等电点、亚细胞定位、保守结构域、保守基序原件等进行预测和分析。利用ClustalX(1.83)和MEGA 6.0软件进行同源序列比对并构建系统进化树。运用实时荧光定量RT-PCR分析番茄UEV基因组织表达特异性及高盐、干旱、高温、低温胁迫下UEV基因的表达差异。【结果】从番茄基因组数据库中共鉴定到5个UEV家族基因,分别命名为SlUEV1~SlUEV5,分布在1、4、10三条染色体上。SlUEV的CDS长度为441~855 bp,氨基酸数目为146~284 aa,分子量在16.2~33.14 kD。SlUEV分为3个亚家族,其中SlUEV1、SlUEV2、SlUEV5属于UEV1亚家族,且其基因结构、保守结构域和保守基序均相似;SlUEV4属于COP10亚家族,而SlUEV3在拟南芥中并未找到同源蛋白,表明UEV家族在进化过程中发生分离。组织表达分析发现SlUEV1、SlUEV3分别在番茄花和茎中表达量高,SlUEV4在果实发育期表达量高,而SlUEV2和SlUEV5呈组成性表达。非生物胁迫试验发现SlUEV对干旱胁迫较为敏感,多个SlUEV基因上调表达,表明SlUEV基因响应番茄干旱胁迫。【结论】番茄基因组中包括5个UEV家族基因,系统进化树分析将其分为3个亚家族。多个SlUEV基因在番茄不同组织表达存在差异,表明UEV基因影响番茄的生长发育。多个SlUEV基因在干旱胁迫下显著上调表达,推测其在番茄适应干旱胁迫过程中起着重要作用。该研究为后续开展番茄UEV基因调控植物生长和逆境胁迫功能研究提供理论基础。

【Objective】The UEV family genes were identified,and then tissue-specific expression profiles and expression pattern under abiotic stresses were analyzed.This research can lay a foundation for researching the function of UEV family genes in the growth and stress response.【Method】The Arabidopsis UEV protein sequences were used as queries to identify UEV in tomato genome and download all the UEV gene sequences,which information regarding the tomato UEV genes,including CDS sequences,amino acid sequences and chromosome location were obtained from tomato genome.The gene structure,protein molecular weight,isoelectric point,subcellular localization,conserved domain,and conserved element were also prospected and analyzed by tomato genome,Expasy-protparam,Plant-mPLoc,EMBL-EBI,MEME bioinformatics tools.Meanwhile,the homologous sequence alignment and phylogeny were analyzed by ClustalX(1.83)and MEGA 6.0 software.The expression levels of SlUEV genes in different tomato tissues and under high salt and drought stresses were analyzed by real-time fluorescence quantitative PCR.【Result】Five UEV family genes were identified from tomato genome,and designated SlUEV1 to SlUEV5.SlUEV were mapped on 1,4 and 10 chromosome.The length of the CDS ranged from 441 bp to 855 bp,the protein length ranged from 146 amino acids to 288 amino acids,the protein MW ranged from 16.2 kD to 33.14 kD.SlUEV were classified into 3 groups,SlUEV1,SlUEV2 and SlUEV5 belong to the UEV1 subfamily,and their gene structure,conservative structural domain and conservative motif were all similar.SlUEV4 belong to the COP10 subfamily.However,SlUEV3 didn’t find homologous protein in Arabidopsis thaliana,indicating that UEV family was separated during evolution.The expression of five SlUEV genes in different tissues were investigated,SlUEV1 and SlUEV3 were respectively highly expressed in stem and flower,SlUEV4 was highly expressed in fruit ripening stage.While SlUEV2 and SlUEV5 were constitutive expressions.Abiotic stress treated results showed that SlUEV were more sensitive to drought stress,the expression levels of multiple SlUEV genes were upregulated under drought,which indicated that SlUEV genes might be involved in the response of tomato to drought stresses.【Conclusion】Tomato UEV family contained five genes,and SlUEV genes could be classified into threes subgroups.The expression levels of multiple SlUEV genes had specific expressions in tomato tissues.SlUEV genes could respond to drought stresses,therefore we could suppose that SlUEV genes may be involved in the response of tomato to drought stresses.