笃斯越橘E3连接酶基因VuARI2的克隆及其抗寒功能分析  

Cloning and functional analysis of bog bilberry E3 ligase VuARI2 gene in cold resistance

作　　者：张润梅 乌凤章[1] ZHANG Runmei;WU Fengzhang(School of Life and Health,Dalian University,Dalian,Liaoning 116622,China)

机构地区：[1]大连大学生命健康学院,辽宁大连116622

出　　处：《西北植物学报》2024年第9期1420-1432,共13页Acta Botanica Boreali-Occidentalia Sinica

基　　金：辽宁省教育厅面上项目(LJKZ1194)。

摘　　要：【目的】通过克隆笃斯越橘E3泛素连接酶基因VuARI2及分析其在拟南芥中异源表达,探索笃斯越橘VuARI2基因在低温胁迫响应中的功能。【方法】以笃斯越橘为材料,用RT-PCR和RACE技术从茎中克隆获得VuARI2基因,并对其进行系统生物信息学分析;用qRT-PCR技术分析该基因在不同组织中及低温胁迫处理下的表达模式;在拟南芥中异源表达VuARI2并对其进行冷驯化后表达水平、生理指标及冰冻处理后的抗寒性分析。【结果】VuARI2基因编码区长1770 bp,编码589个氨基酸。泛素连接酶VuARI2与野山茶、咖啡树、葡萄和河岸葡萄ARI2蛋白的氨基酸序列同源关系较近。qRT-PCR结果显示,VuARI2基因表达具有组织特异性,在叶和花芽中VuARI2的表达量显著高于根和茎中;茎和花芽VuARI2表达受低温诱导较显著。转基因植物的VuARI2基因表达量在冷驯化后显著高于野生型;总叶绿素含量降幅显著低于野生型;脯氨酸和可溶性糖含量显著高于野生型;超氧化物歧化酶、过氧化物酶和过氧化氢酶活性均显著高于野生型,而相对电导率和丙二醛含量显著低于野生型。冷驯化后转基因植物在冰冻低温下存活率显著高于野生型,相对电导率显著低于野生型,提高了植物抗寒性。【结论】克隆获得了笃斯越橘VuARI2基因,过表达VuARI2基因可以提高植物抗寒性。[Objective]This study aims to explore the function of VuARI2 gene in response to low temperature stress,by cloning and expressing VuARI2 gene in Arabidopsis.[Methods]VuARI2 gene was cloned from the stem of bog bilberry by RACE and RT-PCR and conducted a comprehensive bioinformatics analysis.Expression of VuARI2 in different tissues after cold acclimation was analyzed by RT-qPCR.Furthermore,the transgenic Arabidopsis was analyzed for the expression of VuARI2,physiological indexes,and freezing resistance.[Results]VuARI2 was successfully cloned,with an open reading frame of 1770 bp,encoding 589 amino acids.The multi-sequence alignment and phylogenetic tree indicated that ubiquitin ligase VuARI2 was closely related to the amino acid sequence of ARI2 proteins in wild camellia,coffee tree,grape,and riverside grape.RT-qPCR showed that the expression of VuARI2 in leaves and flower buds of bog bilberry was higher than that in roots and stems,and the expression of VuARI2 in stems and flower buds was induced under low temperature.After cold acclimation,expression levels of VuARI2 gene in transgenic plants were higher in wild type,and chlorophyll content was lower than the wild type.Proline content and soluble sugar content were higher than the wild type.Superoxide dismutase activity,peroxi-dase activity,and catalase activity were higher than the wild type,while relative conductivity and malondi-aldehyde content were lower than the wild type.After cold acclimation,the survival rate of transgenic plants was higher than the wild type,and the relative conductivity was lower than the wild type at freezing temperature,enhancing freezing resistance of plants.[Conclusions]VuARI2 gene is cloned from bog bil-berry and expressed in Arabidopsis.Overexpress of VuARI2 gene enhances the cold resistance of plants。

关 键 词：笃斯越橘 泛素连接酶基因VuARI2 基因克隆 冷驯化 功能分析 抗寒性 

分 类 号：Q785[生物学—分子生物学] S663.9[农业科学—果树学]