茶树Ⅱ型核糖体失活蛋白基因CsRIP1和CsRIP2的克隆与表达分析  

Cloning and Expression of Type Ⅱ Ribosome Inactivating Protein Genes CsRIP1 and CsRIP2 from Camellia sinensis

作　　者：袁红雨[1] 马宁[1] 杨会敏[2] 庞秋芬 谢素霞[1] 程琳[1] 

机构地区：[1]信阳师范学院生命科学学院,信阳464000 [2]红河学院生物科学与技术学院,蒙自661100

出　　处：《林业科学》2015年第2期147-153,共7页Scientia Silvae Sinicae

基　　金：河南省基础与前沿技术研究计划项目(092300410244);河南省教育厅科学技术研究重点项目(12B180032)

摘　　要：【目的】克隆茶树Ⅱ型核糖体失活蛋白基因CsRIP1和CsRIP2,探讨CsRIPs基因的组织表达特异性以及假眼小绿叶蝉和机械伤害处理对其表达的影响。【方法】Cs-Ev2(Gen Bank登录号:GH618807)为一受假眼小绿叶蝉取食诱导的茶树Ⅱ型核糖体失活蛋白基因的c DNA片段,利用RACE技术克隆该基因的全长c DNA,命名为CsRIP1(Gen Bank登录号:FJ648831)。利用RT-PCR技术从茶树子叶中分离出一个新的Ⅱ型核糖体失活蛋白基因的c DNA序列,命名为CsRIP2(Gen Bank登录号:GU951535)。利用PCR技术克隆CsRIPs的基因组序列。设计基因特异性引物,利用Real-time qRT-PCR技术检测CsRIPs的组织表达特异性,以及在叶片中假眼小绿叶蝉和机械伤害处理对其表达的影响。【结果】CsRIP1和CsRIP2的序列一致性为98%,都含有一个1 713 bp的开放阅读框,编码570个氨基酸残基,但是它们具有不同的3'非翻译区。CsRIP1和CsRIP2由信号肽序列、1个RIP结构域、链接肽和2个RBL结构域组成。CsRIPs与其他Ⅱ型RIPs具有较高的序列一致性,Ⅱ型RIPs保守的氨基酸残基,包括构成A链N-糖苷酶活性位点的氨基酸残基、B链中形成寡糖链结合位点的氨基酸残基、形成链间和链内二硫键的半胱氨酸残基以及RBL结构域中的Qx W基序,均出现在CsRIPs相应的位置上。系统进化树分析结果显示,同一物种的Ⅱ型RIPs首先聚类合并,2个CsRIP与樟的Ⅱ型RIPs聚为一支。比较CsRIPs的c DNA序列和基因组序列,发现它们的基因组序列不含内含子。CsRIPs基因的表达具有组织特异性,CsRIP1在叶片中的表达水平最高,在子叶中的表达水平最低,而CsRIP2在子叶中的表达水平最高,在叶片中的表达水平最低。在叶片中,CsRIP1和CsRIP2的表达均受假眼小绿叶蝉取食的强烈诱导,并且其表达水平随着取食时间的增加而逐渐增加,取食48 h后,它们的表达水平大约分别是对照的120和100倍。在叶片中,CsRIPs基因的表达�[Objective]To clone ribosome inactivating protein （ RIP） genes from Camellia sinensis,and to study tissue-specific expression of CsRIPs and effects of Empoasca vitis feeding and mechanical damage on the expression of CsRIPs.[Method]Cs-Ev2 （GenBank accession number： GH618807） is the cDNA fragment of a type Ⅱ RIP gene of tea plants, which was up-regulated by mild infestation of green leafhopper. Its full length cDNA sequence was cloned by RACE method,and designated as CsRIP1 （GenBank accession number： FJ648831）. The cDNA sequence of a new type Ⅱ RIP gene was cloned by RT-PCR method from developing cotyledons of tea plants,and designated as CsRIP2 （ GenBank accession number： GU951535 ） . The genomic sequence of CsRIP1 and CsRIP2 was obtained by PCR method. Their tissue-specific expression pattern and expression characteristics induced by E. vitis feeding and mechanical damage were detected by Real-time qRT-PCR,using gene-specific primers. [Result]Both of CsRIP1 and CsRIP2 contained an open＆nbsp;reading frame of 1 713 bp,encoding a predicted protein of 570 amino acid residues,but their 3＇ UTRs were different. Analysis of the amino acid sequence showed that the predicted precursors of CsRIP1 and CsRIP2 consisted of a signal peptide sequence,a RIP domain,and two RBL domains. CsRIPs had a high identity to other type Ⅱ RIPs at the overall amino acid level. The conserved amino acid residues,including all residues that form active-site of RNA N-glycosidase of A chain,residues that form two sugar-binding sites of B chain,cystein residues that form one interchain and four intrachain disulfide bonds,and QxW motif of RBL domains,are strongly conserved in CsRIP1 and CsRIP2. Phylogenetic analysis showed that different type Ⅱ RIPs from one species form a subgroup first,and two CsRIPs are closely related to type Ⅱ RIPs from Cinnamomum camphora. The comparison of the CsRIP cDNA sequences and their corresponding genomic sequences illustrated that the CsRIP genes have no intron. CsRIPs were express

关 键 词：茶树 假眼小绿叶蝉 Ⅱ型核糖体失活蛋白 CsRIP1 CsRIP2 

分 类 号：S718.46[农业科学—林学]