关键词: 茶树, 假眼小绿叶蝉, Ⅱ型核糖体失活蛋白, CsRIP 1, CsRIP 2

Abstract: 【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-Ev 2 (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 CsRIP 1 (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 CsRIP 2 (GenBank accession number: GU951535). The genomic sequence of CsRIP 1 and CsRIP 2 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 CsRIP 1 and CsRIP 2 contained an open 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 expressed with tissue specificity. The transcript level of CsRIP 1 was the highest in leaves and lowest in cotyledons; while the transcript level of CsRIP 2 was the highest in cotyledons, and lowest in leaves. The expression of CsRIPs was induced by Empoasca vitis feeding dramatically in leaf, and their expression level increased continuously, with approximately 120-fold and 100-fold higher after 48 h of feeding, respectively. Mechanical damage enhanced the expression level of CsRIPs in leaf. The expression level of CsRIP 1 reached maximal value after 6 h treatment. The expression level of CsRIP 2 increased remarkably after 6 h treatment, but reached the maximal value after 12 h. 【Conclusion】 Two type II ribosome inactivating protein genes from C. sinensis were cloned, which presumably play a defense-related role. Further analysis of the promoters will deepen our understanding of the transcriptional regulation of CsRIP genes and our insight into the functions of RIPs in vivo.

Key words: Camellia sinensis, Empoasca vitis, Type Ⅱ ribosome inactivating protein, CsRIP 1, CsRIP 2