Cloning And Expression Of Anti-Empoasca Vitis-related Genes In Tea

Transcriptional changes accompany induced resistance of plants against insect feeding, and monitoring transcriptional reorganization triggered in response to herbivory is an essential step for deciphering the molecular basis of the resistance. Therefore, demonstrating transcriptional changes of plants response to insects is a very effective strategy to understand the mechanism of plant resistance to their insects. The model of transcriptional changes in some plants has been studied when they are infested by herbivories, such as Arabidopsis thaliana, tobacco, Citrus reticulate Banco, Picea asperata Mast and Populus. However, the study of resistance to insects about tea has not been reported. Green leafhopper (Empoasca vitis Gothe) is a typical vascular feeder and the predominant insect pest infesting tea in China. In this study, we order to isolate and identify resistance-conferring genes induced by Green leafhopper.In order to identify genes up-regulated during mild infestation by green leafhopper, a subtracted cDNA library was constructed by using cDNA from leaves under mild infestation after 32h as tester and cDNA from uninfested leaves as driver. The forward-subtracted cDNA was cloned, and the PCR products of 658 randomly selected clones were arrayed on the nylon membrane for differential screening. After differential screening,35 cDNA clones expressed at higher levels in infested than in control leaves were identified. Sequence analysis showed that 35 clones were clustered to 26 independent transcripts. However, insect-induced transcript accumulation was verified only for 21 independent cDNAs by real-time PCR analysis. We refer to these genes as Cs-Ev (Cs and Ev represent Camellia sinensis and Empoasca vitis, respectively).Among 21 transcripts induced by green leafhopper, Cs-Ev2 and Cs-Ev20 classes were isolated twice, Cs-Ev19 class three times, and the remaining cDNAs were a single clone. The 21 up-regulated genes were classified into five functional groups based on their putative roles during Empoasca infestation. These genes encode proteins functioning in defense, involved in the CBL-CIPK pathway, secondary metabolite synthesis, cell wall fortification, and oxidative stress. The library also included the transcripts not previously reported to be induced by insect attack. Our results provide new information about insect-plant interaction.Ribosome-inactivating proteins (RIPs) are rRNA N-glycosidases capable of removing glycosidic bond to a specific adenine residue(A4324) from a conserved loop (ricin/sarcin loop) of the large rRNA in 28S ribosomal subunits in eukaryotic. Currently, RIPs are divided into two groups according to their molecular structure. TypeⅠRIPs are single proteins, consisting of single catalytically active polypeptides. Type 2 RIPs contain at least one ribosome inactivating A-chain, resembling type 1 RIPs, and a corresponding number of carbohydrate-binding B-chains. The two polypeptides remain covalently joined by a disulfide bridge. We isolated a full-length cDNA and genomic DNA sequence of typeⅡRIP from a green leafhopper-resistant tea genotype——Fuzao2's leaves when we study tea responses to green leafhopper. We refer to this gene as CsRIP (GeneBank accession number:FJ648831). This gene contains a complete ORF, encoding a polypeptide of 570 amino acid residues. Sequences analysis illustrated that the conserved residues of typeⅡRIPs are all presented in CsRIP's corresponding satiation, including the amino acid residues of form the active-site of RNA N-glycosidase in A-chain and two carbohydrate-binding sites in B-chain. Our study also found that the expression of CsRIP is not induced by Ectropis oblique hypulina Wehrli.