| Tea plant（Camellia sinensis L.）is an important perennial economic crop in China,and tea leafhopper（Empoasca onukii Matsuda）is one of the most serious pests on tea plant,which has a great impact on tea production and quality.In this dissertation,the role of tea leafhoppers saliva and its related salivary proteins in mediating the interactions between tea leafhopper and tea plant was fully investigated.The feeding perferrence of tea leafhoppers,plant defense-related enzymes,and hormone analysiswere all examined in tea plants treated by tea leafhopper and its salivary proteins.Other technologies such as molecular cloning and protein in vitro expression were also used to take a comprehensive investigation of the salivary proteins of tea leafhopper in mediating plant defense response against pests.The main findings are as follows:（1）The type II ribosomal inactivation protein gene Cs RIP（Gene Bank accession number:FJ648831）could be specifically induced by tea leafhopper feeding on tea plants by PCR amplification.The electrophoresis results showed that tea plants treated with the homogenate supernatant of the salivary glands of tea leafhoppers had the same bright bands as that treated by tea leafhopper,while the control group tea plants applied with phosphate-buffered tea plants did not produce any significant bands.It was proved that the application of the homogenized salivary glands of tea leafhopper to the tea plants can induce plant defense responses.（2）The perferance bioassay was conducted using a Y-type olfactory device,and the results showed that the tea leafhopper had obvious avoidance of the tea plant either fed by the tea leafhopper or application of homogenized salivary glands of the tea leafhopper.Both application of the homogenized salivary glands of tea leafhopper and the feeding of tea leafhopper significantly induced the activities of polyphenol oxidase（PPO）,phenylalanine ammonia-lyase（PAL）and peroxidase（POD）in tea plants,and the induction of three enzymes reached a peak when tea plants were treated for 24 h.In additon,the homogenized salivary gland of tea leafhopper induced the expression of jasmonic acid（JA）pathway related genes such as lipoxygenase（LOX）and allene cyclase gene（AOC）,and triggered the expression of salicylic acid（SA）pathway-related gene,phenylalanine ammonia-lyase（PAL）at 24 h.However,the up-regulation of LOX and AOC was much higher than that of PAL.Moreover,the expression of oxidase gene（PPO）and trypsin inhibitor gene（PI）that are also JA-related genes were significantly increased,while the SA pathway downstream genes（pathogenesis-related genes PR1,NPR1）only showed a downward trend without any significant difference.The results of hormone analysis showed that the content of jasmonic acid in tea leaves treated by tea leafhopper was much higher than that in tea leaves without treatments.These results demonstrated that the feeding of tea leafhoppers and applying salivary homogenate mainly induced the JA signal pathway of the tea plant,and inhibited the expression of downstream genes of SA pathway through the antagonistic effect of JA and SA signal pathway.（3）The proteome of the salivary gland of tea leafhopper was analyzed using the LC-MS/MS technology,and the information of specific proteins were predicted by the transcriptome of the tea leafhopper as a protein database.Mass spectrometry results showed that the salivary glands of the tea leafhopper had at least 1097 proteins,including hydrolases,transporters,oxidoreductases,ATP synthetases,ligases,and transferases.The signal peptide prediction software Signal P and the protein transmembrane region analysis software TMHMM were used to predict the potentially secreted proteins in the salivary glands.Based on the transcriptome and proteome data of tea leafhopper salivary gland,we screened out some proteins including trypsin,β-glucosidase,and laccase,which may interfer the defense responses of tea plants,The effects of three proteases,trypsin,β-glucosidase,and laccase on tea plants were further verified.The preference bioassay by using the Y-type olfactory instrument showed that tea leafhoppers had obvious avoidance of tea plants treated with pure commercial enzymes.Additionally,the application of commercial trypsin induced changes in tea resistance-related enzymes and genes,which was consistent with the results of tea plants treated with homogenized salivary glands of tea leafhopper.Thus,trypsin was considered to be one of the key secreted proteins in the saliva of tea leafhopper,which mediates the defense responses of the tea plant.（4）The transcript sequence of the trypsin gene Eosep gene fragment was obtained from the proteome database of salivary gland of the tea leafhopper by Blastp software,and the full-length c DNA sequence was obtained using the PCR technique.The full-length c DNA sequence of the tea leafhopper trypsin Eosep gene includes a 1377bp ORF（open reading frame）,which encodes a total of 458 amino acids,has a signal peptide consisting of 17 amino acid residues,and has no transmembrane domain.It can be identified to be a classic secreted protein.The molecular formula is predicted to be C2158H3350N608O688S24.The molecular weight,the theoretical isoelectric point,and the instability coefficient of the protein is estimated to be 49.59 k D,4.90,and 37.23 respectively,which indicated that the protein is stable.The total average hydrophilic coefficient is-0.524,indicating that the protein is hydrophobic.Motif Scan analysis results showed that 204-445 aa is the serine protease domain.According to NCBI prediction,this protein may be trypsin in serine protease with high possibility.Trypsin protease was obtained using the molecular methods of cloning,expression,and purification in vitro,and it significantly induced the expression of defense-related enzymes and genes in tea plants.In summary,defense responses in tea plants induced by the salivary proteins of the tea leafhopper was similar to that induced by the tea leafhopper.Furthermore,induced defense response in tea plants treated by the salivary glands homogenate of the tea leafhopper,and its salivary proteins was closely related to the jasmonic acid signaling pathway,and the salivary trypsin of the tea leafhopper may be a key elicitor secreted by the tea leafhopper to mediate the plant defense response.These results in this paper not only revealed the important role of saliva and its related proteins in mainpulating the interactions between tea plant and tea leafhopper,but also provided a theoretical basis for using insect salivary elicitors to implement the integrated pest management on tea plants. |
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