Identification Of Salivary Protein And Functional Study Of NlCBP1&NlCBP2 In Brown Planthopper Nilaparvata Lugens

The rice brown planthopper,Nilaparvata lugens（St?l）is a major rice insect pest in Asia.It secretes saliva during the process of sucking and feeding rice and participates in plant-insect interaction.Recently,there are few studies on the identification and function of the salivary protein of BPH（Brown planthopper）.In this study,the artificial diet feed method was used to collect the saliva protein of BPH and the salivary protein was identified by Shotgun LC-MS/MS mass spectrometry technique,the molecular characteristics of Ca²⁺binding protein were studied in order to further analyze its function in plant-insect interaction.（1）Identification of salivary protein in BPH:A total of 107 proteins were identified in the watery saliva of N.lugens,over 80%of which showed significant similarity to known proteins.When annotated by the Blast2GO suite,29 proteins had catalytic activity and 24 proteins were binding proteins.The saliva enzymes included oxidoreductases,hydrolases,phosphatases,peptidases（proteases）,kinases,transferases,and lyases.Binding proteins in N.lugens watery saliva included ATP-binding,lipophorin,calcium-binding,actin-binding and DNA-,RNA-,and chromatin-binding proteins.Other non-enzymatic proteins,such as ubiquitins,heat shock proteins,ribosomal proteins,and immunoglobulin proteins were also found in N.lugens watery saliva.The qualitative analysis of BPH saliva protein was able to provide a basis for the study of BPH-rice interaction.（2）Functional study of NlCBP1&NlCBP2:Studies have shown that calcium binding protein plays an important role in aphids and other pierce-sucking insects-plant interaction.So we selected 8 calcium-binding proteins for study.First,the relative expression levels of NlCBP1 and NlCBP2 in the different tissues（head/midgut/ovary/salivary gland）of N.lugens were compared by real-time fluorescence quantitative PCR.It was found that NlCBP1 and NlCBP2 were highly expressed in the salivary glands,and then NlCBP1 and NlCBP2 was detected by in situ hybridization shows they were highly expressed in the salivary gland.NlCBP2was verified by Western blotting that it was a protein secreted into rice by BPH feeding on rice.In order to explore the functions of NlCBP1 and NlCBP2 further,dsNlCBP1 and dsNlCBP2 were used to inject the 3rd instar BPH.After 3 days of injection,the mortality of BPH significantly increased compared with the control,even the mortality of BPH was significantly different from the control after 7-8 days interference.The EPG results showed that the time taken by BPH to feed on the phloem became a significant decrease in the total feeding time.These conclusions may indicate that NlCBP1 and NlCBP2 are the key factors for the BPH feeding on rice.Interfering with NlCBP1 and NlCBP2 may affect the survival and feeding of N.lugens.This article identified the water saliva protein of BPH and made a study for further research on the function of the salivary protein of BPH.It lays the foundation for the future study of the BPH-rice interaction.