The Functional Study Of Salivary Protein Gene NlSRP In The Brown Plant Hopper Nilaparavata Lugens(St?l)

Rice(Oryza sativa L.)is one of the most important food crops in the world.The brown planthopper(Nilaparvata lugens St?l)is an essential kind of pest on rice.Jiangxi Province is a necessary migration of brown planthoppers from north to south,and the damage caused by brown planthoppers is common and serious.At present,the control of brown planthoppers in China is mainly based on chemical pesticides.The long-term and large-scale use of pesticides not only increases production costs,damages the ecological environment of farmland,but also develops pests’ resistance to pesticides.Therefore,it is particularly important and urgent to research and explore accurate,environment-green,and sustainable methods and technologies for controlling brown planthopper.RNA interference technology is currently one of the potential technologies that can be applied to green pest control.Salivary proteins secreted by sucking-piercing mouthparts are crucial for their feeding and reproduction.Therefore,salivary proteins are one of the important targets of RNAi for pest control.In previous studies,full-length transcriptome and RNA-seq sequencing were performed on the heads of brown planthoppers(mainly including salivary glands)that respectively feed on susceptible and resistant rice.Through analysis of the expression levels,gene annotation,and sequence characteristics of differentially expressed genes,six candidate salivary protein genes were selected.Through RNAi microinjection experiments,one of the genes annotated as uncharacterised protein(we named it NlSRP,N.lugens serine-rich protein)was found.It is significantly reduced in the survival rate of brown planthopper after inhibition of expression.However,the function of NlSRP in brown planthopper is unknown and has not been reported in the literature.Therefore,this study conducted a preliminary study on its function in brown planthopper salivary protein NlSRP.The main research results are as follows:(1)We obtained 315 bp full-length cDNA sequence of NlSRP by RACE technology.In the NCBI database,using the amino acid sequence of NlSRP for Blast P comparison,it was found that the homologous protein RZF33770.1 was only found in the small brown planthopper(Laodelphax striatellus),with a functional annotation of "hypothetical protein".It is speculated that the gene NlSRP may only exist in the Delphacidae family.Analysis showed that the amino acid sequence of NlSRP is rich in serine(25.0%)and short repeats(-MGSA-),without transmembrane domains,and contained signal peptides.Expression pattern analysis reveal that NlSRP was highly expressed in salivary gland tissue.(2)We validated the secretability of NlSRP protein.We acquired the saliva protein NlSRP from brown planthopper by prokaryotic expression,purified to obtain protein and prepared antibodies against NlSRP.Further,through Western blot experiments we did,NlSRP protein is detected in both the total saliva protein secreted by the brown planthopper and the total protein in the rice leaf sheath tissue fed by the brown planthopper,indicating that NlSRP protein can enter the rice leaf sheath tissue with the feeding and secretion of the brown planthopper.(3)We have preliminarily verified that the NlSRP gene can affect the survival of brown planthoppers.We used the nucleotide of NlSRP to perform sequence alignment in the NCBI database.The blastn alignment result found that the nucleotide sequence of NlSRP was more than 97% similar to the nucleotide sequence of the annotated salivary sheath protein,suggesting that its function may be related to the salivary sheath formation and feeding behavior of the brown planthopper.After inhibiting the expression of the NlSRP gene through dsRNA microinjection technology,the survival rate,insect weight gain,and honeydew excretion of the brown planthoppers are significantly reduced,and the number of salivary sheaths and the lengths of salivary sheathes secreted by the brown planthoppers are also significantly reduced.It is so concluded that that NlSRP may have an important role in feeding and survival of the brown planthopper by influencing the formation of the salivary sheaths.(4)We performed identification experiments on the T2 generation of NlSRP-RNAi transgenic rice constructed in the laboratory through PCR amplification technology.It is laying a foundation for further research on the function of NlSRP in the interaction between brown planthoppers and rice.At same time,it is exploring the application of transgenic plant-mediated RNA interference technology in the green control of brown planthoppers.This study identified and cloned a serine rich protein NlSRP secreted by the brown planthoppers,which plays an important role in the formation,feeding,and survival of the salivary sheath of the brown planthopper.The results of this study can provide new targets and experimental references for green prevention and control of brown planthoppers.