Molecular Cloning Of NcPGRP Gene And Its Transcript Level Influenced By RDV Infection In The Green Rice Leafhopper,Nephotettix Cincticeps(Uhler)

The green rice leafhopper,Nephotettix cincticeps?Fabricius??Hemiptera:Cicadellidae?,is one of the most important rice pests in China which cause serious crop damages by feeding directly and vectoring various plant virus,for instance rice dwarf virus?RDV?,serious endangered the safty of rice production and it needs to be controlled by safe control measures.Insect innate immunity is the first line of defense against pathogenic microorganism and peptidoglycan recognition protein is one of the most important recognition protein in innate immunity.As the vector insect of RDV,the interaction effect of RDV and PGRPs of green leafhopper still need to be deeply studied.In this study,peptidoglycan recognition receptopr?PGRP?genes were annotated using the transcriptomic data of the green rice leafhopper,Nephotettix cincticeps?Uhler?,and screened PGRP genes,whose transcript levels influenced by RDV infection.The characteristics of molecular and protein of NcPGRP was studied by molecular cloning and prokaryotic expression.The function of NcPGRP in the immune system of green leafhopper was studied by real-time PCR and RNAi.The main results from this thesis are listed here as follows.1.Annotation and screening of the PGRP genes,of which the transcripts levels were influenced by RDV infection in N.cincticeps.Using a bevy of known insect PGRP genes as reference sequences,12 of PGRPs genes were anotated and they possessed high E-value with the reference sequences,using transcriptomic data from N.cincticeps and BLAST method.Three of PGRP genes,of which the transcripts levels were influnced by RDV infection,were screened from12 of annotated PGRPs,using qPCR approach.2.We cloned the full length cDNA nucleotide sequence encoding the whole open reading frame of NcPGRP.The length of this cDNA was 552 bp,encoding a 19.9^kDa expression product.There is no signal peptide in its deduced amino acid sequence by the prediction in silico.In the amino acid sequence of NcPGRP,there were three Zn²⁺-binding and amidase activity residues and one DAP-type peptidoglycan recognition site.Polygenetic tree analysis results showed that NcPGRP and other species PGRPs did not clustered into one group.NcPGRP could be detected in all tissues including head,gut,fat body,malpighian tubes,ovary,cuticle and testis,but the transcript level was highest in the head.We detected an alternative splicing form of the NcPGRP transcript.The exon 2 of NcPGRP was cleaved after alternative splicing.However,we did not detect the alternative splicing form in every sampling N.cincticeps body.Therefore,it is considered as a random splicing without practical function.3.The prokaryotic expression of NcPGRP and preparation of polyclonal antibody against its expression product.0.1 mM IPTG was able to induce the recombinant expression of NcPGRP in the Rosetta strain of Escherichia coli with His-tag.The recombinant products were in the inclusion bodies of the E.coli cells,and the molecular mass of this recombinant product was 20^kDa.The polyclonal antibody was prepared against NcPGRP.Western blot analysis results showed that the molecular mass of the recombinant proteins was correct,and the expression level of NcPGRP was very low in the N.cincticeps body,which was not able to be detected.4.The qPCR results indicated that the transcript levels of NcPGRP were significantly up-regulated after the injection of E.coli DH 5?and Micrococcus luteus.There was no significant difference in the transcript levels of NcPGRP post immune inductions by these two kinds of bacteria.On the contrary,the transcript levels of NcPGRP were sharply down-regulated in N.cincticeps infected with RDV.The double-strands RNA of dsPGRP was injected into N.cincticeps body between the second and third abdominal segments,since the wounds generated by the needles were smallest and it was able to increase the survival rate of N.cincticeps post injection.The optimum RNA interference efficiency was occurred when the injection dose was10ng of the dsPGRP,with a good interference efficiency until on 4 days after injection.The mortality of green leafhopper injected by pathogenic bacteria after RNA interference.Mortality of green leafhopper of which injected with pathogenic bacteria was detected while the expression of NcPGRP was interferenced and using the mortality of green leafhopper which injected with dsGFP as control.The results indicated that after RNA interferencing of NcPGRP,both of E.coli DH5?and M.luteus can not influence the mortality of green leafhopper,and there was no difference between the lethal ablity of E.coli DH5?and M.luteus.