Cloning And Functional Analysis Of Ribosomal Protein Genes NlRPL5 And NlRPS8 In The Brown Planthopper

Brown planthopper,N ilaparvata lugens St?l,is one of the most destructive inset pests in the subtropical areas of Asia.N.lugens feeds on rice crop and can cause hopperburn on rice plant,whenever its density is high enough.N.lugens can also transmit the grassy stunt disease,which will further reduce rice yield.Various types of resistant rice varieties have been used to control this insect pest as an effective and environment-friendly strategy for protecting rice.However,several N.lugens strains have recovered their virulence to these resistant rice varieties due to a continuous cultivation of planthopper-resistant varieties for a long time.Therefore,it is important to develop the strategy for controlling N.lugens,including identifying some new genes as targets.In order to screen suitable genes as targets,we previously analyzed the gene expression profile of N.lugens,and found the expression pattern of some ribosome protein-related genes showed significant difference between the Rh colony reared on resistant rice variety Rathu Heenati(RHT)and the Tn colony reared on susceptible variety TN1.In this study,the complete open reading frame(ORF)of NlRPL5 and NlRPS8 was cloned,and the physicochemical properties of the protein sequence were predicted.The RT-qRCR analysis was conducted to detect the relative mRNA expression levels of N lRPL5 and N lRPS8 in different stages and different populations of N.lugens.RNA interference(RN Ai)was performed to explore the effect of NlRPL5 knockdown on N.lugens.Hunger treatment was performed to explore the effect of NlRPS8.The results are reported as below: 1.Molecular cloning,expression and functional analysis of ribosomal proteinrelated gene Nl RPL5 in the rice brown planthopperThis section aimed to explore the function of Nl RPL5(GenBank No.KX379234),a ribosomal protein L5 gene,in the brown planthopper N.lugens.The open reading frame(O RF)of Nl RPL5 was cloned from N.lugens based on a previous transcriptome analysis.The results revealed that the open reading frame of NlRPL5 is 900 bp,encoding 299 amino acid residues.The phylogenetic tree of Nl RPL5 showed that N.lugens has the closest relationship with Laodelphax striatella and Diuraphis noxia among species analyzed.The reverse transcription quantitative PCR(RT-qPCR)results suggested that the expression of Nl RPL5 gene was stronger in gravid females,but was relatively low in nymphs,males and newly emerged females.The expression level of Nl RPL5 in the ovary was about 2 folds of those in the head,thorax,or fat body.The relative expression levels of NlRPL5 in resistant rice varieties ASD7 and RHT were 1.66 and 1.85 times of that in susceptible rice variety TN1.RN Ai of dsNlRPL5 resulted in a significant reduction of mRNA levels,about 50% decrease in comparison with the ds GFP control at day 6.Treatment of ds Nl RPL5 significantly restricted the ovarian development,and decreased the number of eggs laid on the rice plants.This study provided a new clue for further study on the function and regulation mechanism of RPL5 in N.lugens.2.Molecular cloning,expression and functional analysis of ribosomal proteinrelated gene Nl RPS8 in the rice brown planthopperThis part aims to study the expression pattern and function of a ribosomal protein S8 gene,Nl RPS8(GenBank No.KU341337)in the rice brown planthopper,N.lugens.The full length cDN A of Nl RPS8 was amplified from N.lugens by RT-PCR and rapid-amplification of cDNA ends according to the RNA-seq analysis of transcriptome and the gene expression profile information.The results showed that the open reading frame of Nl RPS8 is 627 bp,encoding 208 amion acid residues.The phylogenetic tree of Nl RPS8 showed that N.lugens has the closest relationship with Maconellicoccus hirsutus among species analyzed.The q RT-RCR analysis revealed that the mRN A level in gravid female adults was the highest,and relatively low in male adults,newly eclosed female adult,and nymgps.The qRT-RCR analysis was applied to measure the relative expression levels of NlRPS8 in different tissues(head,thorax,fat body,ovarian,mid gut)of N.Lugens,and the highest expression was in the ovarian.The relative expression levels of Nl RPS8 in resistant rice varieties ASD7 and RHT were 1.99 and 2.14 times of that in susceptible rice variety TN1,respectively.The relative expression levels of Nl RPS8 in the group fed with water was 1.6 times of that fed with TN1 rice.indicating that NlRPS8 probably participated in responding to the starvation stress.In conclusion,this study makes contribute to understanding the function of ribosomal protein-related genes in N.lugens,and it also provided new clues for further study on the adaptation of N.lugens to resistant rice varieties.