Knockdown Of Midgut Genes By RNAi In The Brown Planthopper

The brown planthopper, Nilaparvata lugens Stal (Hemiptera:Delphacidae), is the most destructive insect pest of rice crops. The brown planthopper damages rice plants by directly sucking the phloem sap and by acting as a vector for the transmission of the rice grassy stunt virus. Although insecticide control of N. lugens has been a convenient option, indiscriminate usage has resulted in resistance, leading to a resurgence of the insect, besides creating serious environment pollution. Hence, genetic improvement of rice host resistance is a preferred alternative. Plant-mediated RNAi is a potential approach for controlling this insect pest of rice.dsRNA-mediated gene-silencing is a conserved mechanism in many eukaryotes,in which Dicer RNase Ⅲ type enzymes bind and digest cytoplasmic dsRNAs into small interfering RNAs(siRNAs),duplexes composed of approximately21to23dsRNA nucleotides.These small RNA cleavage products then function as sequence-specific interfering RNA in transcript turnover, cleavage, and translational control.double-stranded RNA(dsRNA)mediated RNAi has emerged as one of the most powerful strategies for the rapid analysis of gene function,particularly in organisms for which stable transgenesis is not available,such as insects.Plant-mediated RNAi has been reported in lepidopteran and coleopteran plant pests.However, there is a more urgent need to develop this technique for application in insects for which no effective Bt(Bacillus thuringiensis)toxins are known.Insects in this category include phloem-sucking hemipteran pests,such as planthoppers,aphids and whitefly,which are highly destructive agricultural pests worldwide,causing millions of dollars’ worth of yield loss and control costs.In this work, we cloned the sid-1,Argonaute genes and other RNAi genes and verified their expression in N.lugens. SID-1is a multispan transmembrane protein that is expressed on the cell surface, thereby mediating a systemic RNAi effect. RISC-associated Argonaute(Ago) proteins play an essential role in mediating distinct assembly and cleavage steps of the RNA interference catalytic cycle. Pasha could help to orient the pri-miRNA in the Microprocessor, contributing to the specific positioning of the Drosha cleavage site. RISC-loading complex subunit tarbp2is a part of the miRNA processing machines, it is crucial to miRNA loading into RISCs. The full-length cDNA of the sid-1gene is2,119bp long and contains an open reading frame(ORF)of1,875bp(GenBank accession no.JF915743),encoding a protein of624amino acids. The phylogenetic tree of deduced amino acid sequences showed that the N.lugens SID-1protein is most closely related to the A.mellifera SID-1like protein. The full-length cDNA of this N.lugens Argonaute gene is3,446bp long, including a2,085bp ORF. Server identified the presence of typical PAZ and Piwi domains,which are signature motifs of the Argonaute family proteins. The developmental expression pattern revealed that Nlsid-1and Nlaub transcripts were present at all development stages. Nlsid-1and Nlaub were expressed in all six tissues tested. The full-length cDNA of NlRLC is1,673bp long, including a1,008bp ORF. The full-length cDNA of Nlpasha is1,399bp long, including a903bp ORF.We obtained EST sequences from the midgut CDNA libraries of N.lugens, N.lugens EST sequences were highly expressed in midgut, including7ESTs: conting00295、conting2656(Possible gene type:ribosomal protein L8mRNA)、 conting10483(cytochrome b)、conting7979(16S ribosomal RNA)、conting10523(18S ribosomal RNA gene)、conting11875(cytochrome c oxidase subunit Ⅲ)、 conting15241(16S ribosomal RNA). The EST sequences were ligated into L4440vector, and the vectors were transformed into strain HT115. dsRNA were expressed using IPTG.We also isolated genes encoding a hexose transporter(N1HT1),a carboxy-peptidase(Nlcar)and a trypsin-like serine protease(Nltry),which are highly expressed in the midgut of N.lugens. each gene is transcribed in all developmental stages from1st to5th instars, female adults and male adults.The three genes show primary expression in midgut tissues with limited transcription in salivary glands,fat body and head. dsRNA constructs targeting these genes were developed and transformed into rice plants. RNA blot analysis showed that the dsRNAs were transcribed and some of them were processed to siRNAs in the transgenic lines. Ingestion of transgenic rice plants producing NlHT1, Nlcar and Nltry dsRNA was shown to suppress the expression of the three genes in N. lugens. These experiments were repeated several times. Reduction of mRNA expression by40%to70%was observed,demonstrating the effectiveness of delivering dsRNA through the feeding of transgenic plants for the brown planthopper.The gene knockdown technique described here may prove to be a valuable tool for further investigations in N.lugens.The results demonstrate the potential of dsRNA-mediated RNAi for field-level control of planthoppers,but appropriate target genes must be selected when designing the dsRNA-transgenic plants.