Molecular Cloning And Function Analysis Of Development-related Genes NlICP And Nlcdc2in The Rice Brown Planthopper, Nilaparvata Lugens

The rice brown planthopper, Nilaparvata lugens St l, is one of important insectpests in the areas of Aisa. It causes severe damage to rice not only by intensive sapsucking, spawning in sheath of rice but also by transmitting viral disease. Theadaptability, fast growth and the capacity of high reproductive are the difficulty toprevent the population of BPH. The identification of valid new targets for pestcontrol will be great signification in prevention and treatment of N. lugens. Therefore,two development-related genes NlICP and Nlcdc2were screened according to theRNA-seq data of transcriptome. In order to study the importance of NlICP andNlcdc2in the development of the rice brown planthopper, the full-length cDNAs ofNlICP and Nlcdc2were cloned, and the development expression pattern wasanalysed. RNA interference technique was used to study the function of genes. Thisstudy will not only lay a foundation for the deeper research of development ofpesticide but also may serve as a potential target gene for RNAi-mediated controllingN. lugens. The results are reported as below:1. Molecular cloning and function analysis of cuticular protein gene NlICP inthe rice brown planthopperInsect cuticular protein gene, named as NlICP, was chose according to theRNA-seq analysis of transcriptome. Partial cDNA sequences encoding NlICP wereamplified by RT-PCR. Based on the specificity primers, the5’ and3’ cDNA endswere synthesized by Rapid Amplification of cDNA Ends PCR （RACE-PCR）. Thenall cDNA fragments were spliced into a full-length cDNA through DNAMAN.Phylogenetic analysis showed that the deduced amino acid sequence of NlICP wasclosely matched to the ICP of other species.The complete cDNA sequence of NlICP contains a585bp open reading frame andincluded a5’ untranslated region （UTR） of112bp, a3’ UTR of126bp, whichencoding a protein of194amino acid residues with a predicted molecular weight of21.49kDa and theoretical isoelectric point of5.96. A consensus region of R&R consensus: CHIT_BIND_RR₂and CHIT_BIND_RR₁were found in this protein.SignalP3.0analysis showed that the predicted protein contains a length of16aminoacid signal peptide.Quantitative real time RT-PCR experiments were used to evaluate the differentdevelopment of expression patterns of NlICP in N. lugens. The transcripts of NlICPwere detected only in the nymphal stage of N. lugens, and its expression reached thehighest level in the3rd instar nymph but no expression in adults, suggesting that thecoded protein of NlICP belongs to larval cuticular protein.Feeding-based RNAi was used in NlICP of N. lugens, RNAi analysis revealed thatN. lugens nymph fed with dsNlICP was disturbed, and the expression levels of NlICPdecreased by58.8%and45.6%after the continuous feeding for6and8d,respectively, significantly lower than those in the control group （P<0.01）. Somenymphs died because of incomplete molting after RNAi, and the survival ratedecreased by26.7%compared to the control group after continuous feeding for5d.The results suggest that NlICP is associated with N. lugens nymphal ecdysis anddevelopment, and may serve as a potential target gene for controlling N. lugens.2. Molecular cloning and function analysis of cuticular protein gene Nlcdc2inthe rice brown planthopperCell division cycle gene, named it Nlcdc2, according the RNA-seq analysis oftranscriptome. Partial cDNA sequences encoding Nlcdc2were amplified by RT-PCR.The5’ and3’ cDNA ends were synthesized by RACE-PCR, based on the specificityprimers. Then all cDNA fragments were spliced into the full-length cDNA throughDNAMAN. Phylogenetic analysis showed that the deduced amino acid sequence ofNlcdc2（P34cdc2） was closely matched to other species CDK1.The complete cDNA sequence of Nlcdc2contains a909bp ORF and included a5’UTR of182bp, a3’ UTR of124bp with a poly A tail, which encoding a protein of302amino acid residues with a predicted molecular weight of34.70kDa andtheoretical isoelectric point of7.60. Protein kinase sites are the characterized of theprotein: ATP binding GXGXXGXV; catalytic sites DFG and Lys33; P34cdc2kinaseconserved region PSTAIR region; phosphorylation sites Thr14, Tyr16, Thr161andfour tryptophan residues base Try168, Try188, Try228, Try244Quantitative real time RT-PCR experiments were used to evaluate the different development of expression patterns of Nlcdc2in N. lugens. Nlcdc2mRNA weredetected in all stage of N. lugens, and reached the peak in the developmental stage in3,5-year-old brachypterous female adults. So Nlcdc2have an important relationshipwith ovarian development and reproduction.Five instar nymphs was used for RNAi, the mRNA expression of Nlcdc2wassignificant difference with the control after feeding6days. Knocking-down ofNlcdc2disrupted ovarian development, and the development of ovarian was slowerthan the control, with no obvious mature oocyte. Then, the brown planthopper whichfed with dsRNA were placed on rice plant to spawning, and there was a significantdifference compared with the control through counting the number of individualsafter spawning for6days. However, there was no difference compared with thecontrol a few days later. It seemed that the RNAi was a transient interference. Theseresults might suggest that Nlcdc2play a crucial role in the reproductive anddevelopmental of BPH, and may also serve as a potential target gene for controllingbrown planthopper.