Characterization And Functional Analysis Of The Genes Involved In Chitin Synthesis Pathway In The White-backed Planthopper,Sogatella Furcifera(Horváth)(Hemiptera:Delphacidae)

The white-backed planthopper,Sogatella furcifera(Horváth)(Hemiptera:Delphacidae),is a serious insect pest that affects rice crops in some Asia-Pacific countries.Until now,the use of insecticides has still being the primary strategy employed to control S.furcifera.However,due to the specific characteristics,such as long-distance migration,short development period and high fecundity,as well various objective factors including the unreasonable spraying chemical insecticides,S.furcifera has developed different level of resistance to various insecticides.Therefore,it is an urgent need to develop a novel pest control strategy to achieve the goal of continuous control of the pest.In insects,chitin has been verified as a crucial structural constituent of the cuticle,alimentary canal,tracheal system and genital ducts.Chitin biosynthesis,catalyzed by a series of enzymes,is a complex biochemical and physiological process.Since chitin is absent in higher mammals,the genes involved in chitin biosynthesis pathway are considered as potential targets for screening and designing new biological pesticides.Based on the results of transcriptome sequencing of S.furcifera,the molecular characteristics and functions of the chitin synthesis pathway gene were systematically studied.The main results are as follows:1 The transcriptome sequencing and unigene functional annotation of S.furciferaThe transcriptome of S.furcifera was sequenced on Illumina Hi Seqsequencing platform,and about 6.68 Gb bases are generated in total.After assembling all samples together and filtering the abundance,a total of 66,425 unigenes were obtained.And then by aligning with 7 functional databases,25,059 Unigenes were annotated by each of the 7 functional databases.By searching in the nonredundant(NR)NCBI protein database using Blastx,and 19,564 unigenes were annotated successfully.In additon,there were 3,915 unigenes successfully annotated in GO,14,850 in KOG,and 14,854 in KEGG.2 Molecular cloning and sequence analyses of chitin biosynthesis pathway genes of S.furciferaOn the basis of S.furcifera transcriptome data,The cDNA sequences of chitin synthesis pathway genes were cloned by using PCR and RACE,and deposited in Gen Bank under the following accession numbers:SfTre1(MG869613),SfTre2(MG869614),SfHK(MF964940),SfG6PI(KY695112),SfGFAT(MF964939),SfGNA(MH672691),SfPAGM(KY381946),SfUAP(MF964941),SfCHS1a(KY350143),and SfCHS1b(KY350144).The open reading frames(ORFs),conserved domains,amino acid structure,and transmembrane regions were predicted and annotated by using relevant molecular bioinformatic softwares,such as SeqMan,MEGA,NetNGlyc,Prot Param and TMHMM,and their classification status were clarified as well.3 Temporal and spatial expression patterns of chitin biosynthesis pathway genes of S.furciferaS.furcifera at every stage from egg to adult were sampled to determine expression profiles in developmental stage by quantitative real-time PCR(qPCR).The results revealed that these genes were constitutively expressed at 18 examining time points in the developmental stages.Specifically,SfTre1,SfG6PI,SfGFAT,SfPAGM,SfUAP and SfCHS1 highly expressed after molting,lowly during the inter-molting phases and then elevated again before the next molt,wich indicated these time points were associated with the requirement of chitin.For SfTre2,its expression levels were relatively high in egg and first-instar nymph,and then gradually decreased from second-instar to fifth-instar nymphs.The expression pattern of SfHK showed a trend of first increasing and then decreasing from egg to fifth-instar nymph,and then slightly increasing,a minor peak appeared,in 2 days after eclosion.The different expressions of chitin synthesis pathway genes imply that they may play multiple physiological functions during the development of S.furcifera.Five different tissue samples,integument,fat body,gut,ovary and head,were dissected from first-day fifth-instar nymphs and third-day adults to examine tissue-specific expression.The expression of all these genes were detected in five tissues,but the expression patterns of each gene were different.It is worthy note that The expression level of SfTre1 was high in the integument,ovary and head,whereas the expression of SfTre2 was high in the ovary and head,which suggested that two genes are involved not only in the synthesis of chitin but also in energy metabolism.Furthermore,SfG6PI,SfGFAT,SfPAGM,SfUAP,SfCHS1 and SfCHS1a were also highly expressed in the integument,indicated these genes probably play important roles in insect growth and development.4 Functional verification of chitin biosynthesis pathway important genes of S.furciferaRNAi was applied to explore the functions of these genes in molting and metamorphosis of S.furcifera.Sequence-specific ds RNAs for SfTre(SfTre1 and SfTre2),SfG6PI,SfGFAT,SfPAGM,SfUAP,and SfCHS1(SfCHS1a and SfCHS1b)were prepared in vitro and injected into first-day fifth-instar nymphs.The results indicated that the expression levels of corresponding target genes were markedly down-regulated.Moreover,RNAi lead to abnormality of phenotype and high lethality.The malformed phenotypes observed were as follows:dual cuticles,the old cuticle produced underneath the new one,covered whole body and old cuticle only slightly splitted on the head and thorax;old cuticle could not be completely detached from the body and form cuticle tail;shrunken abdomen that was smaller than that of normal insects;nymphs molted successfully but the new cuticle crimpled and the wings malformed.The results suggested that these genes are indispensable for the growth and development of S.furcifera.5 Sublethal effects of buprofezin on development,reproduction,and chitin synthase 1 gene(SfCHS1)expression in S.furciferaRice stem dipping method was used for testing toxicity of buprofezin to 3th instar nymph of S.furcifera.The results showed that the LC50 of buprofezin to 3rd-instar nymphs were 0.89 mg L^-1.When 3rd-instar nymphs were exposed to the LC₁₀ and LC₂₅(0.10 and 0.28 mg/L)of buprofezin,the duration of juvenile development was significantly prolonged in the F₀ and F₁generations.The fecundity(eggs per female)of the F₁ females was reduced by 5.29%and 12.34%,respectively.Though there were no significant diferences,the decreasing of survival rate,emergence rate,copulation rate,and hatchability were still clearly observed in the treatments with buprofezin at LC₁₀ and LC₂₅,compared with the control group.The relative fitness of S.furcifera in LC₁₀ and LC₂₅ treatments was reduced by 47%and 63%,respectively.Furthermore,the expressions of SfCHS1 and its two variants in the nymphs of S.furcifera exprosed to buprofezin increased compared to the control.Increased expression of these genes m RNA may result from compensation response of the SfCHS1,SfCHS1a,and SfCHS1b gene at the transcritional level that is caused by the retarded chitin synthesis.The results indicated that the influence of buprofezin on chitin synthesis pathway may be the main mechanism for its inhibition of population growth of S.furcifera.In summary,eight cDNAs of genes involved in chitin biosynthesis pathway were cloned on the basis of the S.furcifera transcriptome sequencing and analysis,and the characterization and function of these genes was detailedly analyzed.The qPCR was employed to analyse the expression pattern of these genes at different developmental stages and in different tissues.Furthermore,RNA interference(RNAi)was performed to verify the functions of six important genes.Additionally,bioassay technology was utilized to illuminate the effects of buprofezin on the development and reproduction of S.furcifera,but also evaluate the effects of buprofezin on expression profiles of SfCHS1.Those results obtained in this study would be beneficial to promote and evaluate the potential of chitin synthesis pathway as pest control targets,and provide theoretical foundation for facilitating the research and developement of green biopesticides.