Study On The Mechanism Of White-backed Planthopper Molting Regulated By MiRNA Targeting Chitinase Gen

Sogatella furcifera is a major rice pest and a common vector of Southern rice black-streaked dwarf virus（SRBSDV）.Currently,control of S.furcifera has primarily been controlled using chemical insecticides;however,their irrational use has led to insecticide resistance and agricultural pollution.Therefore,there is an urgent need to develop new pest control strategies.Micro RNAs（mi RNAs）is a class of endogenous non-coding RNAs with a length of about 22 bp in organisms,which regulate gene expression at the post-transcriptional level and participate in the regulation of various biological process.Chitin is a major component of insect exoskeleton.its synthesis and degradation is vital for the successful molting in insects.It is a good pest control strategy to interfere with chitin metabolism in insects by mi RNA,thus disrupting the normal growth and molting process of insects.Therefore,this study used S.furcifera as the research object,focusing on the biological function of the chitinase gene,and comprehensively used RT-q PCR,RNAi,and dual luciferase reporter systems to reveal the mi R-33-Sf Cht5 and mi R-142-Sf Cht7 molecular mechanism of regulating S.furcifera molting development.The main results are listed as follows:1 Construction of transcriptome and mi RNA libraries and comparative analysis of different eclosion stagesThe transcriptome and mi RNA libraries for high-throughput sequencing were constructed in triplicate using nine samples from three developmental stages:72 h before eclosion of 5^th instar nymphs（L5N）,molting stage（MS）,and early adult（EA）.A total of 17,757 genes were annotated and identified in the transcriptome library,including 15,543 known genes and 2,214 novel genes.Comparative gene expression analysis revealed 1,105,1,373,and 36 genes in L5N vs MS,L5N vs EA,and MS vs EA,respectively.Gene Ontology functional enrichment analysis showed that the differentially expressed genes were primarily enriched in the structural constituent of the cuticle,structural molecular activity,amino sugar metabolic process,and four membrane groups.A total of 253 mi RNAs,including 134 known mi RNAs and 119 novel mi RNAs,were identified in the three different developmental stages of S.furcifera.A total of 12differentially expressed mi RNAs（DEMs）were identified by comparing the expression levels.The corresponding target genes were predicted and found to be mainly enriched in the signal transduction pathway,which was consistent with the characteristics of insect growth and development.The expression patterns of 12 DEMs in different developmental stages and tissues were further analyzed by RT-q PCR to further understand the mi RNAs with potential biological functions.For mi R-2a-2 and mi R-305-5p-1 highly expressed in the 5^th instar nymphs before eclosion and in the integument,injection of mi R-2a-2 and mi R-305-5p-1 mimic/antagomir into 1-d-old 5^thinstar nymphs significantly increased the mortality rate and defective molting phenotype,suggesting that these mi RNAs are involved in S.furcifera nymph–adult transition.2 Identification,expression,and functional analysis of chitinase genes from S.furciferaBased on genomic and transcriptome data of S.furcifera,the full-length c DNA sequences of 10 chitinase genes（Sf Cht2,Sf Cht3,Sf Cht4,Sf Cht5,Sf Cht6-1,Sf Cht6-2,Sf Cht10,Sf IDGF1,Sf IDGF2,and Sf ENGase）were amplified by RT-PCR and RACE-PCR.Combined with the Sf Cht7 and Sf Cht8 genes previously published by the research group,phylogenetic analysis showed 12 chitinase genes of S.furcifera were clustered into 9 groups,and the closest parental relationship was observed with the brown planthopper Cht gene.Spatiotemporal expression patterns analysis revealed that the expression levels of eight genes（Sf Cht3,Sf Cht5,Sf Cht6-1,Sf Cht6-2,Sf Cht7,Sf Cht8,Sf Cht10,and Sf IDGF2）with similar transcript levels peaked prior to molting of each nymph and were highly expressed in the integument,while Sf Cht3,Sf Cht5,Sf Cht7,Sf Cht10,and Sf IDGF2 were highly expressed in adult wings.Sf Cht4 was highly expressed in adult testis,while Sf Cht2,Sf Cht6-2,Sf IDGF1,and Sf ENGase were highly expressed in the ovary of adults.The expression levels of Sf Cht5,Sf Cht6-1,Sf Cht6-2,Sf Cht7,Sf Cht10,and Sf IDGF2 were significantly upregulated with the 20E treatment while the remaining genes exhibited no significant changes.The functional analysis of 10 chitinase genes of S.furcifera was conducted using RNAi.After injecting the ds RNA of Sf Cht2,Sf Cht3,Sf Cht4,Sf Cht5,Sf Cht6-1,Sf Cht6-2,Sf Cht10,Sf IDGF1,Sf IDGF2,and Sf ENGase into the nymphs on the first day of the5^th instar,the expression levels of all target genes decreased significantly compared with the control group.Among them,silencing the expression of Sf Cht5,Sf Cht10,and Sf IDGF2 genes blocked molting and led to high mortality in S.furcifera,and affect the transcription level of key genes in chitin metabolism pathway of S.furcifera.Silencing the expression of Sf Cht2,Sf Cht3,Sf Cht4,Sf Cht6-1,Sf Cht6-2,Sf IDGF1,and Sf ENGase did not affect the normal development into adults and morphology of S.furcifera.3 Identification and spatiotemporal expression pattern analysis of binding sites of Sf Cht5 and Sf Cht7 regulated by mi RNABased on the mi RNA database of S.furcifera,the targeting relationship between mi R-33 and Sf Cht5 and between mi R-142 and Sf Cht7 was predicted using RNAhybrid and mi Randa software.Injecting mi R-33 and mi R-142 mimic into 1-d-old 5^th instar nymphs significantly reduced their corresponding target gene expression than the control,indicating that mi R-33-Cht5 and mi R-142-Cht7 have a negative regulation mode,which was further confirmed by the dual luciferase reporter system.When the dual luciferase reporter vectors of Sf Cht5 and Sf Cht7 were constructed and co-transfected with mi R-33 and mi R-142 mimic,the fluorescence activity decreased significantly compared with the control,indicating that the mi R-33 and Sf Cht5 pair and mi R-142 and Sf Cht7 pair have a targeted regulation relationship and that their regulation region is located in the open reading frame of the sequence.Spatiotemporal expression pattern analysis showed that Sfu-mi R-33 and Sfu-mi R-142 had opposite expression patterns with Sf Cht5 and Sf Cht7,respectively.4 mi RNA-mediated Sf Cht5 and Sf Cht7 gene regulation of key genes of the chitin metabolic pathway involved in moltingOverexpression or inhibition of mi R-33 blocked molting development and resulted in high mortality in S.furcifera.Similarly,overexpression of mi R-142 significantly reduced the survival rate of S.furcifera and molting failure and abnormal wing development were observed.Inhibition of mi R-142 resulted in normal eclosion of nymphs into adults without affecting phenotype and survival rate.In addition,injecting mimic-33 or mimic-142 reduced the chitinase content in S.furcifera.Based on the S.furcifera genome and transcriptome data,four chitin deacetylase genes（Sf CDA1,Sf CDA2,Sf CDA3,and Sf CDA4）and twoβ-N-acetylhexosaminidase genes（Sf NAG1and Sf NAG2）were identified.The spatiotemporal expression characteristics showed that all six genes were highly expressed in the integument.Except for silencing Sf CDA3expression,Sf CDA3 had no significant effect on the phenotype and mortality of the insect.After interfering with the expression of Sf CDA1,Sf CDA2,Sf CDA4,Sf NAG1,and Sf NAG2,the molting development of 5^th instar nymphs was blocked,the survival rate of S.furcifera decreased significantly,and the expression of key genes Sf CHS1 and Sf TRE of the chitin synthesis pathway and of Sf Cht5 and Sf Cht7 decreased significantly.In addition,the overexpression of mi R-33 and mi R-142 or RNAi Sf Cht5 and Sf Cht7significantly downregulated the expression of key genes in the chitin metabolic pathway.Further,treatment with 20E hormone significantly downregulated the expressions of mi R-33 and mi R-142 as well as significantly upregulated the expressions of the corresponding target genes Sf Cht5 and Sf Cht7 and key genes in chitin metabolic pathway.Therefore,mi R-33-Cht5 and mi R-142-Cht7 regulate the transcription levels of key genes in the chitin metabolic pathway to affect the molting development of S.furcifera.In conclusion,this study identified and annotated various transcripts and mi RNAs from three developmental stages of S.furcifera.The functions of Sf Cht5,Sf Cht10,and Sf IDGF2 during molting development in S.furcifera were revealed.Furthermore,the molecular mechanism of mi RNA-mediated regulation of Cht5 and Cht7,key genes involved in the molting development of S.furcifera,was revealed.The results not only enrich the molecular data of the nymph–adult developmental stage of S.furcifera,but also provide a comprehensive understanding of the molecular mechanism of mi RNA-mediated regulation of Sf Cht5 and Sf Cht7 in the molting development of S.furcifera.The results of the present study will be helpful in the subsequent development of insecticide targets.