Molecular Mechanism Of Interspecific Competitive Advantage And Temperature Tolerance Of Liriomyza Trifolii

Liriomyza spp.(Diptera:Agromyzidae)represent a great variety of polyphagous insects and has disseminated globally with rapidly competition and substitution among different closely related species.Liriomyza trifolii,L.sativae and L.huidobrensis are the most insidious leafminers on vegetables grown in China.Although the three closely related species have similar morphological characteristics and overlapping niches,but their competitiveness is different.Previous studies have shown that L.trifolii tends to be more invasive and competitive than its closely related species.There are many factors that affect the competitive substitution of three congener invasive Liriomyza species,and among those the role of temperature tolerance should not be ignored.Relevant experiments have been used to study the species competitiveness and temperature adaptability of Liriomyza spp.,but there is a lack of research on the its molecular mechanism and related gene regulation mechanism.In this study,we first compared the transcriptomes and the Hsps genes of three closely related Liriomyza spp.to explore the molecular evidence of the interspecific competition of those three invasive leafminer flies;Then,the transcriptome of the competitive advantage species-L.trifolii under temperature stress was sequenced to select the thermal tolerance related genes and pathways;At the same time,the key gene family,small heat shock protein genes(sHsps)and heat shock transcription factor gene(Hsf1)under temperature stress were studied;finally,the molecular mechanism of transcription regulation of sHsps(Hsp20.8)was discussed.The main results are as follows:(1)Transcriptomes for three closely related leafininers,L.trifolii,L.sativae and L.huidobrensis,were established by RNA-Seq to investigate competition and displacement mechanisms.Results showed that these three leafminers have distinct highly-expressed gene clusters.Furthermore,genes related to competitive factors showed that L.trifolii and L.sativae share a similar expression pattern that differs from L.huidobrensis.In addition,the comparative study on the gene characteristics and expression patterns of five different Hsp genes showed that both the number of TATA-box-like elements in 5'UTR and the values of Ton and Tmax in the gene expression pattern of L.trifolii showed that it had higher temperature adaptability than L.sativae and L.huidobrensis.(2)In this study,we identified and annotated a number of unigenes(>3000)were expressed in L.trifolii under thermal stress.GO,COG and KEGG pathway enrichment analyses indicated that DEGs related to "post-translational modification,protein turnover,chaperones" and "lipid transport and metabolism" were both enriched after L.trifolii pupae were exposed to heat and cold stress.Genes encoding Hsps and cuticular proteins were modulated in response to heat and cold stress in L.trifolii,respectively.In Top10 differentially expressed gene enrichment analysis,the sHsps is one of the gene families with relatively high expression level and number under high temperature stress.(3)The full-length cloning and expression patterns of sHsps and Hsf1 genes were studied by RACE and qPCR.Five sHsps and one Hsf1 were obtained in L.trifolii.The expression patterns showed that the prepupa and pupa were the highest expression at developmental stages,and the expression of sHsps was significantly induced in L.trifolii pupae under different high and low temperature stress.In adults,Hsf1 expression pattern in L.trifolii generally decreased with rising temperatures,whereas expression of the five sHsps showed an increasing trend that correlated elevated temperatures.Expression of Hsfl and sHsps in L.trifolii increased with the increase of treatment time and exposed to 40? without a recovery period was higher than levels where a recovery period was incorporated after thermal stress.To elucidate potential interactions between Hsf1 and sHsps,double stranded RNA(dsRNA)was synthesized to knockdown Hsf1 in L.trifolii by RNA interference(RNAi).The knockdown of Hsf1 by RNAi decreased the survival rate and gene expression of Hsp19.5,Hsp20.8 and Hsp21.3 under heat stress.(4)The recombinant protein of Hsf1 was constructed by pET28a vector.The recombinant protein was expressed and purified in Escherichia coli.The target protein was also verified by Western blot.Genome Walker method was used to amplify the promoter sequence of Hsp20.8.The JASPAR website was used to analyze and predict the location of HSE elements in Hsp20.8 promoter and primers were designed for electrophoretic mobility shift assay(EMSA)experiment.The results showed that Hsf1 protein could bind to third HSE fragments in the Hsp20.8 promoter.Meanwhile,the transcriptional activity of different regions of Hsp20.8 promoter was analyzed by dual luciferase reporter gene method.The results showed that all four fragments of Hsp20.8 promoter have transcriptional activity,but PGL3-20.8-2+PRL-TK fragment had the highest activity.In addition,the Hsp20.8 promoter sequences of different geographical populations were also amplified and compared.The results showed that neighbour-Joining(NJ)phylogenetic tree of Hsp20.8 promoter could be divided into two branches according to latitude,which was similar to the geographical distribution pattern of sampling points.In addition,the mutation sites of each geographical population were more in Hsp20.8 promoter-2 region and Hsp20.8 promoter-3 region.In general,the more mutation sites in promoter fragments,the higher transcriptional activity of promoter.Therefore,the difference of Hsp20.8 promoter in different geographical populations of L.trifolii may lead to the difference of temperature tolerance among different geographical populations,which may contribute to the differentiation of geographical populations and the continuous expansion northward of L.trifolii.In this study,we compared the transcriptomes and Hsps of three closely related species of Liriomyza spp.,and chose competitive advantage species-L.trifolii as the target species.Temperature as a competitive factor,using transcriptome sequencing analysis and the function and regulation mechanism of important stress tolerance genes(sHsps and Hsf1)to reveal the molecular mechanism of interspecific competition and temperature tolerance of L.trifolii under temperature stress.The results have important theoretical significance for revealing the molecular mechanism of competition and substitution among Liriomyza species and the mechanism of tolerance to temperature stress in the invasion and diffusion of L.trifolii.In addition,based on the analysis of potential differences in competitiveness and temperature stress tolerance of three Liriomyza species,which has important practical significance for the invasion prevention and control of Liriomyza species.