Expression Profiles Of Chemoreceptor Genes From Three Major Families And Functional Analysis Of PxylGR1 In Plutella Xylostella

Plutella xylostella is a specialised insect that feeds on cruciferous plants with great harmfulness and strong drug resistance.Glucosinolate（GS）is a characteristic secondary metabolite of cruciferous plants,but also an important insect defense material.A special sulfate esterase in the digestive tract of P.xylostella larvae can detoxicate GS and protect them from its harmful effects.Meanwhile,GS can also be used as an important taste signal to select cruciferous plants for larval feeding or adult oviposition.However,the molecular mechanism of the perception of GS by P.xylostella is still unclear.Gustatory receptor（GR）plays a central role in taste perception.In this study,we analyzed the spatiotemporal expression characteristics of three major chemoreceptor genes of P.xylostella,and attempted to use CRISPR/Cas9 gene editing technology to analyze the function of GR gene,so as to provide candidate genes and method references for further elucidates the molecular mechanism of the taste of GS and other compounds in P.xylostella.In this study,transcriptome sequencing and analysis were performed on the head of larvae,male and female antennae and foreleg of adult,respectively.Temporal and spatial expression dynamics of three major chemoreceptor genes（GR,OR and IR）were obtained.Then,the expression profiles of 62 GR genes in larvae head and more tissues of male and female adults were detected by semi-quantitative PCR.Finally,GR1 gene with significantly higher expression in female antennae and forefeet than that in males was selected and its function was analyzed using CRISPR/Cas9 gene editing technology.The main results are as follows.1.Transcriptome analysis and GR gene expression profile analysis of P.xylostellaIn addition to GR,which mainly plays a role in taste,there are OR and IR receptors.OR mainly plays a role in the sense of smell,while IR plays a role in taste,smell,temperature and humidity and other senses.The time,tissue and sex specificity of these genes may provide important information for further functional studies.A total of 48 OR,8IR and 15 GR were found to be expressed in different insect states,tissues and genders,showing different expression characteristics The expression levels of the three receptor genes were generally high in the antennae,among which 27,38 and 37 OR genes were expressed in the the larval heads,the antennae and foreleg of adult.Especially,the expression levels of co-receptor Orco（OR2）and three sex pheromone receptors（OR1,3,6）in male antennae were the highest,and the expression levels of co-receptor Orco in female antennae were also the highest.Five pheromone receptor genes（OR1,3,5,6,7）were highly expressed in males,and six OR genes（OR18,22,25,38,49,59）were highly expressed in female antennae.Among the 8 IR genes,3（IR8a,25a,76b）belong to co-receptors,and IR25a has the highest expression level in all tissues.In addition,the expression level of IR76b in antennae and leg and IR8a in antennae is significantly higher.10,11 and 15 GR genes were expressed in in the the larval heads,the antennae and foreleg of adult,and the sugar receptor gene GR37 was highly expressed in females in both antennae and foreleg.2.Tissue expression profile of GR gene based on RT-PCR analysisIn view of the fact that glucosinolates are non-volatile and are detected by GR,we further studied the GR gene of P.xylostella.By integrating genomic and transcriptome data,62 GR genes were identified,48 of which had complete open reading frames.The phylogenetic tree analysis showed that 3 of the 48 full-length GRs belonged to CO₂receptors,10 belonged to sugar receptors and 35 belonged to bitter receptors.In addition,the expression specificity of 62 GR genes in the larvae head and more tissues（antennae,head,leg,wing and female oviposition）of male and female adults was determined by semi-quantitative PCR.The results showed that 21 of the 62 GR genes were expressed in larval head,19,22,27 and 21 in adult head,antennae,leg and wings,and 24 in female ovipositor,respectively.GR2,20,and 61 were highly expressed in the head of female moths,GR5,23,34,and 37 were highly expressed in the antennae of female moths,GR37,53,and 54 were highly expressed in the feet of female moths,and GR17,21,and 22 were highly expressed in the wings of male moths.3.Knockout of GR1 by CRISPR/Cas9 technique and strain screeningGS stimulated both the feeding of P.xylostella larvae and the oviposition of adult.In order to find the GR that could detect glucosinolates,transcriptome analysis and existing quantitative results showed that GR1 expression level was high in each tissue,and there was a significant difference between male and female expression level.In order to explore its possible function in taste sensing hosts,GR1 was knocked out as a candidate receptor gene.After verifying the of GR1 sequence,an effective target site was selected on the second exon of GR1 according to the corresponding principle of sgRNA target site design.sgRNA was designed and synthesized according to the target site.The proportionally mixed sgRNA and Cas9 proteins were injected into the fresh fertilized eggs within 2 hours by microinjection system.24 hours later,20 fertilized eggs were randomly selected,and the genomic DNA was extracted and mutated.The results of monoclonal sequencing showed that many types of mutations were found at the target site.The individuals of P.xylostella were paired and a homozygous strain of of GR1 with a deletion of 7 bases at the target site was obtained in G4 generation.The mutation caused the premature termination of GR1translation,only 52 amino acids were retained（454 of the wild type）,and the original transmembrane domain was completely lost（6 of the wild type）,so the function of the gene was presumed to be lost.The strain was used for subsequent behavior determination.4.Determination of oviposition and feeding behavior of GR1 mutant homozygous strain to crude extract of sinapine.Previous studies have found that glucosinolate are signal substances for host localization of P.xylostella,which stimulate oviposition and feeding.The primary extract of glucosinolate was obtained by using radish seeds as raw materials.The behavioral response of GR1 mutant homozygous strain to the initial extract of sinapidin was determined by spawning selection test and feeding selection test.Oviposition selection experiments showed that the GR1 mutant homozygous strain still maintained the same preference as the wild strain,and the oviposition selection index after 24 h and 48 h had no significant difference with the wild strain.The results of larval feeding selection experiment showed that there was no significant difference in larval feeding selection ratio between GR1 mutant homozygous strain and wild strain at 0.5 h,1 h,3 h,5 h,7 h,9 h and 24 h.This suggests that GR1 is not a taste receptor for glucosinolate.