| The diamondback moth,Plutella xylostella,is a Crucifer specialist.Glucosinolates are characteristic secondary substances of cruciferous plants and an important stimulant to oviposition of P.xylostella adults,but the molecular mechanism of the glucosinolate perception is unknown.In-depth study on the sensory mechanism of P.xylostella against glucosinolate and identification of its taste tissue and receptor are of great significance for in-depth understanding of the interaction between cruciferous plants and their specialized insects and for the development of new taste-based pest control techniques.In this paper,the oviposition stimulating effect of glucosinolate on female P.xylostella was first confirmed and clarified.Then through the oviposition preference experiment after the ablation of specific tissues,the importance of the foreleg tarsus and antennae in glucosinolate perception was clarified.Finally,according to the results of transcriptome and q PCR assays,the GR7 gene with significantly high expression in the foreleg of female adults was selected,and the possible function of GR7 in glucosinolate perception was explored using CRISPR/Cas9 gene editing technology combined with oviposition preference determination.The main results are as follows:1 The role of foreleg tarsus and antennae in the oviposition preference stimulated by glucosinolate in P.xylostellaThe purpose of this study was to confirm the stimulating effect of glucosinolate on the oviposition of female P.xylostella,and further to clarify the role of the foreleg tarsus and antennae in glucosinolate perception.To determine the stimulating effect of glucosinolate on the oviposition of female P.xylostella,the oviposition preference(indicated by preference index)between glucosinolate(crude extract)treated and control parafilms was calculated using the small cage method with parafilm as oviposition medium.To determine roles of antennae and foreleg tarsus in the glucosinolate perception,the oviposition preference indices of treated female moths(with ablation of foreleg tarsus,antennae,or both antennae and foreleg tarsus)were compared with that of control female moth,using the same small cage method with parafilm and plant leaves as oviposition medium,respectively.Glucosinolate(crude extract)showed significant oviposition-stimulating effect on female P.xylostella,in a dose dependent pattern within the range of test concentration(0.01–5%),with the preference indexes of 0.23,0.41,0.59 and 0.69,respectively.Removal of foreleg tarsus and antennae significantly reduced the oviposition preference for glucosinolate,but there was no significant difference between the two treatments(antenna removal and tarsus removal).When both foreleg tarsus and antennae were removed,the oviposition preference was further significantly reduced,with the preference index decreasing from 0.94 in control moths to0.42 in the tissue removed moths using plant leaves as oviposition medium,and from 0.59 to about zero using parafilm as oviposition medium.Glucosinolate significantly affected the oviposition preference of female P.xylostella,in which foreleg tarsus and antennae played similar important roles.2 Knocking out GR7 gene by CRISPR/Cas9 system and screening the homozygous strainThe transcriptome and q PCR results of GR gene of P.xylostella in the early stage showed that GR7 gene of P.xylostella was highly expressed in the foreleg tarsus of adult female moth,which may play a role in glucosinolate perception.Therefore,the GR7 gene was knocked out by CRISPR/Cas9 gene editing technology to obtain the knocked-out strain and confirm its function in vivo.According to the GR7 genomic DNA sequence of P.xylostella,a suitable target site was selected on the first exon,then sg RNA was designed and synthesized according to the target site sequence,and the fresh eggs within 2 hours after delivery were collected and placed on glass slides.The suitable concentration of sg RNA/Cas9 protein mixture was injected into the eggs by microinjection.After 24 hours,20 genomic DNA were randomly selected for mutation detection.The sequencing peak map showed that the nested peak appeared at the target site,indicating that the target site had a mutation.In order to further determine the insertion or deletion of the mutation,monoclonal sequencing was carried out.Compared with the wild type sequence,it was found that there were 6 common base deletion and insertion mutation types and 6 different mutation sequences.The G0 adults were reared in single-pair strains,and after spawning and hatching,the maternal genomic DNA,was extracted to retain the effective mutant offspring which could lead to protein termination.The G1 generation continued to be reared in single-pair strains,and the genomic DNA of the female parent was extracted after spawning and hatching.Homozygous heterozygous strainswith deletion of 2 bases and 82 bases were obtained in G2 generation,and a large number of GR7 homozygous strains were obtained in G3 generation.Base deletions in both strands of the GR7 gene in this strain resulted in early termination of translation by protein,with only 27 and 60 amino acids(448 amino acids in the wild type)remaining,presumed that normal function has been lost,which is used for subsequent functional analysis.3 Effect of GR7 knockout on oviposition and larva feeding preference for glucosinolate of P.xylostellaIn order to explore the possible role of GR7 gene in the glucosinolate perception of P.xylostella females,the obtained GR7 gene knockout strains were used to further determine the oviposition and feeding preference of wild and GR7 knockout strains to glucosinolate(crude extract).The results showed that there was no significant difference between the two strains in oviposition selection index,among which the selection indexes of wild strains at24 h and 48 h were 0.58 and 0.45 respectively,and those of knockout strains were 0.58 and0.43 respectively.In addition,the results of determining the feeding selectivity of larvae showed that there was no significant difference in the selection rate of artificial feed added with crude extract of glucosinolate between the two strains 5 h after inoculation,among which the selection rate of wild strain was 0.71 and that of knockout strain was 0.69.Based on the above results,GR7 is not involved in the feeling of glucosinolate in P.xylostella,and other GR genes with high expression in the foreleg of female moth need to be further studied. |
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