Functional Study Of General Odorant Binding Protein 2 Gene In Spodoptera Litura

Spodoptera litura is a notorious agricultural pest in the world.Long-term chemical control of S.litura results in many problems such as pesticide resistance,environmental pollution and pesticide residues in agricultural products,so it is urgent to study green and efficient control measures.General odorant binding proteins(GOBPs)and pheromone binding proteins(PBPs)are two subclasses of Lepidoptera odorant binding proteins(OBPs).For a long time,the former is thought to be mainly involved in the perception of plant volatiles in insects,while the latter is important in the perception of sex pheromones.However,more and more studies have found that GOBPs also have strong binding ability to sex pheromone components,and the genes are localized in sex pheromone-sensitive sensilla,indicating that it may be involved in the perception of sex pheromone components.However,there is a lack of in vivo evidence due to technical limitations.CRISPR/Cas9 system provides an opportunity to obtain direct evidence of the function.In this study,CRISPR/Cas9 system were successfully used in genome editing of SlitGOBP2,and the homozygous mutant strain was obtained.With the mutants,the feeding selectivity of larvae in the presence of sex pheromone components,electroantennography(EAG)responses to sex pheromone components and plant volatiles and behavioral responses were determined.It is confirmed that SlitGOBP2 has dual functions in the perception of sex pheromones and plant volatiles.The main results are as follows:1.Knocking out SlitGOBP2 gene by CRISPR/Cas9 systemAccording to the genomic DNA sequence of SlitGOBP2,the target sites were selected on the first exon,and then sgRNA were designed and synthesized in vitro.The mixed sgRNA and Cas9 protein was injected into fertilized eggs within 2 h after oviposition.At 24 h after the injection,20 injected eggs were randomly selected,and the genomic DNA was extracted for mutation detection.The enzyme digestion results showed that GOBP2 gene was mutated.Further TA cloning detection showed that there were 12 different genome types in 20 positive clones.With the confirmation of the mutations,the remaining injected eggs were reared into adluts,which were mated with wild-type adults in two groups(mutated ♀ × wild type ♂;mutated ♂ × wild type ♀).The larvae(generation G1)gDNA and exuviates of the final instar G1 larvae were extracted to check the genotypes,and the adults with the same mutated genotype were in-crossed.The genotypes of G2 adults were checked using the same method,and the same in-crossing method was used.Finally,a genotype of 4 bp deletion(GAGG)and 5 bp insertion(ACCAC)were obtained in generation G3.The mutation resulted in premature termination in translations of the GOBP2 gene,leading to function lost,providing an important foundation for follow-up functional analysis.2.The perception of pheromone components in S.litura larvae and the role of GOBP2Previous studies have found that the sex pheromone components of some moths can attract larvae to better food.In S.litura,however,PBPs are not expressed in larvae but GOBP2 gene is expressed in the 1-4th instars of larvae.In order to explore the possible role of GOBP2 gene in the sex pheromone perception of S.litura larvae,the 1-4th instar larvae were selected and artificial diet was added with different doses(2-15 ng)of the main component of sex pheromones(Z9,E11-14:Ac).The effects of sex pheromone component on larval feeding selection and the differences between wild type and GOBP2 knockout strains were determined.The results showed that when added with Z9.E11-14:Ac at moderate doses(5 and 10 ng).the diet attracted significantly more wild-type larvae for feeding,but had no effect on larvae of GOBP2 knockout larvae.Therefore.GOBP2 is involved in the perception of sex pheromone components in larvae.3.Involvement of GOBP2 in electrophysiological responses in adult S.lituraSex pheromone communication is an important prerequisite for mating and reproduction between moths.In order to confirm the role of GOBP2 gene in male sex pheromone perception,EAG responses of homozygous SlitGOBP2 mutations and wild-type 3-day-old moths to sex pheromone components and analogues,single plant odor and cabbage leaves were measured.Compared with wild males,the responses of GOBP2 mutant males were lower to 500 ng Z9,E11-14:Ac.Z9,E12-14:Ac,Z9-14:Ac and Z9-14:OH,while GOBP2 mutant males showed no significantly different to 5000ng farnesol,2-pentadecanone and linoleic acid.The responses of GOBP2 mutant males to high-dose cabbage leaves were significantly lower than those of wild males.Compared with wild females,the responses of GOBP2 mutant females were lower to Z9,E12-14:Ac,Z9,E11-14:Ac,Z9-14:Ac,and the responses to Z9-14:OH was significantly decreased.The responses to farnesol,2-pentadecanone and linoleic acid were significantly decreased,and the responses to different doses of cabbage leaves were significantly decreased.According to the results,GOBP2 gene may be involved in the perception of sex pheromone components and plant volatiles of S.litura.4.Involvement of GOBP2 in behiavioral responses in adult S.lituraThe electrophysiological study confirmed that GOBP2 has the dual function in perception of plant odors and sex pheromones,and further behavioral tests were carried out.The Y-tube olfactometer and direct stimulation method were used to determine the response of males to sexual pheromone component Z9,E11-14:Ac,and the oviposition assay was conducted to determine the ovipostion preference to the bean plants by female moths.The results of Y-tube olfactometer showed that there was a significant decrease in the number of pheromone selection,hairpencil displaying and mating attempt of GOBP2 mutant males to 1000 ng Z9,E11-14:Ac compared with the wild males.The results of direct stimulation assay showed that there was also a significant decrease in the number of moths showing hairpencial displaying in GOBP2 mutant males than the wild type males to 50,500 and 1000 ng Z9,E11-14:Ac.The results of oviposition experiment for females showed that the number of eggs laid by mutant females on the leaves of host plants was significantly lower than that of wild females.According to behavioral experiments,it is confirmed that GOBP2 plays an important role in perception of sex pheromone components and plant volatiles of S.litura.