CRISPR/Cas9 genome editing system is a genome editing technique derived from an acquired immune mechanism found in bacteria and archaea.Although this system has been become a flourishing genome editing technology these years and successfully applied in many model organisms(such as Drosophila and Bombyx mori)and non-model organisms,it has been rarely applied in important agricultural pests.Chilo suppressalis(Walker)(Lepidoptera:Pyralidae)is a notorious pest of a variety of crops including rice,wheat and sugarcane.The control of C.suppressalis for a long time has been dependent on chemical insecticides,which result in various problems such as insecticide resistance by the pest and high level of insecticide residue in the rice.Therefore,alternatives are urgently anticipated to control this pest.Pheromone binding proteins(PBPs),a sub-class of odorant binding proteins(OBPs),are mainly responsible for detection of sex pheromones and then helping to transport hydrophobic pheromones from external environment to ORs in the dendritic membrane.Therefore,PBPs play an important role in perception of sex pheromone and can be used as a target gene for behavioral regulation of pest.Considering that Lepidoptera insects usually contain multiple PBP genes,is there any functional differentiation between PBPs?Is there any difference in the role of perception of sex pheromone?Although many indirect evidences have been presented,there are rarely functional studies in vivo for direct evidence,due to technical limitations.In present study,the CRISPR/Cas9 system were successfully used in genome editing of two CsupPBPs and the CsupPBPl and CsupPBP3 homozygous mutants were obtained,respectively.With these mutants,EAG responses of PBP 1-mutant males,PBP3-muatant males and wild-type males were measured and compaired.The results demonstrated that PBP1 plays more important roles in the sex pheromone perception in C.suppressalis,and implied that a compensation effect might be existed among PBPs.Our study also provides an important reference for in vivo functional study of olfactory genes by CRISPR/Cas9 system.The main results are as follows:1.Knocking out of CsupPBPl and CsupPBP3 by CRISPR/Cas9 systemAccording to the cDNA sequences of CsupPBP1 and CsupPBP3,target sites on exon2 were selected,respectively,and then sgRNA and Cas9 mRNA were synthetized in vitro.Fertilized eggs were collected within 4h after oviposition and used for microinjection of sgRNA and Cas9 mRNA.Mutationswere detected using gDNAs from injected eggs at 24h after the injection.Based on the relative intensities of the 3 bands on the gel,the indel frequency of CsupPBPl or PBP3 gene was calculated as 21.3%and 19.5%,respectively.To gain detailed information of the indel sequences,TA cloning was operated and 15 clones for each gene were successfully sequenced.As a result,7 different indel mutations were detected in PBP1-sgRNA-injected eggs,while 6 different mutations in PBP3-sgRNA-injected eggs,suggesting that different mutations occurred at both target sites.2.Screening of CsupPBPl and CsupPBP3 homozygous mutantsBy single pair mating stragegy,PBP 1 mutant offsprings with 17 bp deletion and PBP3 mutant offsprings with 7 bp deletion wereobtained,respectively,after 3 generations of screening.Both deletions resulted in premature termination in translations of the PBP gene,leading to a truncated protein in PBP1 and PBP3 of 82 and 69 amino acids length,respectively.Compared with wild type of PBP1 167 amino acids length and PBP3 166 amino acids length,both PBP mutants were highly truncated,and in particular,5 of 6 conserved cysteineswere absent in both PBPs.Therefore,it is believed that the two mutants completely lost the normal function of PBP 1 or PBP3.3.Determination of CsupPBPl or PBP3mutations by electroantennographyassayThe electroantennogram(EAG)responses of heterozygous and homozygous from CsupPBP1 and CsupPBP3-G3 mutants males and wile-type moths to the three sex pheromones(Z11-16:Ald?Z13-18:Ald?Z9-16:Ald)at different dosages(1ng,10ng,100ng,1000ng)were measured.Compared with wild males,homozygous PBP 1-mutant males showed significantly lower EAG responses to all three sex pheromone components at all tested dosages,while PBP3 homozygous mutant males displayed significantly decreased EAG responses at 1 ng,100 ng and 1000 ng dosages for the major component Z11-16:Ald,but only at higher dosages(10-1000ng)for the two minor components.This indicated that CsupPBPl plays more important role than CsupPBPs.In addition,EAG results between PBP1 and PBP3 mutant males were gernally agreeable with the differences in relative expression in male antennae,however,an interaction or compensation effect among PBPs was implied regarding the sex pheromone perception.As for the two heterozygous mutants,their EAG responses to 3 phermone components were also reduced compaired to the wild type males,but the degree of reduction was much smaller than that of homozygous mutants.PBP1 heterozygous mutants showed significant EAG reduction only at 100ng for Z11-16:Ald,at 1000ng for Z13-18:Ald,and showed no significant reduction at any dosages for Z9-16:A1d.Similarly,PBP3 heterozygous mutants showed significant reduction only at the highest dosage for Z11-16:Ald and Z13-18:Ald,and showed no significant reduction at any dosages for Z9-16:Ald. |