Effects Of Serine Protease Gene Cluster Knockout On Global Gene Expression And Fitness Costs In Helicoverpa Armigera

Serine proteases(SPs)are a large group of proteolytic enzymes,with important roles in a variety of physiological processes,such as cell signalling,defense and development.SPs contain trypsin,chymotrypsin,thrombin and elastase.Enzymatically active SPs feature a high specificity catalytic triad in their catalytic domain,composed of histidine(His),aspartic acid(Asp)and serine(Ser).Primary protein-digestion in Lepidopteran larvae relies on SPs.Helicoverpa armigera(Hübner),the cotton bollworm,is one of the most significant Lepidoptera pests of agriculture.SPs such as trypsin and chymotrypsin dominate the larval gut environment and contribute to about 95%of the total digestive activity in H.armigera.SPs are very important to the host adaption,pathogen defense and development of H.armigera.Previous studies have indicated that H.armigera can regulated the expression of SPs to avoid the damage from foreign protein such as plant protease inhibitors and Bt toxin.CRISPR/Cas9 system now has become a precise and highly efficient technology to modify or manipulate genome.In this study,the CRISPR/Cas9 based reverse genetics approach were used to identify the function of SPs in global gene expression and fitness costs in H.armigera.Based on the H.armigera genome data,133 SPs genes have been indentified and the phylogeny and structure of these genes were analysed;then two SPs gene clusters were knocked out on chromosome 5 and 18 respectively using CRISPR/Cas9 and homozygous strains were obtained;RNA sequencing was conducted to detect the effects to global gene expression induced by two gene clusters knockout;in addition,the sensibility of gene cluater knockout strains to Bt toxin was detected;finally,life tables on artificial diet and different host plants of gene cluater knockout strains were established.Our study provided a framework of information about SPs and a foundation for future attempts to elucidate the function of these proteases in H.armigera.These results also demonstrated the utility of CRISPR/Cas9 for Lepidoptera in large genomic fragment deletions.The presentation of gene cluater knockout strains faced with environmental stress such as Bt toxin and plant protease inhibitors might provide new idea for pest control.1.Identification and analysis of SPs in H.armigeraAccording to the characteristics of SPs,133 SPs genes were identified including 54 trypsin genes,34 chymotrypsin genes and 45 serine protease homolog genes in H.armigera based on genome data.These genes distribute on 23 chromosomes unevenly.Among these chromosomes,chromosome 5 has the largest trypsin gene cluster and chromosome 18 has the largest chymotrypsin gene cluster.12 of the 133 SPs have one or more special domains indicated these SPs might participate in complex physiological and biochemical process.Results above demonstrated the SPs in H.armigera is surprisingly large and diverse.This framework of genomic information about SPs sequences revealed some interesting evolutionary features and could point to directions for future experimental studies on SPs genes/proteins in H.armigera and other invertebrate species.2.Two strains homozygous for deletion of two SPs gene clustersChromosome 5 contains 29 trypsin-like genes and 28 genes of them form the largest trypsin cluster,chromosome 18 contains 37 chymotrypsin-like genes and 26 genes of them form the largest chymotrypsin cluster.So we chose these two clusters as target.Finally a 68 kb genomic fragment was deleted covering 18 trypsin-like genes on chromosome 5 through CRISPR/Cas9 system and this homozygous strain was called Tryp-KO.In the same way,another 105 kb genomic fragment was deleted covering 26 chymotrypsin-like genes on chromosome 18 and this strain was called Chym-KO.Our results demonstrated the possibility of large genomic fragment deletions in Lepidoptera using CRISPR/Cas9 system.3.Effects of two gene clusters knockout on global gene expressionTo explore the effects of gene clusters deletion to the expression of global genes,RNA sequencing was conducted.In general,transcriptome analysis revealed 1492 upregulated and 461 downregulated DEGs in Tryp-KO strain and 993 upregulated and 1349 downregulated DEGs in Chym-KO strain compared to SCD.Then we did GO and KEGG enrichment analysis of DEGs.Briefly,for Tryp-KO strain,the dominant DEGs were related to the response to stimulus and were all upregulated,it meant the trypsin gene cluster knockout brought some pressures to this strain but it gave a positive response to adapt to the oppression.;for Chym-KO strain,the DEGs of "ribosome" and its related category were dominant in both GO and KEGG enrichment analysis and were downregulated almost which might influence the development and fecundity of this strain.We used the GO and KEGG annotation to screen DEGs related to SPs.The results showed 47 DEGs related to SPs in Tryp-KO strain including 35 upregulated genes and 12 downregulated genes,and 38 DEGs related to SPs in Chym-KO strain including 26 upregulated genes and 12 downregulated genes.The results demonstrated the possible genetic compensation induced by CRISPR/Cas9 knockout.4.Effects of two gene clusters knockout on sensibility to Bt toxinThe sensibility from larvae of knockout strains to CrylAc,Cry2Aa and Vip3Aa protoxin and Cry1Ac toxin was detected.The result showed the sensibility of Chym-KO to Cry1Ac protoxin increased 13 fold compared to SCD,and the sensibility to other three Bt toxins kept unchanged.The sensibility of Tryp-KO to these four Bt toxins did not change compared to SCD.Then we tested the proteolytic activity of midgut juice from larvae of knockout strains and SCD.For total enzyme activity,no significant difference was detected between these two strains compared with SCD,but the total enzyme activity of Tryp-KO increased slightly compared with Chym-KO.The trypsin and chymotrypsin enzyme activity of Tryp-KO strain kept unchanged compared to SCD.Chym-KO strain displayed significant decreasing chymotrypsin enzyme activity and the trypsin enzyme activity of this strain remained the same compared to SCD.Then we incubated midgut protease from larvae of these strains with Cry1Ac,Cry2Aa and Cry2Ab protoxin respectively.The bands of SDS-PAGE showed there was no difference of activation and degration to protoxin between these strains.We speculated that the knockout of trypsin gene cluster induced genetic compensation because of the large number of trypsin genes,but the knockout of chymotrypsin gene cluster did not have genetic compensation because the majority of the chymotrypsin genes have been deleted.We also suggested that there were one or more proteases deleted in this chymotrypsin gene cluster may have the ability to precipitate Cry1Ac protoxin,so knockout of such proteases caused the increased sensibility of Chym-KO to Cry1Ac pro toxin.5.Effects of two gene clusters knockout on vitalityTo detect the effects to vitality induced by two gene clusters knockout,the life tables on artificial diet and three host plants for knockout strains and SCD were established.In general,the vitality of Tryp-KO strain was comparable to SCD on artificial diet,but the vitality of Chym-KO strain was significantly reduced.On different host plants,the vitality of two gene cluster knockout strains was lower than SCD.Tryp-KO strain lived better on cotton than Chym-KO strain,but worse on corn than Chym-KO strain.Both Tryp-KO and Chym-KO strain could not have next generation on soybean meanwhile SCD could reproduce as usual.Above results indicated that the knockout of SPs gene cluster caused survival pressure to H.armigera especially to Chym-KO strain.Our results also provided a new idea of combining CRISPR/Cas9 system and protease gene clusters in pest control strategy.