Study On The Function Of Wnt1 Gene In Two Important Lepidopteran Pests

The Wnt signaling pathway is highly conserved in the process of biological evolution.It is involved in regulating embryonic development,balance of energy metabolism,tissue stability and stem cell maintenance and other biological processes.It is one of the current research hotspots.Some progress has been made in the research of Wnt signaling pathway in insects,among which the research on Drosophila melanogaster is the most thorough.In Drosophila,the developmental process of segmengtation,appendages,pigmentation production and wings are controlled by Wnt signalling.At present,there is no report about the study of Wnt signaling pathway in the embryonic development of Spodoptera frugiperda and Helicoverpa armigera.Thus,the gene editing technology CRISPR/Cas9 system was used to edit the Wnt1 of two pests to explore the physiological functions of the Wnt signaling pathway in the embryonic development of the two pests in our experiment.The main research results are as follows:(1)Bioinformatics analysis of Wnt1 gene:The open reading frame sequence of Spodoptera frugiperda Wnt1 gene is 1179bp in length,contains 3 introns and 4 exons,and encodes 392 amino acids.The predicted molecular weight and isoelectric point are 44.035KD and 9.39,respectively.According to the analysis of amino acid sequence homology and the phylogenetic tree,the genetic relationship between Spodoptera frugiperda and Spodoptera litura Wntl is the closest,followed by the Helicoverpa armigera,and the amino acid sequence homology is as high as 99.5%and 97.4%,respectively.The open reading frame sequence of Helicoverpa armigera Wntl gene is also 1179bp in length,contains 3 introns and 4 exons,and encodes 392 amino acids.The predicted quantum and isoelectric point are 44.057KD and 9.39,respectively.The genetic relationship of the Wnt1 gene between Helicoverpa armigera and Spodoptera litura is the closest,and the amino acid sequence homology is as high as 97.7%.The Wnt1 genes of Spodoptera frugiperda and Helicoverpa armigera both contain 26 cysteines.(2)Analysis of Wnt1 gene expression profile of Spodoptera frugiperda:The expression profile of Wnt1 gene of Spodoptera frugiperda was detected by qRT-PCR.The results showed that Wnt1 was expressed throughout the life cycle of Spodoptera frugiperda.During embryonic development,the expression level is highest when the embryo develops to 6h,and the expression level gradually decreases as the development progresses.In different developmental stages from egg to adult,the expression level of Wnt1 in the egg stage is higher than that in the larval,pupal and adult stages.The expression of Wnt1 in female moths is second only to eggs and much higher than that of male moths.By detecting the expression of Wnt1 in the head,thoracic segment,abdominal segment,antennae,external genitalia and wings of female and male moths,conclusions can be drawn that the difference in Wnt1 expression between female and male moths is mainly reflected in the abdominal segments,and expression in female are much higher than in male,while there is almost no difference in Wnt1 expression in the other five tissues.(3)Knockout of Wnt1 gene of Spodoptera frugiperda:The targeted knockout of the Wntl gene of Spodoptera frugiperda by injection of sgRNA and Cas9 protein produced mutant phenotypes of segment fusion,appendage defects and tissue specialization,and resulted in a high embryonic lethality rate.The lethality increased with the increase of injection concentration.The expression levels of genes related to embryonic development in the mutants were detected by qRT-PCR,the results showed that the expression levels of eight Hox genes(Abd-a、Abd-b、Ubx、Lab、Dfd、Antp、Pb and Scr)and Notch、Dpp、Dsx、Hth、Smo、Dll、Dac、Yellow、Tra2 were down regulated.(4)Analysis of Wnt1 gene expression profile of Helicoverpa armigera:The expression profile of Wnt1 gene of Helicoverpa armigera was detected by,and the results showed that Wntl was expressed throughout the life cycle of Helicoverpa armigera.During embryonic development,the expression level is highest when the embryo develops to 6h,and the expression level gradually decreases as the development progresses.In the different developmental stages from egg to adult,the expression level in the egg stage is the highest,while the expression level in the larval stage,pupal stage and adult stage is relatively low.(5)Knockout of Wnt1 gene of Helicoverpa armigera:The targeted knockout of the Wntl gene of Spodoptera frugiperda by injection of sgRNA and Cas9 protein produced mutant phenotypes of segment fusion,appendage defects and abnormal pigmentation,and resulted in a high embryonic lethality rate.The expression levels of genes related to embryonic development in the mutants were detected by qRT-PCR,the results showed that the expression levels of six Hox genes(Abd-a、Abd-b、Ubx、Lab、Dfd and Antp)and Hth、Pygo、Yellow were down regulated,while the expression levels of Notch、Dpp、Dsx、Yki and Sgf were upregulated.The above results indicate that the Wnt signaling pathway is indispensable in the embryonic development of Spodoptera frugiperda and Helicoverpa armigera.The research of the function of Wnt1 gene lays the foundation for the study of the body development pattern of Spodoptera frugiperda and Helicoverpa armigera,and also provides candidate genes for constructing conditional lethal genetic system to control pests.