Functional Analysis Of Key Mutated Genes In Nm2(Non-Molting In The 2^ndInstar)Silkworm,Bombyx Mori

Silkworm（Bombyx mori）is an important economic insect,which belongs to the Lepidoptera completely metamorphosis insect.It is often used as a model organism for lepidopteran insects in related basic research.Molting is one of the important physiological processes of silkworm growth,development and metamorphosis.It is a genetic characteristic formed during its long-term evolution.The subject of this study was a non-molting in the 2^nd instar（nm2）mutant.Most of the mutants showed no sleep at the end of the 2^nd instar,no molting,and rarely ate food,and died one after another after maintaining this state for a week or so.Previous studies have determined that BmCPG10 is the main gene responsible for the nm2 mutation.Based on the previous research,this study used CRISPR/Cas9 technology,digital gene expression（DGE）and two-dimensional electrophoresis（2-DE）technology to analyze the function of BmCPG10 gene and related genes in a more systematic and detailed way,so as to elaborate the molecular mechanism of the mutation.The main research results are as follows:First,the function of the BmCPG10 gene was analyzed using CRISPR/Cas9editing technology.In the exon 1 of BmCPG10 gene,a suitable target site was selected to synthesize sgRNA in vitro,and mixed with Cas9 protein injected into early embryos of silkworm.After the examination,2 silkworms showed 4 base deletions at the target site.One of the silkworms died at the end of the 2^nd instar,and the other was a male silkworm,which developed normally and moths.The male moth mated with the normal wild-type female moth,and the eggs were mostly unseminated,suggesting that loss of BmCPG10 function not only affects the silkworm’s growth and development,may leads to sperm activity reduce.The BmCPG10 gene was up-regulated in both of the edited silkworms,which was consistent with the expression trend of BmCPG10 in the natural mutant nm2.Second,DGE and qRT-PCR were used to analyze the expression of head related genes in nm2 mutant and the original mutant C603 at the 2^nd instar.It was found that in the nm2 mutant,the genes involved in the ecdysone signaling pathway were significantly different,among which neverland,spook and sad were significantly up-regulated,while CYP314A1 and CYP18A1 were significantly down-regulated;some epidermal protein genes were significantly different,and the up-regulated epidermal protein genes were more than the down-regulated epidermal protein genes.There were also differences in the expression of nuclear receptor genes in the ecdysone-mediated signaling pathway,in which the expression of key nuclear receptor geneβFTZ-F1 was significantly up-regulated,and the expression of early and late genes HR3 and HR4 were significantly down-regulated,and other nuclear receptor genes were not significantly changed.Third,two-dimensional electrophoresis（2-DE）analysis was carried out using the nm2 mutant and the C603 wild-type silkworm’s prothoracic gland which containing the first pair of tracheal plexus（PG^W）at the 2^nd instar as materials.It was found that one of the epidermal protein genes BmCPR41,was significantly up-regulated in the nm2 mutant,and the detection results of qRT-PCR at the mRNA level was consistent with the protein level,and was consistent with the results of the expression profile.Further analysis revealed that the BmCPR41 gene was up-regulated in parallel with the BmCPG10 gene in both of the edited silkworms.Based on the above results,we speculated that although the BmCPR41 gene is not the main gene responsible for the nm2 mutant,it may cooperate with the BmCPG10 gene to participate in the formation of the nm2 mutant.Through the above analysis,we further confirmed that the main gene causing the nm2 mutant character is the BmCPG10 gene,and confirmed that the BmCPG10 gene regulates the physiological function of silkworm growth and development through the ecdysone signaling pathway.At the same time,a downstream gene of the ecdysone signaling pathway was discovered.The epidermal protein gene BmCPR41 may cooperate with the BmCPG10 gene to cause the nm2 mutation.This study provides evidence for further elucidation of the formation mechanism of nm2 mutant,and also helps to understand the regulation mechanism of ecdysone in the growth and development of silkworm.