Characterization Of Several Diapase-related Genes In Silkworm,Bombyx Mori And Functional Studies Of The Akr2e-like Gene

Insect diapause is a biological process influenced by genetic control and external environmental factors.The study of insect diapause mainly focuses on the effects of external environmental signals such as temperature,humidity,photoperiod and food on diapause,and the mechanism of diapause hormone and its receptor on diapause.However,the molecular mechanism of diapause in silkworm is not fully understood,and the regulation network of diapause-related genes remains unclear.In this study,a molecular model for diapause research of the bivoltine silkworm Qiu Feng was established.The transcriptome was sequenced in the ovary of the diapause diapause-induced group（DEP）and the non-diapause induced group（NDEP）,and a batch of genes related to diapause preparation were screened.Identification and functional analysis of the candidate genes were conducted by RNAi and CRISPR/Cas9 knockout techniques,and the molecular mechanism of silkworm diapause preparation was explored.The main results are as follows:1.Transcriptome analyses identified 183 genes with higher expression,and 106 with lower expression under diapause-inducing conditions.In the peroxisome pathway,5 DEGs encoding fatty acyl-CoA reductase 1（FAR-1）showed up-regμLation in DEP.FAR-1catalyzes the reduction of saturated fatty acyl-CoA to fatty alcohols,the high amount expression of the Far1 homolog in DEP oocytes is very likely to have arisen from the rapid accumulation of fatty alcohols,wax-esters and ether lipids in the cells,which in turn lead to the difference of the composition,structure,and physical properties of cellular membranes between the two groups being compared.In the glycerolipid metabolism pathway,BGIBMGA009801 encoded Aldo-keto reductases（AKRs）was up-regulated,Lpp and Dagt-1 were also clearly up-regulated in DEP,BGIBMGA000777 encoding esterase FE4-like was the only significant down-regulated gene in DEP;In general,this upregulation of DGEs related to triglyceride synthesis likely reflects the up trend of TAG accumulation,the concurrent suppression of its catabolism during diapause preparation.In the life-regulating pathway,Bigflp was down-regulated in the DEP group,while the Pepck gene was up-regulated,which was contribute to the occurrence of diapause fate.2.The differentially expressed genes involved in the above pathways were silenced by RNAi technology.The results showed that there was the synergistic up-regulation of the genes related peroxisome pathway,after BGIBMGA011147,BGIBMGA002907 were interfered,but which could not change the diapause status of individuals;the interference of BGIBMGA009801 resulted in a consistent down-regulation trend of other gene related triglyceride synthesis pathway;while the interference of BGIBMGA000777 caused the up-regulation of the other four genes in DEP,and the up-regulation of BGIBMGA009801reached a very significant level.After the interference of BGIBMGA000777 in NDEP group,the diapause of offspring eggs was changed,and the non-diapause eggs were transformed into diapause egg.This transformation may be related to the high expression of BGIBMGA009801 during this period.Therefore,BGIBMGA009801 was selected as the key gene for subsequent experimental research;after BMGA0123057 related the longevity regulatory pathway was interfered,the progeny eggs was changed from non-diapause to diapause in the NDEP group.3.In the present study,one cDNA encoding AKR2E protein was isolated from pupae cDNA library of Qiufeng,a bivoltine silkworm strain.The cDNA encodes a 343 amino acids protein showing 43%and 38%identity with AKRs from Drosophila melanogaster and Homo sapiens,respectively.Molecular phylogenetic analysis suggests that this protein phylogenically belong to an AKR2E subfamily of the AKR2 family,the amino acid sequence showed over 30%identity with AKR2E proteins,so this protein was named as AKR2E-like.The ORF segment of the AKR2E-like was cloned into the plasmid pET-28a（+）to construct a recombinant expression plasmid.SDS-PAGE and Western blot results revealed that His-tagged fusion protein was successfully expressed.qRT-PCR analysis demonstrated that the expression of ark2e-like gene was developmentally regulated and showed a constant increase in early stage of pupation in diapause-destined group.The expression of akr2e-like was highest in the ovary,followed by the fat body,and the expression of the head was the least.In the DEP group,the expression of the akr2e-like in the ovary was more than 2 times higher than that in the NDEP group.4.The G0 mutant mosaic of the akr2e-like gene was obtained by CRISPR/Cas9 system,and a C base deletion and an A base insertion were found near the target of AKR3A（2）;G₁hybridization was obtained through crosing strategy WT×mosaic,and selfing in G₁ got G₂ generation,mutant homozygous laid non-diapause eggs was screened within G₂;but progeny eggs（G₃）from G₂ was not all non-diapause eggs,86.4%of progeny eggs were still diapause eggs,non-diapause eggs only account for 13.6%;selfing in G₃ produced G₄generation offspring eggs,the proportion of non-diapause eggs increased to 26%;From the G₂ to the G₄ generation,the diapause rate of group had been decreasing.Duncan’s multiple range test results show that the diapause rate of the G₄ generation is significantly decreased,indicating that the homozygous mutation of the akr2e-like gene did affect diapause rate of the progeny eggs.