Identification Of The BmGNβ1 Gene And Its Effect On Bombyx Mori Nucleopolyhedrovirus Infection

Heterotrimeric GTP-binding protein,abbreviated as G protein,consists of three subunits:α,β,andγ.It predominantly serves as a signaling molecule between G protein-coupled receptors（GPCRs）on the cell membrane and in the cytoplasm for signal transduction.G proteinβsubunit 1（GNβ1）has multiple functions,including control of second messengers,regulation of cell growth,and switching of ion channels.Herein,we identified 22 GNβgenes in the complete genome of the Bombyx mori,and cloned the full-length sequence of BmGNβ1 to further explore its functions.Cell transfection,overexpression,and RNA interference（RNAi）techniques were employed to verify the functions of BmGNβ1 at the cellular level and individual level.The main findings are as follows:Firstly,identifying the genes of G protein family members and perform bioinformatic analysis.We use HMM in Pfam database to select all candidate G protein from silkworm genome,and screen for G protein family member genes using NCBI Conserved Domain Search Database（CDD）,SMART database,and Pfam database,including 5 Gα,22 Gβ,and1 Gγsubunit genes distributed on 17 chromosomes were identified.Most of the Gαgroup members show high homology,followed by the Gγgroup and Gβgroup.Additionally,10conserved motifs were identified,with the Gαgroup containing 5 motifs,the Gβgroup consisting of 4 motifs,and the Gγgroup having only one conserved motif 9.Secondly,cloning and temporal-spatial expression characteristics of the BmGNβ1 gene.The full-length BmGNβ1 was cloned from the midgut tissue of the silkworm BY using the Rapid amplification of c DNA end（RACE）technique.Nucleic acid sequence analysis showed that the full length m RNA of the gene consists of 288 bp of 5′UTR,935 bp of 3′UTR,and 1023 bp of CDS region.It is composed of a total length of 2243 bp and encodes340 amino acids（Genebank accession number OP899565）.Phylogenetic tree analysis showed that BmGNβ1 is most closely related to the Papilio polytes and Papilio machaon.Temporal-spatial expression profile showed that BmGNβ1 had the highest expression level during the larval stage and the lowest during the egg stage.Tissue expression profile showed that BmGNβ1 expression significantly decreased in the midgut,head,hemolymph,and Malpighian tubules of the silkworm 48 hours after BmNPV infection compared to the control group.Thirdly,functional identification of BmGNβ1 gene.Subcellular localization of BmGNβ1 showed BmGNβ1 is located in the cytoplasm.By constructing BmGNβ1-p IZ/V5-His-m Cherry overexpression vector and transfecting it into the BmN cell line with synthesized si RNA,BmGNβ1 was overexpressed or silenced at the cellular level.By detecting the gene expression level of the BmNPV late stage expression 39K protein gene VP39,it was found that in the cell line overexpressing BmGNβ1,the positive cells infected with the BmNPV（labeled with EGFP）and the expression level of VP39 gene were significantly lower than those in the control group at 24h,48h,and 72h after BmNPV infection.The si RNA results were the opposite.The results showed that overexpression of BmGNβ1 can inhibit BmNPV proliferation,while knockdown BmGNβ1 promotes BmNPV proliferation.The results of si RNA interference on BY variety at the 5^th instar showed that RNA interference of BmGNβ1 promotes BmNPV proliferation.This further indicates that high expression of BmGNβ1 can reduce the replication and proliferation of BmNPV after infection in Bombyx mori.Fourthly,the BmGNβ1 gene triggers immune mechanisms via the PI3K-AKT signaling pathway and inhibits BmNPV proliferation.RT-q PCR was used to detect the m RNA transcript levels of key genes and their downstream genes in the signaling pathway involved in BmGNβ1 overexpression system cells 48 h after BmNPV infection.The results showed that compared to the control group,the expression levels of PI3K,AKT,Bmp21,BmPKN and BmCREB decreased,while the expression levels of BmFOXO,Bmp53,and Caspase-1increased.The results suggest that BmGNβ1 induces the expression of genes（Bmp53,BmFOXO,Caspase-1,Bmp21,BmPKN,and BmCREB）through the PI3K-AKT pathway to induce cell apoptosis and thereby inhibit BmNPV proliferation.In summary,this study identified 28 G protein family member genes in the silkworm genome and cloned the full-length sequence of BmGNβ1.Both cellular and individual experiments showed that overexpression of the BmGNβ1 gene can reduce the expression of viral genes and virus replication and proliferation after BmNPV infection,potentially by inducing cell apoptosis through the PI3K-AKT pathway to inhibit BmNPV proliferation.This study provides potential targets and theoretical support for breeding silkworm varieties resistant to BmNPV.