Analysis Of Molecular Chaperone Activity Of The BmGRP78 In Bombyx Mori,the Silkworm And Its Function In Response To Stressful Condition

Bombyx mori is an important economic insect and a promising model organism in biological studies,health safety and environmental pollution assessments.However,due to the continuous domestication,its tolerance to environmental fluctuations has decreased,which has seriously affected the economic benefits of sericulture.Glucose-regulated protein 78(GRP78)is an important regulator of endoplasmic reticulum homeostasis.When cells are exposed to stress conditions such as extreme temperature,viral infection,environmental toxins,hypoxia,and changes in calcium ion concentration,ER homeostasis is disrupted,resulting in accumulation of damaged or unfolded proteins in the ER lumen,while GRP78 plays an important role in degrading unfolded protein and restoring endoplasmic reticulum homeostasis.Therefore,it is of great significance to explore the molecular mechanism of Bm GRP78 in the process of biotic and abiotic stress.In this study,Bm GRP78 gene was cloned by PCR and analyzed by bioinformatics.Bm GRP78(NM＿001043372.1)contains an open reading frame(ORF)of 1977 base pair(bp),encoding a polypeptide consisting of 658 amino acids.Using bioinformatics methods,its protein molecular weight is predicted to be about 73.1 k Da,and it is composed of three conserved domains of HSP70 protein family,namely Nucleotide binding domain(NBD),Substrate binding domain(SBD),C-Terminal domain(CTD)with a signal peptide sequence of 20 amino acids at the N-terminus and a KDEL endoplasmic reticulum retention tag at the C-terminus.Then,a recombinant p ET-32a(+)-Bm GRP78 prokaryotic expression vector was constructed,and the recombinant plasmid p ET-32a(+)-Bm GRP78 was transformed into Escherichia coli(E.coli).After induction and expression by IPTG,a large amount of recombinant Bm GRP78 protein was obtained.After purification and ultrafiltration concentration,New Zealand white rabbits were immunized,and polyclonal antibodies were finally obtained with high titer and good specificity.The spatiotemporal expression of this gene was analyzed by quantitative real-time PCR(q RT-PCR)method,and the results showed that Bm GRP78 is ubiquitously expressed in various tissues and developmental stages.Secondly,the molecular chaperone activity of Bm GRP78 was evaluated by thermal aggregation assay of citrate synthase and malate dehydrogenase and ATPase activity detection.The results showed that Bm GRP78 has the function of preventing protein aggregation and has stable molecular chaperone activity at high temperature.Heat shock and heavy metal stress strongly affected the protein expression of Bm GRP78 in Bm N cells,but the effect was small at the transcriptional level.Bm NPV infection did not affect the transcription and translation levels of Bm GRP78,but resulted in the translocation of part of Bm GRP78 into the nucleus.Finally,a CRISPR/Cas9 all-in-one expression vector was designed and modified,and the Bm GRP78 was knocked out,which provided a basis for studying the biological function of the Bm GRP78 in silkworm.This study shows that the Bm GRP78 protein has stable molecular chaperone activity both in vitro and in vivo.When Bm N cell is exposed to environmental stress,the expression of Bm GRP78 protein increases.Therefore,we speculated that it might help to remove misfolded proteins in cells and protected cells.The above research results lay a foundation for elucidating the involvement of Bm GRP78 in regulating the immune defense mechanism and toxicological mechanism of silkworm.