Cloning,Identification And Functional Study Of BmClC-2 Gene In Silkwrom,Bombyx Mori

Voltage-gated chloride channel protein(voltage-gated chloride channel,Cl C)belongs to a large family of chloride ion channel proteins,which are ubiquitous in eukaryotes and prokaryotes,mainly located on the plasma membrane or organ membrane of various cells.It can regulate different physiological processes and cell functions.Chloride channel protein 2(Cloride channel protein 2,Cl C-2)is a member of the voltage-gated chloride channel family.So far,although some progress has been made in the study of Cl C-2 gene and protein molecular structure,its structure,function,and regulatory mechanism are still not fully understood.The current research on Cl C-2 is mainly concentrated in humans,mice and plants,and almost no reports in insects.Bombyx mori is the earliest lepidopteran insect to complete genome sequencing.The complete genome,expression profile and proteome database provide an important platform for the functional study of silkworm genes.In this study,bioinformatics technology was used to identify the genes of the silkworm Cl C family and explore the function of the Cl C-2 gene.1.The silkworm genome was screened,and three members of the Bm Cl C gene family were identified for the first time,located on chromosomes 1,17 and 27;and preliminary clustering was performed based on the protein sequence of Bm Cl Cs.The exons and introns of these 3 genes The number is different.The three Bm Cl C proteins are hydrophobic proteins and have similar protein conserved domains,but there is still a diversity of motifs.They may be used as potential proteinprotein interaction domains to assist Cl C proteins.effect.Using q PCR technology to analyze the expression of 3 Bm Cl Cs in 9 tissues of Bombyx mori,the results showed that 3 Bm Cl Cs had the highest expression in fat body.It is speculated that Bm Cl Cs may play a key role in the nutritional transport of Bombyx mori.2.The Bm ClC2(BGIBMGA004625)gene in the Bm Cl C gene family located on chromosome 27 was selected for full-length cloning,and the gene was 4808 bp in the silkworm BY variety,2865 bp in open reading frame,and the start codon was in full length 310-312 bp,the stop codon is located at 3170-3172 bp,consisting of 954 amino acids coding region;the full length of the Bm Cl C-2 gene in the XBY variety is 4774 bp,the open reading frame is the same as the BY variety,the start codon Located at positions 284-286 bp of the full length,the stop codon is located at positions 3144-3146 bp,encoding 954 amino acids.The coding length of the Bm Cl C-2 gene in the XBY variety is 4774 bp,and the open reading frame is the same as the BY variety.The start codon is located at positions 284-286 of the full length,and the stop codon is located at positions 3144-3146 bp,encoding 954 amino acids.The full-length sequence of Bm Cl C-2 gene amplified with BY was submitted to NCBI with Gen Bank accession number MT353899.The sequence of the Bm Cl C-2 gene of the silkworm was analyzed.The Bm Cl C-2 coding regions of the two silkworm varieties were found to have a sense mutation at the 933 th amino acid position,but the secondary structure of the protein did not change.The non-coding regions of each variety have missing and increased parts,and RNAi and overexpression can be continued for further research.The sequence of the Bm Cl C-2 gene was analyzed by bioinformatics.Bm Cl C-2 encodes 954 amino acid residues with a molecular weight of 105.76 k D,a theoretical isoelectric point of 9.48,and an average hydrophilicity value of 0.11127.The protein is a hydrophobin.There are Cl C-1-like,Vottage-Cl C,Clc A,PRK05227,CBS and other domains.It is localized on the cell membrane and has no signal peptide.There are 12 transmembrane structures.Prediction of the locus,through comprehensive analysis,640-855 aa as the antigen segment.RT-PCR and q PCR studies on the whole tissue expression of Bm Cl C-2 found that the Bm Cl C-2 gene was expressed to varying degrees in 9 tissues of the two varieties and was expressed in the midgut,marshall duct and gonad At higher levels,after 24 hours of Bombyx mori Nuclear Polyhedrosis Virus(Bm NPV),the difference in Bm Cl C-2 gene expression between the two varieties was the midgut,gonad and tracheal plexus.3.Construction of p ET-28a(+)-Bm Cl C-2 prokaryotic expression vector,efficient expression of recombinant fusion protein,and purification of recombinant Bm Cl C-2 protein containing His tag,with good specificity and high titer Polyclonal antibodies laid the foundation for analyzing the function of this protein in silkworm.4.By studying the expression of Bm Cl C-2 in various developmental stages of silkworm and two species of fifth-instar larvae after Bm NPV treatment,it was found that the protein was hardly expressed in the silkworm moth stage,and the highest expression level was the egg stage The varieties were infected with Bm NPV and uninfected,and the protein had varying degrees of change in various tissues.Expression profiling analysis showed that Bm Cl C-2 may play a large role in the nutrient transport during embryonic egg development.Bm NPV will affect the expression of this protein in various tissues.After RNA interference,XBY blood cells were observed under a fluorescent microscope to show green fluorescence,indicating the presence of Bm NPV in their blood cells,indicating that the resistant variety XBY lost its resistance to Bm NPV after Bm Cl C-2 was interfered.The individual survival rate survey also proves that individuals of the XBY variety infected with Bm NPV are eventually infected by the virus and die,which initially confirms that Bm Cl C-2 may play a role in the anti-Bm NPV in silkworm.