Cloning And Functional Analysis Of BmABCD₂ Gene In Silkwrom,Bombyx Mori

ATP-Binding Cassette Transporter?ABC-Binding Transporter?,abbreviated as ABC transporter,is a class of transmembrane proteins.The ABC transporter contains a common core structure—two nucleotide binding domains?NBDs?and two transmembrane domains?TMDs?in which the NBDs are relatively conserved.According to the conserved structure of NBDs,the silkworms ABC transporters are divided into 8 subfamilies:ABCA,ABCB,ABCC,ABCD,ABCE,ABCF,ABCG,and ABCH.Among them,members of the ABCD subfamily are semi-transporters,but there are few reports on ABCD,especially the function of the ABCD₂ gene in Bombyx mori.We identified two differentially expressed genes belonging to the ABC transporter pathway by transcriptome sequencing analysis of the silkworm anti-BmNPV variety BaiyuN?BYN?and the conventional variety Baiyu?BY?infected by BmNPV.The ABCD₂gene,in order to further clarify the sequence information and related functions of ABCD₂,we used the RACE technique to clone two strains ABCD2 gene?named BmABCD2?and analyzed the gene sequence by bioinformatics analysis.Characteristics and gene family codon usage patterns;detection of BmABCD2 expression patterns after different cultivars,different developmental stages,and after infection by BmNPV;RNAi interference BmABCD₂ identified its function,and GST-Pull Down combined mass spectrometry was used to screen for interaction with BmABCD2 protein.The present studies showed that BmABCD2 may be involved in the inhibition of BmNPV by the silkworm-resistant strain BaiyuN.1.The BmABCD2 gene was cloned in full length,and the BmABCD2 gene of Baiyu with a full length of 3430 bp was obtained,and the open reading frame was 2265 bp.The full length of BmABCD₂ gene of BaiyuN strain is 3258 bp,and the open reading frame is2265 bp.The homology search determined that the fragment belongs to the complete coding region of the ABCD₂ gene of the silkworm,and the gene is located on the chromosome 27of the silkworm.The full-length ABCD2 sequence amplified by BY strain was named BmABCD₂ and submitted to NCBI.The GenBank accession number is MK840981.2.The semi-quantitative and fluorescence quantitative analysis of BmABCD₂ gene expression in the two lines showed that the ABCD₂ gene was expressed in nine tissues of two lines,especially in the midgut,fat body and trachea.After BmNPV infection 48 hours,the most differentially expressed between the two lines of ABCD₂ gene was the gonads and tracheal plexus.3.The coding sequence region of the BmABCD₂ gene of two varieties of Bombyx mori found that there is a sense mutation at the 43th amino acid but the secondary structure of the protein has not changed,and the non-coding regions of both varieties have missing and redundant parts.After sequence analysis,the physicochemical properties of BmABCD₂protein were obtained.The comprehensive prediction results showed that the protein was ATP-binding cassette sub-family D member 2,and there were ABC domain and AAA domain,which were located in the cell membrane,and there were 4 transmembrane structures,and the N-terminal C-terminus is not intracellular.4.Using bioinformatics software to analyze the characteristics and preference of ABC gene family codons,it was found that the coding sequence of silkworm ABC gene family has diversity of base composition but no obvious preference,and preference for 34 codons.Preferencer uses the codons ending in A and G,the expression levels of each gene in the family is different.The codon preference of the ABC gene family in the silkworm is affected by base mutation and natural selection pressure and other factors,but the base composition constraints play a greater role in codon usage preferences.5.The RNA interference of BmABCD₂ gene of BaiyuN strain was observed.After 48h,the blood cells of BaiyuN were observed to form green fluorescence,indicating that the resistant strain BaiyuN lost its resistance to BmNPV after the interference of BmABCD₂gene.The individual survival rate test also proved that the BaiyuN strain individuals were eventually infected by the virus,and it was preliminarily verified that the BmABCD₂ gene has a certain resistance function in the BaiyuN strain.6.GST-Pull Down combined mass spectrometry was used to screen the protein interacting with ABCD₂ protein.Through peptide fingerprinting analysis,more than a dozen proteins that may interact with ABCD₂ protein were found.Such as vacuolar ATP synthase catalytic subunit A,serine protease-2,voltage-dependent anion selective channel subtype X2,glutathione S-transferase?3 subtype X1,glyceraldehyde-3-phosphate dehydrogenase,heat shock protein 70.