Cloning ADAR Gene From Apis Mellifera And Bombyx Mori And Expression Study In Pichia Pastoris And Analysis Of Alternative Splicing And RNA Editing Of Insect Sodium Channel Genes

Before maturing of the eukaryotic mRNA,Pre-mRNA undergoes several processes of post-transcriptional modification,including alternative splicing,editing and polyadenylation.One type of RNA editing is mediated by Adenosine deaminases that act on RNA(ADARs),which is called A-to-I editing.ADARs are RNA editing enzymes that change adenosines to inosines via a hydrolytic deamination reaction on double-stranded(ds)RNA.Like most dsRNA binding proteins,the enzymes will bind to any dsRNA without apparent sequence specificity.However,once bound, ADARs deaminate certain adenosines more efficiently than others.After editing, inosine(I)is translated as guanosine(G),and most enzymes recognize inosine as guanosine.Thus,ADARs change the primary sequence information in a RNA molecule.In addition,because inosine base-pairs with cytidine,ADARs can change the structure of RNA molecule by changing an AU base-pair to an IU mismatch.1.Cloning and anlysis ADAR(adenosine deaminase acting on RNA)gene from Apis mellifera and Bombyx mori and expression study in Pichia PastorisWe have identified a homolog of the ADAR(adenosine deaminases that act on RNA)class of RNA editases from honeybee(Apis mellifera),AmADAR and partial cds(coding sequence)of BmADAR from Bombyx mori,and found that AmADAR has 47.41%identity to dADAR from Drosophila melanogaster and is most homologous to the mammalian RNA editing enzyme ADAR2 by multiple sequence alignment analysis based on protein sequences.We also found that the expression of AmADAR gene was regulated by alternative splicing and expressed differently in four developmental stages(embryonic,laval,pupal and adult),most abundant in the adult, abundant in embryonic,less abundant in larval,and much less level in pupal. BmADAR was expressed most abundant in the pupal,abundant in larval,less abundant in adult,and much less level in embryonic.Taken together,these findings show that both transcription and processing of AmADAR and BmADAR transcripts are under strict developmental control. We constructed the recombinant expression vector pSK-FLIS6-AmADAR and expressed in Pichia pastoris.The recombinant expression vector pSK-FLIS6-AmADAR cotransformed into the genome of electrocompetent cells.The positive yeast transformants His~+ were screened by auxotrophic medium and PCR.Then the Muts phenotypes were screened by Minimal Dextrose Medium(MD) and Minimal Methanol Medium(MM).His~+Mut~s phenotype transformants were inoculated Buffered Glycerol-complex Medium(BMGY)in baffled flask to get enough Yeast cells.Using Buffered Methanlo-complex Medium(MM)including Methanol which concentration is 0.5%to induce the expression.The result suggested that we have acquired Adenosine Deaminases of honeybee which about 70kD by the SDS-Polyacrylamide Gel Electrophoresis and Western blot analysis.2.Alternative splicing and RNA editing of insect sodium channel genesVoltage-gated sodium channels are large transmembrane proteins that initiate action potential in electrically excitable cell,and their transcripts undergo extensive RNA editing and alternative splicing in insect.In this study,we have identified complete coding sequence(CDS)encoding for the para-ortholog0us sodium channel from A.mellifera(Am-para),B.mori(Bm-para),T.castaneum(Tc-paral,Tc-para2) and D.ananassae.RNA editing was found at 10 editing sites(all of the ten editing events result in amino acid changes),5 editing sites(four editing events result in amino acid changes),and 6 editing sites(four editing events result in amino acid changes)in Am-para,Bm-para,Dan-para transcript respectively.In addition,we found six alternative splicing exons in Am-para,in which two are novel optional exons corresponding to D.melanogaster para,nine alternative splicing exons in Bm-para,one alternative splicing exon in Tc-para1 and four alternative splicing exons in Tc-para2,ten alternative splicing exons in Dan-para,in which one alternative splicing exon is evolutionarily conserved in D.melanogaster,Drosophila ananassae, T.castaneum and A.mellifera orthologs of sodium channel.Most interestingly,we identified and characterized two sodium channel genes, Tc-para1 and Tc-para2 in the genome of T.castaneum by analyzing the genomic sodium channel locus.Taken together,these findings show that insect sodium channel can produce various transcripts by gene duplication besides RNA editing and alternative splicing.