To Be Black Spiny Ant Creb Gene Cloning And Prokaryotic Expression

CREB(cAMP response element binding protein) is a central transcription factor,It belongs to a family of bZIP.CREB protein are composed of two major domains:(1) the carboxy terminal basic region/leucine zipper(bZIP) domain,which is involved in sequence specific DNA binding and protein dimerization;and(2)kinase-inducible domain(KID),which can presumably interact with other components of the transcription machinery and with signal transduction pathway,and influence gene expression.CREB binding protein(CBP) is crucial in mediating the transcriptional activity of CREB.CREB have been implicated in a variety of the physiological processes including the cell survival,circadian rhythms and spermatogenesis and also involved in the neuronal plasticity and memory formation.The Polyrhachis vicina roger,as the eusocial insect,has a strict hierarchical division of labor in the group.At the same time,P.vicina is also an very important resource insect,In this paper,the full-length cDNA of CREB gene is coloned from the P.vicina by RT-PCR and RACE.The bioinformatics methods is used to analyse characteristics of full-length cDNA and predict functional motifs in the ORF.The open reading frame(ORF)of CREB gene is subcloned into prokaryotic expressional vector PGEX-5X-1 to construct recombinant plasmid.The recombinant plasmid is transformed into E.coli strain BL21 and induced by IPTG to express.The major experiment results are as follow:1.Gene cloning:The total RNA was extracted from the P.vicina,The full-length cDNA of CREB was obtained by RT-PCR,5'and 3'RACE(Rapid Application of cDNA Ends).The full-length cDNA is1153bp,including an open reading frame of 762bp,5'-UTR of 55 bp and 3'-UTR of 337bp,code the protein that is constituted by 253 amino acides,the molecular weight is 27.5649KD,and theoretical PI is 6.98 and is named PvCREB gene.The nucleotide sequence of PvCREB gene is submitted to GenBank and assigned the accession number EU523223.2.Sequence analysis:Bioinformation analysis indicates that full length cDNA of PvCREB is similar to Apis mellifera carnica,Bombyx mori,Tribolium castaneum,Aeries aegypt and Homo sapiens at 90.8%,65.8%,75.1%,55.8%and 46.7%in turn;The phylogenetic tree based on NJ method shows that the PvCREB clustered as expected with all insect CREB;The sequence of PvCREB contains nuclear locating sequence RKRELRLLKNREAARECRRKKKE,which protein may locate in the nucleus;A series of predicted function motifs are found in the PvCREB protein sequence,including two N-glycosylation sites,one cAMP-dependent protein kinase phosphorylation site,four Protein kinase C phosphorylation sites,eight Casein kinaseⅡphosphorylation sites,three Nutmeg acyl sites and one Leucine zipper site,these sites provide functional information of PvCREB protein;The prediction of the secondary structure of PvCREB using HNN method,show thatα-helix,irregular and extended coiled are mainly structural elements,andβ-sheet spread to the entire protein;The results of tertiary structure prediction using SwissModel First Approach shows that there are very similar in bZIP₁ between the proteins of PvCREB and MmCREB.The difference of Spatial Structure in the bZIP₁ between the PvCREB and the MmCREB is that PvCREBα-helix domain contains more three spiral ring than MmCREB.3.Prokaryotic expression of CREB:By inserting the open reading frame(ORF) of CREB gene into prokaryotic expressional vector PGEX-5X-1 to construct recombinant plasmid,the recombinant plasmid is transformed into E.coli strain BL21 and induced by IPTG to express.The result of SDS-PAGE electrophoresis indicates that CREB gene is expressed in E.coli and the molecular mass of recombinant protein is about 27KD.CREB gene of P.vicina as the first time is cloned and expressed successfully.These results may be useful in further study the important functions of CREB.