Cloning And Expression Analysis Of Ghgme Gene Of Gossypium Hirsuturm L

On the basis of salt stress-related EST sequences of Gossypium hirsuturm L., aGDP-mannose-3’,5’-epimerase (GME) gene, named GhGME, was isolated by the3’,5’-RACE technology. The gene expression in cotton roots, stems, leaves anddifferential expression profile under three treatments with low temperature,salt andGA3by fluorescent quantitative qRT-PCR method. The recombinant plasmid wasconstructed by integrating GhGME gene into pGEM-T vector. Target gene GhGME,derived from double digestion of the recombinant plasmid, was then constructed intothe prokaryotic expression vector pET-23b and eukaryotic expression vector pBin438,and named as pET-23b-GhGME and pBin438-GhGME respectively.pET-23b-GhGME was transferred into E. coli BL21-DE3by electroporation, and theacquired BL21-DE3containing recombinant plasmid was prepared for proteininduction and purification. Transgenic GhGME tobacco plants were obtained byAgrobacterium-mediated transformation containing plasmid pBin438-GhGME. Theresults are as follows:1. According to TaKaRa5’-Full RACE Kit,3’-Full RACE Kit User Manual,5’and3’ end of target gene was respectively amplified with reverse transcription cDNAand primers designed on the basis of salt stress-related EST sequences of Gossypiumhirsuturm L.. The pGEM-T vector containing target fragment was transformed intoEscherichia coli DH5α and positive clones were picked and sequenced afterblue-white screening and PCR test. DNAMAN splicing of the sequencing resultshowed that the full-length cDNA of GhGME was1467bp, containing an1131bpORF which encoded376amino acids. The supposed relative molecular weight ofGhGME protein was42.396KDa, and its isoelectric point (pI) was6.291.2. Fluorescent quantitative qRT-PCR analysis of the gene expression of GhGMEin roots, stems, leaves and three different treatments showed that the relativeexpression of GhGME was higher in roots and stems than that in leaves. And theexpression of GhGME was up-regulated under either the4℃or200mmol/L NaCltreatment, but that was down-regulated under the100μmol/L GA3treatment.3. After enzyme digesting and recycling target gene GhGME and vector, ligatingthem and transforming into E. coli competent cell DH5α, the prokaryotic expression vector pET-23b, named pET-23b-GhGME was constructed. Digestion assays showedthat target gene had been successively ingrated into expression vector pET-23b.pET-23b-GhGME was transferred into E. coli BL21by heat shock and a42.4KDaprotein was induced and purified.4. The eukaryotic expression vector pBin438, named pBin438-GhGME, wasconstructed by fusing gene GhGME fragment and the expression vector pBin438digested with double enzyme digestion, and transferred them into E. coli competentcells DH5α. Enzyme digestion assay showed that target gene had been successivelyintegrated into the expression vector pBin438. Then the expression vectorpBin438-GhGME was transferred into Agrobacterium tumefacious LBA4404byelectroporation method. The putative transformed tobacco plants were obtained byAgrobacterium-mediated transformation. The result from ascorbic acid measurementof seven transgenic tobacco plants identified by PCR and RT-PCR method showedthat the highest content of ascorbic acid of the transgenic plants was2.3fold to thecontrol.