Cloning And Functional Analysis Of VTC2Gene Involved In Ascorbic Acid De Novo Synthesis Pathway In Cotton

Objective: GDP-L-galactose phosphorylase(GGPase), as a key enzyme of ascorbic acid(AsA) regeneration, plays important roles in keeping ascorbic acid balance of plant cell.Here,3cotton GGPase genes were cloned from fiber tissue; GGPase expression profiles wereperformed in transcription and protein levels during fiber development; constructedprokaryotic and eukaryotic expression vectors were used to transformation of Arbidopsis andtobacoo to understand GhGGPase gene function. This study establish a basis to elucidateGGPase gene biological function.Methods: GhGGPase cDNA was obtained through RT-PCR method from cotton fibertissue, and successive bioinformatic method was performed to analyze the structurecharacteristic and function domains of GhGGPase. Constructed expression vectorpCAMBIA2300-GhGGPase and pET28a-GhGGPase were used to transform of Arabidopsisand tobacco with floral dip and leaf disk method respectively. Obtained transgenicArabidopsis and tobacco plants were used to perform stress treatment experiments. Therecombinant GhGGPase protein was achieved by transforming the prokaryotic expressionvector pET28a-GhGGPase into E.coli expression strains BL21(DE3) and incuction of IPTG.The transcription expression profile was analyzed utilizing RNA extract, cNDA obtain afterreverse transcription-polymerase chain reaction and RT-PCR.Results:(1) Cloning of cotton GhGGPase genes. Full-length ORF of three cotton GhGGPasegenes including GhGGPase1, GhGGPase2, and GhGGPase3were cloned with nucleotides of1350,1335and1326bp, respectively.The sequence alignment and domain analysis showedthat GhGGPase proteins, as nuclear localized protein, contain one HxHxQ motif and belongto D-galactose-1-guanosine acyl transfer enzyme (GaiT) family and HIT superfamily,.(2) Expression pattern analysis of cotton GhGGPase genes. The expression patternanalysis of GhGGPase genes during cotton different developmental stages and tissues showedthat, three GhGGPase genes expressed at the highest value in10dpa fiber tissue, expressionof GhGGPase1is higher than GhGGPase2and GhGGPase3. GhGGPase enzyme activityreached the highest value in10dpa and decreased gradually.(3) Expression of recombinant GhGGPase proteins. The recombinant proteins ofGhGGPase1, GhGGPase2and GhGGPase3were obtained with molecular weight of49.5,48.9and48.6kDa after transformation into DE3and induction by IPTG.(4) Functional analysis of GhGGPase gene participating plant stress tolerance.Expression vector pCAMBIA2300-GhGGPase1-3were constructed and transformed intoArabidopsis and tobacco, obtained transgenic plants were used to perform stress treatmentexperiments. The transgenic plants display enhanced tolerances to salt, drought and oxidative stresses coupled with increased enzymatic activity of CAT, POD and SOD, which suggest astrong relationship between stress tolerance and these enzymes activity.Conclusion: Ascorbic acid and its synthesis gene GhGGPase play inportant roles duringcotton fiber development period, and may participate in the fiber elongation. GhGGPase mayperform key function in regulating AsA biosynthesis and content of plant cell.