Cloning And Salt Resistant Analysis Of CDNA Of Glyceraldehyde-3-phosphate Dehydrogenase Gene From Polygonum Sibiricum Laxm

Polygonum sibiricum Laxm.can grow in a high salt stress environment.In this study,the primers which were used to amplify the full length eDNA of the glyceraldehyde-3-phosphate dehydrogenase(GAPDH) were designed according to the expressed sequence tag(EST) sequence of the GAPDH gene acquired from suppression subtractive hybridization library of the stem of P.sibiricum.The full length eDNA ofPs GAPDHgene was cloned by using rapidamplification of cDNA ends(RACE) technology.Then the nucleotide and protein sequence were compared using bioinformation.Northern blotting of Ps GAPDH expression in different tissues of P.sibiricum,which was stressed under 3%of NaHCO₃ was analyzed.The Ps GAPDH gene was inserted into yeast(Saccharomyces cerevisiae) expression vector(pYES2), then the pYES2-GAPDH was transferred into yeast cells for functional analysis.The main results were as follows:(1) The results showed that the cDNA of the Ps GAPDHgene was 1331 bp,encoding 337 amino acids residues and encoding a protein with a predicted molecular mass of 36.672 kDa, and theoretieal pI of 7.66,protein is stable which nonstabilization quotiety is 24.31.The deduced amino acid sequence shows high homology to the eytosolie GAPDH from other higher plants.The highest amino acid identities between sequences of different herbaceous plant species was 96%and of different shrubby plant species was 93%.Ps GAPDH is a tetrameric NAD-binding enzyme involved in glycolysis and glyconeogenesis.C-terminal domain is a mixed alpha/antiparallel beta fold,N-terminal domain is a Rossmann NAD(P) binding fold.(2) The expression of this form of Ps GAPDH in the rhizome,stem and leaves of P. sibiricum under the stress of NaHCO₃ was studied on the mRNA level by northern blot hybridizations.These experiments showed that the Ps GAPDH mRNA levels in leaves was decreased from 4-24 h treated by NaHCO₃ and after 48 h,the mRNA levels became normal. The Ps GAPDH mRNA levels in stems was strongly increased after 2h and became normal after 48 h.The Ps GAPDH mRNA levels in rhizome slightly increased during stress of NaHCO₃.(3) The recombinant plasmid pYES2-GAPDH was constructed by inserting Ps GAPDH gene(cDNA) into yeast expression vector pYES2,pYES2-GAPDH was transferred into yeast, and confirmed by PCR and enzyme digest detection of 3 randomly picked INVScI(pYES2-GAPDH) lines.The expression of foreign Ps GAPDH gene in yeast which is induced by galactose was investigated by RT-PCR,the yeast harboring empty pYES2 vector strain as the control.The results from biofunctional analyses with Ps GAPDH yeast transformants showed that GAPDH yeast transformants had significantly higher resistance to different salt stresses. INVScI(pYES2-GAPDH) had more salt-resistance ability than INVScI(pYES2) and the former survival rate was higher than that of the latter under the stress of 10%NaHCO₃ and 5 mol·L^-1 NaCl.This indicated that the high ability of salt-tolerance of INVScI(pYES2-GAPDH) might be related to the expression of pYES2 gene.