Cloning And Functional Analysis Of GhMDH Gene Of Gossypium Hirsuturm L.

Malate dehydrogenase is widely present in a variety of in vivo organismsglucose metabolism, lipid metabolism and other metabolic pathways. Its mainfunction is the reversible conversion of the catalytic malic acid and oxaloacetic acid.In addition malate dehydrogenase with the resilience of the plant stress-related. Onthe basis of salt stress-related EST sequences of Gossypium hirsuturm L., a malatedehydrogenase (MDH) gene, named GhMDH, was isolated by the3’,5’-RACEtechnology. The GhMDH was then introduced into the prokaryotic expressionvector pET-23b and eukaryotic expression vector pBin438, and named aspET-23b-GhMDH and pBin438-GhMDH respectively. pET-23b-GhMDH wastransferred into E. coli BL21(DE3) by thermal activation, and the acquired BL21(DE3)containing recombinant plasmid was prepared for protein induction. TransgenicGhMDH tobacco plants were obtained by Agrobacterium mediated transformationcontaining plasmid pBin438-GhMDH. The results are as follows:1.Specific primers were designed on the basis of salt stress-related ESTsequences of Gossypium hirsuturm L.. Using3’,5’-RACE technology with the RNAof CCRI35to target fragment5’ and3’end of the amplification.The pGEM-Tvector containing target fragment was transformed into Escherichia coli DH5α andpositive clones were picked up and sequenced by blue-white screening and PCRtesting. DNAMAN splicing result of the sequences showed that the full-length cDNAof GhMDH was1130bp, containing an1014bp ORF which encoded338amino acids.The relative molecular weight of putative GhMDH protein was35.495KDa, and itsisoelectric point (pI) was8.94.2.After enzyme digesting and recycling, then inserting target gene GhMDH intothe prokaryotic expression vector pET-23b, pET-23b-GhMDH was constructed.Digestion and sequencing assays showed that target gene had been successivelyintroduced into expression vector pET-23b. pET-23b-GhMDH was transferred into E.coli BL21(DE3) by heat shock, and a35KDa size protein was induced by IPTG. Then use the reaction system of L-malic acid and NAD reaction under the amount ofNADH is generated thereby measuring the change in NADH absorbance at340nmwith calculated the GhMDH activity is2.58U and specific activity is129U mg-1.3.The eukaryotic expression vector pBin438-GhMDH was constructed byinserting GhMDH gene into plant expression vector pBin438with double enzymedigestion, and transferred into E. coli competent cells DH5α. Enzyme digestion assayshowed that target gene had been successively integrated into the expression vectorpBin438. Then the expression vector pBin438-GhMDH was transferred intoAgrobacterium tumefacious LBA4404by electroporation method. The putativetransformed tobacco plants with overexpressing GhMDH gene were obtained byAgrobacterium-mediated transformation. We use nine transgenic tobacco plants toconfirme by PCR and RT-PCR analysis,and eight of them about tolerance toaluminum toxicity were then assayed. The results showed that the transgenic plantswere obviously higher in tolerance to aluminum than non-transformed plants.