Functional Analysis Of Cytoplast 6-Phosphogluconate Dehydrogenase In Pentose Phosphate Pathway From Rice (Oryza Sativa L.)

Pentose phosphate pathway(PPP) is one of important metaboilism avenues in plants, which has been known to be related to plant growth and development,and resposes to various abiotic stresses.6-phospho-gluconate dehydrogenase(6PGDH) and glucose-6-phosphate dehydrogenase(G6PDH) are two rate-limiting enzymes existing in pentose phosphate pathway.Both of them are widely present in cytosolic and plastidic compartments of higher plants and be diverged into cytoplast 6PGDH1 and G6PDH1 genes and plastid 6PGDH₂ and G6PDH₂ genes.6PGDH could catalyze the third step reaction of PPP,transforming 6-phosphogluconic acid to 5-phosphate nuclear ketose and CO₂,with deoxidizing NADPH.The reaction is not reversible.In our previous astudy it was found that 6PGDH activity and cytosolic Os6PGDH1 gene in rice(Oryza sativa L.) seedling was significantly induced by salinity,cold,drought and ABA treatments.It means that Os6PGDH1 gene could regulate plant responses to the environmental stresses.In order to investigate the function of Os6PGDH1 gene in rice responses to abiotic stresses,cytoplast 6PGDH gene sequence(accession number:AF486280) from rice were used as a query probe to search rice genome database in Genbankthrough BLAST algorithm program.By the specific primers and RT-PCR reaction the full length ORF of Os6PGDH1 gene were amplified and constructed a pET30a(+)-Os6PGDH1.This fusion expression vector pET-30a(+)-Os6PGDH1 were constructed and transformed into BL21 cells.The enzymatic activity of pET-30a(+)-Os6PGDH1+IPTG is 40.06%higher than that both pET-30a(+)-Os6PGDH1 and pET-30a(+).To research the cellular function of Os6PGDH1 gene,the resulting construct of a 35S::Os6PGDH1-GUS gene was introduced into onion epidermal cells by Agrobacterium tumefaciens.The results showed that the fusion protein,under the control of the CaMV35S promoter,was expressed transiently in onion epidermal cells,and the GUS signal was detected exclusively in the cytosol of cells.In a final,the plant expression vectors carrying sense rice Os6PGDH1 gene under the regulation of cauliflower mosaic virus 35S promoter were constructed.The leaf dishes from mature plant were infected by Agrobacterium tumefaciens EHA105 with pBI121-Os6PGDH1.The transformed plants were confirmed by PCR and RT-PCR,and their activity of 6PGDH,and tolerance to high salinity was to some extent increased.