Construction Of The Over-expression G6PDH Plants And Its Mutant Screening In Arabidopsis And The Function Analysis Of Cytosol G6PDH Under Salt Stress

In plants,the pentose phosphate pathway(PPP) is another major glucose metabolic pathway besides glycolysis.PPP is also a key source of reducing power NADPH.As one of the rate-limiting enzymes in PPP,Glucose-6-phosphate dehydrogenase(G6PDH)converts glucose-6-phosphoric acid into delta-6-phosphate glucose acid lactone and release NADP H.G6PDH is encoded by nuclear genes and located in cytoplasm and plasmid.Many studies have proven that G6PDH plays an important role in plant growth and development and adaption to adverse stresses,but the physiological and molecular mechanism remains elus ive.In order to have a deep ins ight of the function and regulation mechanism of G6PDH in plant growth and environmental adaptation,we cloned the G6PDH family genes,constructed over-expression vectors of G6PDH genes,and obtained the over-expression transgenic plants.Meanwhile,we screened and identif ied G6PDH homozygous T-DNA insertion mutant plants.With these mutants,we explored the function of cyt-G6PDH under salt stress with g6pdh5 and g6pdh6 mutant plants.The main results of the paper are as follows:1.Cloned the G6PDH family genes by high fidelity polymerase(G6PDH1,1732bp;G6PDH3,1801bp;G6PDH4,1879bp;G6PDH5,1552bp;G6PDH6,1549bp).PCR products were in the same sizes as predicted G6PDH genes.These products were ligated to the p EASY-Blunt vector to further determine the sequences.2.Used Gateway technology to clone the target genes to binary expression vector p GWB2and transformed the expression constructs into agrobacterium.3.Obtained G6PDH over-expression plants using the method of floral dipping and screened homozygous G6PDH transgenic plants through the Hyg resistance selection and DNA genotyping.4.Purchased Arabidops is mutant seeds of G6PDH family genes from ABRC.Us ing the three primer PCR method,we identif ied homozygous T-DNA insertion mutant plants for five G6PDH genes:G6PDH2(GK319B08,insertion in the sixth exon),G6PDH3(SALK?002754,insertion in the ninth exon),G6PDH4(SALK?131208C,insertion in the second exon),G6PDH5(CS804669,insertion in the tenth exon),and G6PDH6(SALK?016157C,insertion in the thirteenth exon).RT-PCR showed that the expression of corresponding genes was completely knocked out in homozygous mutant plants.5.The analysis of expression pattern of G6PDH genes showed that cytosolic G6PDH5 and G6PDH6 express relatively high in all examined tissues;whereas the expression of plasmid G6PDH(G6PDH1,G6PDH2,G6PDH3,G6PDH4)is low,suggesting that cyt-G6PDH genes may play a more important role in plant growth and development and adaption to environment steesses.6.Germination experiment showed that,under normal condition,the germination of g6pdh5and g6pdh6 was delayed compared to Col-0,but they reached the same germination rate as Col-0at 48 h.The germination lag phenomenon of g6pdh5 was more significant than that of g6pdh6seeds.7.The expression of G6PDH5 and G6PDH6 was induced by 200 m M Na Cl and reached peak at 24 h and 12 h,respectively.G6PDH activity test results showed that salt stress stimulates the G6PDH activity.8.The content of H₂O₂ and membrane permeability measurement showed that under salt stress,the accumulation of H₂O₂ was higher in the g6pdh5 and g6pdh6 leaves than in WT;Ion leakage level also increased signif icantly in g6pdh5 and g6pdh6,suggesting that g6pdh5 and g6pdh6 mutants suffer stronger oxidative stress.These results indicated some important function of cyt-G6PDH under salt stress.Based on the above results,we conclude that this research established the foundation for exploration of the function and molecular mechanism of G6PDH in plant growth,development and salinity tolerance.