Functional Analysis Of NADP-ME Gene In Sweet Sorghum Under Salt Stress

In this study,we found that the NADP-ME gene was associated with salt tolerance by transcriptional sequencing.And we isolated the NADP-ME gene from sweet sorghum leaves and transferred into Arabidopsis and analysized the expression and function of the gene.We also investigated the effect of salt stress on overexpression lines and mutants of Arabidopsis.The main conclusions are as follows:1.In order to study the salt tolerance mechanism of sweet sorghum.We sequenced the sweet sorghum at 0 and 150 mM NaCl stress.By the KEGG pathway,150 genes were annotated into 70 pathways in the M-81 E and 174 genes were recruited into 63 pathways in Roma,most of these genes were involved in photosynthesis and photosynthetic carbon fixation.Under different salt stress,the differentially expressed genes in photosynthetic carbon fixation were expressed in both M-81 E and Roma.However,the expression of NADP-ME gene in salt-tolerance line of M-81 E was higher than that in salt-sensitive line of Roma.From the above results we speculated that NADP-ME might be related to salt tolerance.2.We designed a pair of primers based on the known NADP-ME gene of sweet sorghum.The full length of NADP-ME gene was cloned from sweet sorghum leaves by PCR.The clone contains an open reading frame?ORF?of 1911 bp nucleotides comprising 637 amino acid residues with the predicted molecular mass of 15.7 kDa.The deduced amino acid sequence showed the NADP-ME gene in sweet sorghum leaves was 98% and 96%,respectively,compared with that of Imperata cylindrica and maize.3.The full-length of NADP-ME cDNA was cloned into the expression vector pCAMBIA3300 form constructs,and the transgenic lines was obtained by inflorescence infection.The transgenic lines were screened by the Basta screening marker,and then the phenotypic lines were detected at DNA level.4.Under salt stress,the germination rate,germination potential and root length of wild type Arabidopsis and overexpressing lines were inhibited,but the inhibition degree of different strains was different.The inhibition level of wild type was higher than the overexpression lines.This suggested that the overexpression lines of Arabidopsis had a better ability to resist salt in the germination period.The expression of NADP-ME gene was detected by RT-PCR.The results showed that under the condition of 100 mM NaCl,relative expression level in overexpression lines is the highest.We determined the seedling photosynthetic parameters,the content of chlorophyll,photosynthetic fluorescence parameters and activity of the photosystem I in transgenic line and the wild type Arabidopsis at seedling stage under 0 and 100 mM NaCl.The photosynthetic parameters of the overexpression lines were higher than wild type without salt stress,but other physiological indicators were basically the same with wild type.The contents of Pn,Ci,Tr and Gs in overexpression lines were significantly higher than those in wild type under 100 mM NaCl treatment.The results indicated that the expression of NADP-ME may increase photosynthetic efficiency under salt stress.The content of seedling chlorophyll content,?PSII,Fv/Fm and ?I/Io activity in overexpression lines were significantly higher than those of wild type under salt stress.While Fo,1-qp and NPQ of overexpression lines were significantly lower than wild type.These results indicated that the photosynthetic system of overexpression lines was less inhibited and the photosynthetic ability was stronger under salt stress than that of the wild type.We also measured the biomass under the salt stress.The fresh weight and dry weight of the overexpressing lines were significantly higher than those of the wild type.All these results indicated that the salt tolerance of the overexpressing lines was higher than that of wild type.5.SALK₀64163?At1g79750?,CS855818?At1g79750?,SALK₀36898?At2g19900?,SALK₀73818C?At5g11670?,CS833585?At5g11670?,SALK₁39336C?At5g25880?and wild type Arabidopsis were selected as materials.Results showed that SALK₀64163 and SALK₀73818C mutants had a lower salt-resistant ability than that of the wild type.Under salt stress,the germination rate,germination potential and root length of SALK₀64163 and SALK₀73818C were significantly lower than those of wild type.Therefore,the two salt-sensitive mutants were selected for subsequent experiments.At the seedling stage,the activities of Pn,Ci,Tr and Gs of SALK₀64163 and SALK₀73818C were significantly lower than those of wild type under 100 mM NaCl treatment.Under salt stress,chlorophyll content,?PSII,Fv/Fm and ?I/Io activity of the two mutant lines were significantly lower than those of wild type.While Fo,1-qp and NPQ were significantly higher than those of wild type.The fresh weight and dry weight of wild type were significantly higher than those of mutants.These results indicated that the photosynthetic system of SALK₀64163 and SALK₀73818C were subjected to greater salt damage,and its salt tolerance was significantly lower than that of wild type.The results showed that the sweet sorghum NADP-ME gene could enhance the photosynthetic capacity of Arabidopsis seedlings under salt stress.