Functional Analysis Of The Chloroplast GPAT In Suaeda Salsa Under Salt Stress

Polyunsaturated fatty acid synthesis pathway is divided into eukaryotic pathway and prokaryotic pathway in higher plants. The prokaryotic pathway is existing in the chloroplast and the eukaryotic pathway means endoplasmic reticulum glycerides in the synthesis and transport to the chloroplast. In these two ways, by saturated fatty acids formed PA in this process needs the GPAT catalysis. And PG synthesized from PA. Glycerol-3-phosphate acyltransferase is the first acyl esterifying enzyme in phosphatidyl glycerol synthesis process. In this study, we isolated and characterized chloroplast glycerol-3-phosphate acyltransferase gene?GPAT? from Suaeda salsa.We also investigate the influence of salt stress on arabidopsis GPAT mutant. The main results are as follow:1. According to the homologous sequences from Arabidopsis thaliana,Lycopersicon esculentum, Spinacia oleracea, Beta vulgaris and so on, we designed two degenerate primers to amplify specific DNA fragment using c DNA prepared from Suaeda salsa leaves by PCR. The 5' and 3' fragment of the c DNA was isolated by 5' and 3' RACE. Then the full length was cloned by two specific primers. The clone contains an open reading frame?ORF? of 1167 bp nucleotides comprising 388 amino acid residues with the predicted molecular mass of 43 k Da. The deduced amino acid sequence showed the highest identities with GPAT from Spinacia oleracea and the homology is 87%.2. The full-length of GPAT c DNA was subcloned into the expression vector p B7WG2 D to form sense constructs. The freezing transformation method was used to transform Columbia ecotype type Arabidopsis thaliana by Agrobacterium tumefaciens to get transgenic lines. Extracting leaf DNA as a template, the primer from 35 S promoter on the expression vector and the prime from 3' end sequences were used to authenticate transgenic plants by PCR.3. We measured the germination rate and root length of overexpression lines of Arabidopsis thaliana and the wild type under salt stress. Results showed that the degree of inhibition in overexpression lines was lower than the wild type. This suggested that the overexpression lines of Arabidopsis had a better ability to resist salt in the germination period. We detected the expression of Ss GPAT, found that under the condition of 100 m M Na Cl, relative expression level in overexpression lines is the highest. We determined the content of seedling chlorophyll, chlorophyll fluorescence parameter and PSI activity of transgenic lines and the wild type at seedling stage under 0 and 100 m M Na Cl. The present results showed that the content of seedling chlorophyll, ?PSII, Fv/Fm and PSI activity in overexpression lines were higher than wild type. While Fo, 1-q P, NPQ of overexpression lines were lower than wild type. This suggested that the photosynthetic system of overexpression lines was damaged less by salt stress. By measuring PG fatty acid composition,we found that the PG unsaturated fatty acid content in overexpression lines was higher than the wild type.4. SALK₀60056?ATGPAT2?, SALK₁36675C?ATGPAT6?,SALK₀37660C?ATGPAT1?, SALK₀45942?ATGPAT4? and wild type Arabidopsis thaliana were selected as materials, results showed that SALK₁36675C and SALK₀60056 had a lower salt-resistant ability than the wild type. We measured the germination rate and root length of mutant lines and the wild type under salt stress.Results showed that the germination rate and root length were lower than the wild type. We also determined the content of seedling chlorophyll, chlorophyll fluorescence parameter and PSI activity of SALK₁36675C, SALK₀60056 and the wild type at seeding stage under 0 and 100 m M Na Cl. Results showed that the content of seedling chlorophyll, ?PSII, Fv/Fm and PSI activity in SALK₁36675C and SALK₀60056 were lower than wild type. While Fo, 1-q P, NPQ of SALK₁36675C and SALK₀60056 were higher than wild type. This suggested that the photosynthetic system of SALK₁36675C and SALK₀60056 was damaged more by salt stress. A lower content of unsaturated fatty acid in PG was detected in SALK₁36675C and SALK₀60056 compared to that of wild type.These results showed that Suaeda salsa chloroplast GPAT gene could enhance the salt resistance of Arabidopsis thaliana by improving the unsaturated fatty acid content of PG.