Cloning, Functional Analysis Of P5CS Gene In Caragana Korshinskii Kom And The Changes Of Physiological Index Under Salt Stress

P5CS is a key enzyme of plants synthesis proline. In the way of glutamate synthesis to proline, P5 CS is a bifunctional enzyme, which can catalyze the phosphorylation of glutamate or reduced glutamate gamma semialdehyde, its activity is regulated by feedback regulation of proline. At present, P5 CS gene is already been cloned from cowpea, tomato, bean, apocynum, sugar cane ext. The research showed that the expression of P5 CS gene is significantly affected by water stress.Caragana korshinskii Kom is a shrub, belongs to the Leguminosae Caragana, it has higher resistance ability, including windbreak and sand fixation, soil and water conservation. By the research of cloning, bioinformatics analysis, expression analysis in P5 CS gene of Caragana korshinskii Kom, it can provide the reference frame of osmotic stress tolerance for plants in the desert, saline and alkaline area. The main results of this study are as follows:1. We isolated a new novel P5 CS gene, designated Ck P5 CS, from Caragana korshinskii Kom. The cloned full-length c DNA of Ck P5 CS was 2602 bp with an open reading frame(ORF) of 2322 bp comprising 773 amino acid residues with the predicted molecular mass of 86.2Kd, Its isoelectric point is 8.5.Homology analysis showed that Ck P5 CS and Gm P5 CS had the highest homology. Protein sequence analysis showed that the Ck P5 CS protein is a hydrophilic protein, no signal peptide,no transmembrane domain contain.2. Real time fluorescent quantitative PCR results showed that: The expression of Ck P5 CS gene in Caragana korshinskii had no tissue specificity expression and the leaves have highest expression. The Ck P5 CS gene in Caragana korshinskii Kom expressed under low temperature(4 ℃), salt, and drought stress. With the time of cold stress prolonged, the expression of Ck P5 CS gene in Caragana korshinskii Kom increased first and then decreased, when the processing time to reach 24 h, the expression quantity reached the maximum; with the increasing of salt stress concentration, the expression of Ck P5 CS gene in Caragana korshinskii Kom increased first and then decreased, when salt concentration was 100 m M, the expression quantity reached the maximum; with the increasing of drought stress concentration, the expression of Ck P5 CS gene in Caragana korshinskii showed an increasing trend, under 20%PEG, the expression quantity reached the maximum.So we inferred that Caragana Ck P5 CS gene is involved in abiotic stress response.3. The Caragana korshinskii Kom as the experimental material,under different concentrations salt stress(50m M、100m M、150m M、200m M), with the increasing of salt stress concentration,the MDA contention in leaves of Caragana korshinskii Kom showde overall upward trend, when salt concentration was 100 Mm, the MDA contention reached the maximum; The proline contention increased gradually, when salt concentration was 200 Mm, the proline contention reached the maximum;Chlorophyll contention increased first and then decreased, when salt concentration was 100 Mm, the proline contention reached the maximum; The activity of CAT, APX,GPX showed a trend of first increase and then decrease, when salt concentration was100 Mm, the activity of CAT and APX reached the maximum, when salt concentration was 50 Mm, the activity of GPX reached the maximum, the activity of SOD showed a rising trend, when salt concentration was 200 Mm, the activity of SOD reached the maximum.By the changing of these physiological indices in Caragana korshinskii Kom, that we can speculate Caragana korshinskii Kom could grow normally in the salt stress environment.