Cloning And Functional Analyzing Of SOS1from Salt-secreting Halophyte Limonium Sinense Kuntze

Salt stress is one of serious abiotic factors that affect plant growth and development.Soil salinization causes the excessive accumulation of Na+in most plants, therebyaffecting the growth of plants, but halophytes can grow normally in saline environment.Therefore, cloning the key genes and studying its function can help to furtherunderstanding the mechanism of salt tolerance of plants and using the correlation ofgenetic resources, cultivating genetically modified salt-tolerant plants, effectively improveand make use of the salinization land.In this experiment, we selected a salt-secreting halophyte Limonium sinense Kuntzeas the material. Firstly, the response of Limonium sinense seedings under salt stress andthe expression changes of LsSOS1gene, in addition the antioxidase activity were studied.Secondly, the SOS1gene of Limonium sinense (LsSOS1) was cloned. Then, theoverexpression vector, fusion gene expression vector and interference vector wereconstructed. The LsSOS1was overexpressed in Arabidopsis and the LsSOS1-GFP fusiongene was also expressed in Arabidopsis. In addition, the gene silencing LsSOS1RNAi inLimonium sinense was also been done. The Arabidopsis homozygous overexpressedLsSOS1, Arabidopsis overexpressed LsSOS1-GFP and LsSOS1RNAi resistance plantswere screened. Finally, we studied the salt tolerance of Arabidopsis homozygous andtransgenic Limonium sinense. We also researched the subcellular localization analysis ofthe LsSOS1-GFP fusion protein. These will help us to learn more about the important roleof LsSOS1in salt-tolerance of halophyte Limonium sinense.The main experimental results are as follows:(1) The response of Limonium sinense seedings under salt stress and the expressionchanges of LsSOS1geneIn the experiment of root bending, it showed that the growth of the roots was betterthan the control under low salt stress. The root growth was inhibited gradually along withthe salt concentrations increasing. Limonium sinense seedings was affected by the salt stress, as NaCl concentrations higher than200mmol/L. The expression of LsSOS1geneelevated. The root had higher expression level of LsSOS1than the leaves under salt stress.(2) The changes of antioxidant enzymes activity of Limonium sinense under salt stressThere was no significant change of the antioxidase activity compared with the controlLimonium sinense under low salt treatment. However the enzyme (SOD, POD, APX, CAT),which scavenging the endogenous oxygen radicals, having different degree increases withthe increase of NaCl concentrations. It confirmed that Limonium sinense can maintainnormal growth by adjusting its antioxidant enzyme system and its unique salt tolerantmechanisms to resist damages caused by salt stress.(3) The cloning and sequence analysis of LsSOS1geneDegenerate primers were designed according to the conservative region of SOS1genein other species, using the method of RACE, the LsSOS1gene was cloned. The openreading frame was3456bp. It encoding protein was deduced comprising1152amino acidresidues. LsSOS1had the highest similarity with the Limonium gmelinii SOS1, reaching97%. It encoded a Na+/H+antiporter.(4) The studies of Arabidopsis homozygous overexpressed LsSOS1Ten transgenic lines were screened and three homozygous transgenic Arabidopsiswere selected for the molecular studies of LsSOS1gene expression and physiologyanalysis. The LsSOS1gene was intergrated into the Arabidopsis genome and had normaltranscription and expression by molecular identification and Real-time PCR analysis.Physiological studies at various concentrations of NaCl showed that the germination andgrowth of transgenic plants were better than the wild-type Arabidopsis. The transgenicplants increased more tolerance compared to the wild-type plants under NaCl stress. Thefresh weight, dry weight, and K+/Na+ratio were higher than the wild-type, but thedifference between the transgenic plants was not obvious.(5) The subcellular localization analysis of the LsSOS1proteinArabidopsis homozygous strains overexpressed P35S::Ls SOS1-GFP were screened.Subcellular localization of the LsSOS1-GFP fusion protein was visualized by confocalmicroscope of green fluorescence in the cells of the transgenic seedlings. The green fluorescence of the cell surface reflects the SOS1-GFP expressions on the plasmamembrane or on the cell wall but not on the tonoplast or cytoplasm. To exclude thepossibility of the cell wall association of SOS1-GFP, the root was plasmolyzed bymannitol treatment to further observation.(6) The salt tolerance analysis of LsSOS1RNAi silencing transgenic Limonium sinenseThe conversion rate of explants reached20%by optimizing the genetictransformation system. Thirty LsSOS1RNAi resistance plants were screened.Three transgenic lines were selected random for the studies of LsSOS1geneexpression and physiology and biochemical analysis. Real-time PCR analysis confirmedthat the level of LsSOS1gene in the transgenic plants was lower than in the wild typeplants.The growth of transgenic plants was not good as the wild-type Limonium sinense.The tolerance of transgenic plants decreased compared to the wild-type plants under NaClstress. The fresh weight, dry weight, and K+/Na+ratio were lower than the wild-type。...