Isolation And Characterization Of MsSOS3 From Alfalfa(Medicago Sativa L. )

Soil alkalinity is one of the important environmental factors affecting agricultural production. Currently, the research to the molecular mechanism of plant tolerance to saline-alkaline stress and breeding of salt-alkaline tolerant cultivars has become an important issue. SOS pathway, which consists of SOS family genes SOS1, SOS2 and SOS3 gene, was an important salt response pathway that function as an anti-transporter of sodium regulated by Ca2+ signal pathway to maintain intracellular sodium ion homeostasis. SOS3, a calcium sensor protein, which is important upstream regulator protein in the SOS pathway. Intracellular Ca2+ levels are altered and SOS3 is activated. SOS3 interacts with and activates the serine/threonine protein kinase SOS2, SOS3 combined with SOS2 under the changes of calcium ions concentration response to stress and regulated the activity of sodium anti-transporter SOS1. In this study, MsSOS3 was cloned to generate MsSOS3 over-expression transgenic tobacco to analyze the function of MsSOS3 gene in plant saline-alkaline tolerance, and foundation to the study of the mechanism of plant saline-alkaline stress tolerance.In this study, we present the major achievements: 1. Cloning and analysis of MsSOS3 genePrimers were designed based on the transcriptome of alfalfa in response to saline-alkaline stress, and the full length of MsSOS3 was obtained by using RT-PCR method. The complete open read frame of MsSOS3 was 684 bp, encoding 227 amino acids. By analyzed of BLASTp with homologue genes, MsSOS3 presented high identity to AtSOS3 and MtSOS3, and belonging to members of the CBL gene family, was the homeotic gene of SOS3. 2. Construction of MsSOS3 and Transformation in tobaccoThe pCBM-MsSOS3 over-expression vector was constracted with CaMV35 S promoter and selection gene Bar. The MsSOS3 gene was transformed into wild-type tobacco by Agrobacterium mediated method. 12 transgenic lines were identified by PCR and 9 positive transgenic lines were screened by qRT-PCR further. Among them, transgenic lines 4, 62 and 75 presented higher relative expression level. 3. Analysis of saline-alkaline tolerance in MsSOS3 transgenic tobaccoTransgenic lines, treated with 300 mM NaCl and 30 mM NaHCO3 respectively, were used to evaluate the ability of transgenic line to the tolerance of saline-alkaline stress. Transgenic plants leaves remain green and exhibited normal phenotype under stress, while wild-type turn white in both stress obviously. Moreover, transgenic plant presented lower level in relative electrical conductivity, content of malondialdehyde, O2-and H2O2, meanwhile, higher content concentration in total chlorophyll, soluble protein, soluble sugar, proline and activity of antioxidant enzymes(SOD, CAT, POD) compare to wild-type. Additional, transgenic tobacco accumulated more ion content of K+, which could maintain a higher K+/Na+ and showed higher tolerance to saline-alkaline stress. In summary, overexpression of MsSOS3 in transgenic tobacco increased the plant tolerance to saline-alkaline stress.