Abstract：Abstract The salt overly sensitive (SOS) signal-transduction pathway is a control ion balance signal pathway. This pathway plays a key role in discharge Na+ and ion homeostasis on cellular lever and improves plant salt resistance. It has some realistic significance on enhanced salt tolerance in alfalfa (Medicago sativa), which transformed with muti-gene mediated by Arabidopsis thaliana SOS1, SOS2, SOS3, SOS3-like calcium binding protein 8 (SCaBP8) and bialaphos resistance (Bar) 5 genes of polygenic plant expression vector pSOS. The Agrobacterium-mediated transformation method was used, "Golden Empress" cotyledonary nodes of alfalfa were selected as explants. The best concentration of glufosinate herbicide was identified as 0.6 mg/L, the best concentration of bacteriostat cefotazime was identified as 300 mg/L. 25 independent sources of transformed plants were obtained through screening after the herbicides selection. These plants applied PCR amplification and molecular detection, which showed that there were 6 transformants with the special bands. The positive rate was 24%. Four of positive transgenic plants were randomly selected Southern hybridization identification, and the results showed that there were 3 hybridization signal, and copy number was 1 or 2. The result of RT-PCR analysis demonstrated that the same as expected electrophoretic bands size of Bar gene (463 bp) and SOS1 gene (700 bp) expressed in transgenic plants. It was proved initially that multiple exogenous salt-tolerant genes have been integrated into the transgenic plant genome and expressed at the transcriptional level. After treated with 250 mmol/L NaCl stress, the growth of wild type plants and transgenic plants were inhibited, but the plant height of the transgenic plants were significantly higher than the wild type, the leaf area of the transgenic P1 strain and P2 strain were obviously higher than the control group, the fresh weight of 3 strains were significantly higher than controls, leave chlorophyⅡ content of the transgenic P2 strain and P4 strain were apparently higher than the control group, which demonstrated overexpression of SOS pathway genes could improve the salt tolerance in transgenic alfalfa. The successful construct of transgenic alfalfa provides theoretical basis for selection for saline-alkali soil planting the salt-tolerant alfalfa varieties and alfalfa salt tolerance identification and salt tolerance mechanism research.
[1] 黄文慧，刘自学.概论苜蓿的分布和发展[A].耿华珠.中国苜蓿[M].北京；中国农业出版社，1995:2-7.
[2]Wang Shu jie, Yan Shu zhen, Li Shifang.Callus induction and plant regeneration derived from hypocotyls of alfalfa (Medicago sativa L)[J].Plant Protect ion, 2005, 31(3):502-521
[3] Chen QJ, Xie M, Ma XX, et al.MISSA is a highly efficient in vivo DNA assembly method for plant multiple gene transformation.[J].Plant Physiol, 2010, 153(3):41-51
[4] Yang Q, Chen ZZ, Zhou XF, et al.Overexpression of SOS (Salt Overly Sensitive) genes increases salt tolerance in transgenic Arabidopsis.[J].Mol Plant, 2009, 2(3):22-31
[5] Quan RD, Lin H, Mendoza L, Zhang Y, CaoW, Yang Y, ShangM, Chen S, Guo YSCABP8/CBL10, a putative calcium sensor, interacts with the protein kinase SOS2 to protect Arabidopsis shoots from salt stress.CABP8/CBL10, a putative calcium sensor, interacts with the protein kinase SOS2 to protect Arabidopsis shoots from salt stress[J].Plant Cell, 2007, 19(1):1415-1431
[6] Berres R, Otten L, Tinland B, et al.Transformation of vitis tissue by different strains of Agrobacterium tumefaciens containing the T-6b gene.[J].Plant Cell Rep, 1992, 11(1):192-195
[7] Allen GC, Spiker S, Thompson WF.Use of matrix attachment regions (MARs) to minimize transgene silencing.[J].Plant Mol Biol, 2000, 43(1):361-376
[8] Xue H, Yang YT, Wu CA, et al.a novel strong matrix attachment region isolated from tobacco, increases transgene expression in transgenic rice calli and plants. [J].Theor Appl Genet, 2005, 110(1):620-627
[9] Deak M, Kiss G B, Korz C, et al .Transformation of Medicago by Agrobacterium mediated gene transfer.[J].Plant Cell Report, 1986, 5(1):97-100
[10] 化烨.GsSAMS基因对苜蓿的遗传转化及转基因新株系的培育[D].哈尔滨：东北农业大学，2009
