Functional Analysis Of Salt Tolerance RcSOS1 Gene In Castor(Ricinus Communis L.)

Salt stress significantly affects plant growth and development,geographical distribution and crop yield.Salt-tolerant crop varieties breeding by plant genetic engineering has become effective measurements for saline-alkali soil utilization and development.In this study,we cloned a plasma Na⁺/H⁺antiporter gene?RcSOS1?using rapid amplification of cDNA end PCR?RACE?and performed bioinformatics analysis.The heterologous expression of RcSOS1 gene was carried out in Tobacco.High efficient hairy root induction system in castor bean was established.The hairy roots of overexpressing RcSOS1 gene was used for gene function analysis.This experiment laid the foundation for the research of new germplasm of salt tolerance in castor.The main results of this study are as follows:1.we reported a plasma Na⁺/H⁺antiporter RcSOS1,isolated from Tongbi 5 castor genotype using RACE PCR method.Full length cDNA that consisted of a single open reading frame encoded a putative polypeptide of 1134 amino acids.Multiple sequence alignment showed that RcSOS1 was 74.18%identical to other plasma membrane Na⁺/H⁺antiporter proteins.Structural analysis revealed that RcSOS1 contains 12 transmembrane domains and 101 phosphate binding sites.2.Subcellular localization using onion epidermis displayed that RcSOS1 was located in the cell membrane.Transgenic tobacco hairy roots were used for the detection of the regenerated plants,RcSOS1 has been successfully inserted into the tobacco genome and can be normally transcribed by PCR and RT-PCR analyses.The content of sodium and potassium,proline and malondialdehyde and relative electrical leakage were compared between RcSOS1 transgenic lines and non-transgenic plants under 200mM NaCl treatment for 45 days.The results showed that Na⁺content of transgenic plants was significantly lower than that of non-transgenic plants;whereas the K⁺content in transgenic plants was significantly higher than that in non-transgenic plants.Na⁺efflux to increase the ratio of K⁺/Na⁺might make the contribution to the improvement of salt tolerance and maintain the intracellular homeostasis in transgenic plants.At the same time,the increased proline content,the decreased MDA content and electrolyte leakage rate contribute to eliminate excess reactive oxygen species and maintain the stability of cell membrane.3.In this experiment,hairy roots of castor were induced by three different Agrobacterium rhizogenes?R1000,C58C1 and ATCC15834?.The results showed that the highest induction rate of R1000 strain,the concentration of OD₆₀₀=0.5,explant culture 3d,bacterial infection 10min,MS medium supplemented with 50 mg/l acetosyringone had the best culture conditions.Establishment of hairy root induction system to provide the basis for obtaining establishment of transform system in castor.The overexpressing hairy roots of RcSOS1 gene was treated under 200mM NaCl.The overexpression of RcSOS1 gene increased the Proline content and decreased the MDA content,decreased the Na⁺content and increased the K⁺content.The preliminary analysis of RcSOS1 gene could improve plant salt tolerance.