| SOS(Salt Overly Sensitive)signal transduction pathway was one of the clearest study on salt-tolerant pathways.The important physiological function of SOS pathway in plant is that regulating ion steady state and improving plant tolerance to sodium under salt stress.The ThSOSs family genes were mainly included SOS1,SOS2,SOS3,SOS4 and SOS5.And the SOS1,SOS2 and SOS3 genes were involved in the SOS signal transduction pathway in cells.Ca2+ levels were changed in the cells under the stimulation of high salt signals.It was used as a second messenger which binded with SOS3 initiating the SOS signaling pathway to transmit the salt signal.In this study,four full-length SOS signal pathway genes(named ThSOSl,ThSOS2,ThSOS3-1 and ThSOS3-2)were screened by analysising of transcriptomes sequences of T.hispida.ExPASy-PROSITE,multi-sequence alignment and phylogenetic were analysis.The expression patterns of four ThSOSs genes in T.hispida were moninterd by Real-time quantitative RT-PCR under different stress treatments.Furthermore,the resistance functions of ThSOS3-1 and ThSOS3-2 genes were analyzed by transformation in Arabidopsis thaliana and transient transformation in T.hispida.The results were as follows:The results of bioinformatics analysis of four ThSOSs genes showed that ThSOS1 was Na+/H+ transporter,encoding 873 amino acids and with i28 KD the relative molecular weight.The C-terminal of ThSOSl was a hydrophobic tail in the cytoplasm.And the N-terminal was a hydrophobic region consisting of 10-12 transmembrane lipid regions.The ThSOS2 was a Ser/Thr protein kinase encoding 446 amino acids and predicted sizes with 51.4 kD.Its N-terminal was a catalytic region with 26 amino acids.And the C-terminal was a regulatory region with 21 amino acids(FISL structure),which binds to its own N-terminal catalytic region induced self-inhibition of SOS2 kinase activity.The ThSOS3 protein encodes a Ca2+ binding protein,containing three EF-arms configurations,with a high affinity and Ca2+.Its N-terminal contains a tetradecanoylated(myristoylated)site.Atypical FRQ1 Multi-domain was in the ThSOS3-1 gene.While The ThSOS3-2 gene was with a Ca2+ binding site,EFh super family domain,and an FRQ1 Multi-domain.The results of multi-sequence alignment showed that the sequence consistency of the four ThSOSs genes was low,but the amino acid sequence identity of the two ThSOS3 genes(ThSOS3-1 and ThSOS3-2)was 65%,together with AtSOS3 gene as a group.To analyze the salt and drought stress response of ThSOSs gene,the expression patters of ThSOSs genes in T.hispida under 0.4 M NaCl,20%(w/v)PEG6000 and 100 ?M ABA treatments were moniter by using the real time RT-PCR.The results showed that four ThSOSs genes can respond to these stresses,but the patterns of expression were different in different tissues of T.hispida at different times.These genes were upregulated in roots and downregualted in the leaves.Intresintings,the expression patterns were similar in roots under NaCl and mannitol stress treatment and the expression of the ThSOSs gene was more sensitive to the salt signal and ABA signal.Since the SOS3 gene is the starting signal gene of the SOS signaling pathway.At the same time,the maximum of the expression level among each gene was similarity in the root.Therefore,the stress resistance functions of 2 ThSOS3 genes were further analysised.The plant overexpression vector pROK?-ThSOS3-1/ThSOS3-2 was constructed and transformed into Arabidopsis thaliana.To analysis the Arabidopsis thaliana morphological and physiological differences between ThSOS3-1/ThSOS3-2 gene and wild type Arabidopsis thaliana(named WT),the comprehensively assessed the stress resistance function of ThSOS3-1 and ThSOS3-2 genes were studied under the stress treatments(NaCl and mannitol).The results showed that the germination rates and fresh weights of ThSOS3-1 and ThSOS3-2 transgenic lines were significantly higher than those of in control under salt and mannitol stress.And the plants of overexpression growth were also stronger than the wild type.The physiological staining and physiological indexes of transgenic plants and control plants were further compared and analyzed.NBT,DAB and Evans blue chemical staining showed that the leaf coloring of the transgenic pROKII-ThSOS3-1/ThSOS3-2 plants were lighter than that of in control under the stresses conditions.The activities of SOD,POD and the content of chlorophyll were significantly higher than those of wild type plants,and MDA content and relative conductivity were significantly lower than those of in control.These results indicate that the overexpression of ThSOS3-1/ThSOS3-2 gene Arabidopsis thaliana can maintain higher protective enzyme activity after salt and drought stress,reduce the content of cell reactive oxygen species and the degree of cell damage.In totally,the overexpression lines were a higher resistance than the WT to drought and salt.In order to verify the results of heterologous Arabidopsis expression,the plant overexpression vector and knockdown vector further were constructed and transient transformed into T.hispida.At the same time,the vector pROKII was also transient infected into T.hispida as a control.And the stress resistance functions of two ThSOS3 genes in T.hispida and control were analysised.The results of NBT,DAB staining and SOD,POD acitvities showed that overexpression plants stainning was lighter than that of the control plants under 150 mM NaCl stress,and the inhibition plants stainning were deeper than that of the control plants.The activities of SOD and POD in overexpression plants were higher than that of in control plant,and the activities of SOD and POD in inhibition plants were lower than those of in control plant.The results of Evans blue staining and MDA content showed,compared with control plants,that the overexpression plants stainning was lower and the content of MDA was lower,the inhibition plants stainning was deeper and the content of MDA was higher under NaCl stress.The results showed that SOS3 gene could increase the activity of protective enzymes and enhance the ability of intracellular scavenging of reactive oxygen species,reducing the accumulation of O2-· and H2O2.These results indicated that the ThSOS3 genes were enhanced plants salt tolerance,and there were excellent salt tolerance gene. |
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