Cloning And Function Analysis Of Unknow Genes TaSST And Assumptions Membrane Protein Genes TaSSA In Wheat

Soil salinization was a global ecological problem, and it was one of major factors which cause land desertification and cultivated land degradation. Soil salinization brought huge negative impacts over agricultural production and ecological environment. In recent years, with the rapid development of molecular biology, more and more attention was paid to the improvement of salt tolerance for plants by transgenic technology. The mechanism of wheat salt tolerant gene was investigated in this paper, which provided a theoretical basis for cultivating salt-tolerant wheat varieties in the future. The main contents were as follows:1.Study on cloning and function of wheat salt tolerance gene TaSSTThrough the analysis of gene microarray of wheat salt-tolerant mutant RH8706-49 under salt stress, Cluster10-30 probe sequence whose expression increased with salt stress for 12 h was selected and successfully cloned into the full-length cDNA sequence of this probe by elcetronic cloning technology and RT-PCR. The gene was 950bp long, which contained 594bp complete open reading frame and encoding 198 amino acids. By NCBI-Blast comparison, it could be found that the gene was a putative protein gene with a 89% homology to rice gene protein （Os09g0307300）. The gene was named as TaSST. The gene has landed on Genebank.Analyzing the relative expression level of wheat gene TaSST under salt stress by real-time fluorescence quantitative PCR method, it was found that the expression of TaSST was enhanced after it was induced by salt, ABA and PEG in wheat salt-tolerant mutant. Through comparion it was indicated that TaSST gene expression in salt-tolerant mutant was higher than that of salt sensitive mutant. The results demonstrated the positive correlation between and TaSST gene and wheat salt tolerance gene in molecular level,Constructing plant binary expression vector and transforming into Arabidopsis, salt tolerance in the obtained homozygote transgenic Arabidopsis plant was analyzed. The results showed that the Arabidopsis seed germination with over-expression of wheat gene TaSST was significantly better than that of the control plant after the treatment with different concentrations of NaCl, as well as the overall surviving status of plant. These indicated that TaSST involved in Arabidopsis response to salt stress.With the construction of TaSST-GFP fusion protein expression vector, transgenic Arabidopsis root distribution of GFP fluorescence was observed by fluorescence confocal microscopy in order to determine the subcellular localization of TaSST protein. The result showed that fluorescence mainly located in the cell membrane in the transgenic Arabidopsis. Thus, it is speculated that wheat TaSST protein located in the cell membrane of wheat TaSST.To determine the function of TaSST gene in abiotic stress signaling pathway, nine kind of known salt tolerance related gene in Arabidopsis including ADH, FRY1, SOS2, SOS3, SAD1, P5CS, COR15a, RD29B and KIN2 was analyzed quantitatively by real time fluorescence quantitative PCR. In transgenic Arabidopsis with over-expression gene, the expression of TaSST, ADH, P5CS, RD29B and COR15a increased, and the expression of SAD1, SOS2 and SOS3 did not change obviously, whereas the expression of KIN2 and FRY1 significantly decreased. It was speculated that TaSST might be located in the upstream of ADH, P5CS, RD29B and COR15a and involved in the regulation of osmotic stress by CDPK, ABA, SOS pathway. P5CS was a key enzyme in the synthesis of proline, proline assay results consisted with and the expression increase of P5CS in signal pathway. The result further verified TaSST was related to wheat salt tolerance.2. Study on cloning and function of wheat tolerance gene TaSSAThe same method was successfully used to clone into the full-length cDNA sequence of the Cluster2-48 probe whose expression increased with salt stress for 12 h. The gene was 915bp long, contained 699bp complete open reading frame and encoding 233 amino acids. By NCBI-Blast comparison, it was found that the gene was a putative gene containing five transmembrane structure domain and it showed high similarity with rice （OsI₀1272） homology protein with identity of 78%. The gene was named as TaSSA. The gene has landed on Genebank.Analyzing the relative expression level of wheat gene TaSSA under the stress of salt ABA and PEG by real-time fluorescence quantitative PCR method, it was found that the expression of TaSSA in salt-tolerant mutant was higher than that of salt sensitive mutant. It was speculated that TaSSA had TRAM-LAG1-CLN8 superfamil conserved domain by bioinformatics.Constructing TaSSA-GFP fusion protein expression vector, it was found that fluorescence mainly lied in the cell membrane in transgenic Arabidopsis by laser confocal microscopy. It is speculated that wheat TaSSA protein located in the cell membrane.Constructing plant binary expression vector and transforming into Arabidopsis, salt tolerance analysis result showed that the germination early cutting quantity of Arabidopsis seed with over-expression of wheat gene TaSSA was more than that of wild type with different salt concentration treatment. Meanwhile, although the root length of seedlings was basically the same as that of the control plant, the lateral root number was more, and the overall growth status of plant was better. It indicated that TaSSA might involve in Arabidopsis response to salt stress and played a positive regulatory role.Some known gene in various stress signaling pathway in Arabidopsis was analyzed by quantitative PCR analysis. The results showed that the expression of SOS3, SOS2, KIN2 increased and COR15a, and the expression of SAD1, ADH, P5CS, RD29B and FRY1 decreased in the transgenic Arabidopsis with over-expression TaSSA. It was speculated that TaSSA might in the upstream of SOS3, SOS2, KIN2 and COR15a.