Study The Regulation Function Of SmWRKY28 Proteins In Salix Mongolica Of NaHCO₃ And NaCl Stress

WRKY gene family is a typical families of gene encoding transcription regulators.Has been widely reported the function involved in plant response to adversity stress and plant protection of the structure of the development process of WRKY gene. WRKY gene expression can be induced in different levels by various biotic and abiotic stresses. But it has no been reported about the function and the relativity on saline-alkali of Salix mongolica WRKY transcriptional and regulatory proteins. In this study, has been cloned an open reading frame （ORF） of 1425bp, it encoded 475 amino acid sequences by RT-PCR technique from Salix mongolica cDNA,compared by BLAST found a WRKY structure, phylogenetic analysis showed it has the highest homology with PtWRKY28, so named SmWRKY28.Real-time PCR detected SmWRKY28 gene was expressed in Salix mongolica’s roots, stems, leaves, flowers, male and female flowers. Northern blot analysis showed SmWRKY28 in 150mM NaCl、30mM NaHCO₃、100μMMe-JA with prolonged the stress time the expression also increased. But at a time when the stress time on 48h of 30 mm NaHCO₃ there has the most obviously expression quantity.The 35S-WRKY28-GFP fusion gene by gene gun introduced into onion epidermal cells,Fluorescence microscopy showed that the fusion protein in the cell nucleus which to exercise its function. Optimization the expression conditions of small amount induced of pGEX-6p-3-SmWRKY28 in the host BL21 Central nucle protein, showed at 1mM IPTG optimal induction time 5h.GST - SmWRKY28 induced purified fusion protein of stability in the form of inclusion body,is 78 kD,which would be a prerequisite for making antibodies, it also provide the conditions for further co-immunoprecipitation.By over-expression Arabidopsis SmWRKY28 gene late germination experiments resistance analysis showed. On 80,100,150mM NaC1 and 2,4,6mM NaHCO₃ and 15,30,75μMMe-JA of 1/2MS flat over-expressing SmWRKY28 growing better than wild-type Arabidopsis thaliana, it showed a stronger saline-alkali resistance, under NaCl stress the over-expression SmWRKY28 gene Arabidopsis thaliana which the fresh weight is higher than wild type, however the MDA （malondialdehyde） content is less than the wild-type, descripted SmWRKY28 gene was related with saline-alkali resistance and improve the antioxidant capacity of plants. The chlorophyll content and fresh weight are better than the wild type of the over-express SmWRKY28 gene Arabidopsis under the stress of NaHCO₃. Further explained the SmWRKY28 transcription factors is related to the saline-alkali resistance.By agrobacterium mediated the gus gene into Salix mongolica, and gus staining show that transgene Salix mongolica have β-galactosidase activity. Establishing Salix mongolica genetic transformation system, Prepared material for further analytical SmWRKY28 gene function can be used for molecular plant breeding, laid the foundation for creating new strains of resilience foundation.