| Cerasus humilis(Bge.)Sok.is a kind of perennial shrub of Rosaceae Cerasus in China.It has good resistance to saline alkali,drought and low temperature.The fruit can be eaten fresh and has important ecological and economic value.The current research focuses on new variety breeding and fresh fruit processing,but the potential molecular mechanism of Cerasus humilis to salt stress is not clear.Taking “Nongda No.4” Cerasus humilis variety as experimental materials,this paper studied the characteristics and salt resistance function of ChDREB2 C transcription factor,The main results are as follows:1.The ChDREB2 C gene was cloned from the leaves of Cerasus humilis.The total length of the gene was 1167 bp,including a typical AP2/ERF conserved domain.Phylogenetic analysis showed that the Cerasus humilis ChDREB2 C had high homology with Prunus mume Pm DREB2 C and Prunus avium Pa DREB2 C.The expression patterns in different tissues of the ChDREB2 C were analyzed by q RT-PCR,and the expression of this gene was the highest in leaves.Under Na Cl stress on three week old Cerasus humilis,it was found that ChDREB2 C was significantly induced by Na Cl,and the expression was the highest at 3 h.2.The subcellular localization was carried out by Agrobacterium tumefaciens infecting the of onion inner epidermis and tobacco leaves.The results showed that ChDREB2 C protein was localized in the nucleus.Transcriptional activation test showed that ChDREB2 C had transcriptional activation activity and the transcriptional activation region was at the C-terminal.Yeast two hybrid system showed that ChDREB2 C could form homodimer by itself.3.Arabidopsis thaliana was transformed by Agrobacterium tumefaciens flower soaking method and treated with 0 m M,50 m M,100 m M and 150 m M Na Cl stress,respectively.By observing the leaf phenotype,it was found that the growth of overexpressed Arabidopsis was better than that of wild type.By measuring the physiological and biochemical indexes,it was found that the germination rate,root length,relative water content,proline content and antioxidant enzyme activities of overexpressed Arabidopsis were significantly higher than those of wild type,while the electrolyte leakage and malondialdehyde content were significantly lower than those of the wild type.Indicating that overexpression of ChDREB2 C could improve the salt tolerance of Arabidopsis under Na Cl stress.4.The expression of related genes was analyzed by q RT-PCR.It was found that the expression of salt-related genes such as At SOS1 and At NHX1 were significantly up-regulated,and the expression of ABA-related genes such as At RD29 A,At RD29 B,At KIN1,At ABA4 and At ABF2 were significantly up-regulated.The p YSE2-ChDREB2 C vector was constructed and transformed into Y1 H gold yeast competent cells.It was found that overexpression of ChDREB2 C gene could improve the salt stress tolerance of yeast under Na Cl stress.5.Through yeast two hybrid and firefly luciferase complementary imaging experiment,it was found that ChDREB2 C interacted with ChABF2.Moreover,the results of subcellular localization showed that ChABF2 protein was located in the nucleus,cytoplasm and cell membrane.The above results lay a foundation for further analyzing the biological function of Cerasus humilis ChDREB2 C transcription factor in resisting salt stress,and provide a practical reference for improving Cerasus humilis varieties by using molecular biology. |
