Preliminary Identification Of Salt Tolerance Function Of Sm WRKY40 Transcription Factor From Eggplant

Eggplant（Solanum melongna L.）is an annual plant of Solanum in Solanaceae,which is rich in vitamin P and other nutrients.As one of the main vegetables in China,it is widely planted throughout the country.Each stage of eggplant growth will be affected by many environmental factors,among which soil salinization has a bad impact on the yield and quality of eggplant.The WRKY family of transcription factors plays an important role in plant stress response,and many studies have been conducted on plant response to salt stress.Through plant genetic engineering,transcriptional factors with salt tolerance function were introduced into eggplant to obtain new eggplant varieties,providing a new idea for germplasm innovation and molecular improvement of eggplant.However,it is difficult to obtain transgenic plants by conventional genetic transformation methods due to high lignification degree of eggplant.In this study,we obtained eggplant SmWRKY40 gene by homology comparison,and established the Agrobacterium-mediated genetic transformation system of eggplant.The SmWRKY40 gene was overexpressed in Arabidopsis thaliana and eggplant respectively.The function of SmWRKY40 gene under salt stress was initially verified by using overexpressed lines.The main results of the study are as follows:1.Using the multiple bioinformatics website and software were used to analyze the transcription factor of SmWRKY40 in eggplant.The results showed that the maximum open reading frame（ORF）of SmWRKY40 is 1020 bp,encoding 339 amino acids,and the conserved domain of SmWRKY40 was predicted.SmWRKY40 protein belongs to the GroupⅡsubfamily of WRKY transcription factors.By comparison with homologous sequences of other species and phylogenetic tree analysis,it is found that SmWRKY40 is closely related to Sc WRKY40 in wild tomato（Solanum chilense）.The theoretical molecular weight of SmWRKY40 protein is 37641.80 k D,and the instability coefficient（Ⅱ）is 45.17.The theoretical isoelectric point（theoretical p I）is8.84,indicating it’s a weakly acidic hydrophilic protein,with no signal peptide and transmembrane structure.Subcellular localization results show that the protein is most likely located in the nucleus.Three abscisic acid response elements（ABRE）are predicted in the upstream 2 KB region of the promoter of SmWRKY40 gene.Yeast single hybrid experiment showed that SmWRKY40 has transcriptional activation activity.The Real Time PCR show that SmWRKY40 gene is specifically expressed in the mature roots of eggplant,and is induced by abiotic stress（drought,salt,high temperature）and ABA in different degrees,among which salt stress is the most significant.2.The plant overexpression vector p CAMBIA2301-KY-SmWRKY40 was constructed,and each step of the eggplant genetic transformation process mediated by Agrobacterium was explored.The optimal explant materials and tissue for eggplant genetic transformation were screened.Kanamycin（Kan）concentration for sifting non-transformed explants and Timentin（Tim）concentration for antibiotic were screened.Each step on the efficiency of explant transformation in the genetic transformation process was studied.The results showed that’Prika’hypocotyl was the best explant material for the genetic transformation of eggplant.75 mg/L Kan could effectively screen out resistant buds in eggplant,and 200 mg/L Tim could effectively inhibit the regeneration of Agrobacterium and protect the explants from being polluted.After 3days of pre-culture,the explants had the best differentiation effect.The callus rate of resistant explants was 65.0%,and the bud rate of resistant explants was the highest,which was 15.0%under the condition.OD₆₀₀ of the bacterial solution being 0.8 and infecting for 15 min,the optimal transformation effect was reached.It was preliminarily proved that 22 SmWRKY40 positive eggplant seedlings were identified by DNA test.On this basis,q RT-PCR test was carried out on the positive seedlings to detect the expression of SmWRKY40 gene.3.SmWRKY40 overexpression vector was heterologically transformed into Arabidopsis Col-0,and it was found that the seed germination rate and root elongation of the OE line under Na Cl,mannitol and ABA treatment were significantly better than that of the wild type.It was found that the over-expressed Arabidopsis thaliana lines were less damaged by Na Cl than the WT lines,and their growth status was significantly better than that of WT.ROS and MDA contents in OE lines were lower than those in wild-type plants under salt stress,suggesting that overexpression of SmWRKY40 caused less oxidative damage to Arabidopsis thaliana.However,the activities of antioxidant enzymes（SOD,POD,CAT and APX）and the contents of osmoregulatory substances（soluble protein and proline）were the opposite.The activities of antioxidant enzymes and the contents of osmoregulatory substances in the OE lines were higher than those in the control plants.After salt treatment on the OE lines of SmWRKY40 and WT lines of eggplant,it was found that the damage to the SmWRKY40 OE material was less and the chlorophyll content was higher than that of the WT.In conclusion,SmWRKY40 plays a positive regulatory role in salt tolerance of eggplant.