The Mechanisms Of CdbZIP23 Regulated CdZAT8 Cascade In Bermudagrass Response To Abiotic Stress

Abiotic stresses have adverse effects on plant growth and development.Among them,drought,high temperature and salinity are of high frequency during plant growth and development.As sessile organisms,plants have evolved precise mechanism to perceive multiple and fluctuating environmental cues.Bermudagrass（Cynodon dactylon L.）is a warm-season turfgrass with economic and ecological values.It is widely used in sports fields,courtyards,ecological management and other aspects.Bermudagrass exhibites tolerance to diverse abiotic stresses.However,the molecular mechanisms in response to abiotic stress have not been fully illustrated.Research on it can provide a theoretical basis for the later analysis of the molecular mechanism.In this dissertation,two stress induced genes,Cd ZAT8 and CdbZIP23,were identified and cloned based on bermudagrass transcriptome data.The expression patterns of two genes and subcellular localization of encoded proteins were characterized.Gene functions were dissected through overexpression of both genes in Arabidopsis thaliana under different stresses.Additionally,regulatory networks between Cd ZAT8 and CdbZIP23 were elaborated to provide a theoretical basis for the molecular mechanism in response to adverse stress in bermudagrass.It will also provide new ideas for breeding tolerant bermudagrass varieties with high yield and quality.The results of the study are as follows:1.Cd ZAT8 and CdbZIP23 were induced by various stresses.According to the bermudagrass transcriptome data（GSE140362）after treatments with drought,high temperature,salt and water stress,Cd ZAT8,encoding a protein with one C₂H₂-type zinc finger motif,was significantly up-regulated by multiple stresses.Expression level of Cd ZAT8 was determined in bermudagrass’Common’variety under drought,high temperature and salt treatment conditions by q RT-PCR.The results showed that the relative expression level of Cd ZAT8 was significantly up-regulated after treatments with drought,high temperature and salt,which was consistent with the transcriptome data.Additionally,the promoter of Cd ZAT8 was cloned by FPNI-PCR and the cis-acting elements of the promoter were predicted.The motif analysis result showed that ABRE,MBS,CGTCA-motif,G-BOX,TCT-motif and other elements are present in the region of promoter.Totally 8 ABRE elements,known as the binding site of the b ZIP transcription factor,are present in the promoter region at 500 bp upstream of the initiation codon of Cd ZAT8.We then further analyzed the expression level of b ZIPs family genes.The results revealed that the relative expression level of CdbZIP23 was up-regulated under drought,high temperature and salt treatments,which was in accordance with expression level changes of Cd ZAT8.2.Cd ZAT8 and CdbZIP23 localized in the nucleus.The nuclear localization signal of Cd ZAT8 and CdbZIP23 were observed by constructing a fusion expression vector for subcellular localization and performing transient transformation experiment in tobacco.The results showed that Cd ZAT8 and CdbZIP23 were predominantly localized in the nucleus.The transcriptional activation activity assay showed that Cd ZAT8 had no transcriptional activating activity by yeast two-hybrid method.3.Response of transgenic Cd ZAT8 and CdbZIP23 plants to multiple stresses.Transgenic Arabidopsis plants with overexpression of Cd ZAT8 and CdbZIP23 were generated to further analyze the function of Cd ZAT8 and CdbZIP23.The T2 generation seeds of the two transgenic lines were obtained by hygromycin screening and PCR vertification.For seed germination assays,the seeds of Cd ZAT8,CdbZIP23 and wild-type were treated with ABA,mannitol and Na Cl to compare germination rate in response to different abiotic stresses.The results indicated that there was no significant difference in seed germination under control condition.However,the seed germination rate of transgenic plants decreased significantly when compared with wild type under stress conditions.When treated with different concentrations of ABA,the germination rate of CdbZIP23 was higher than that of wild type.Moreover,the Cd ZAT8,CdbZIP23 OE lines and wild type seedlings with similar root length were selected for ABA and Na Cl treatment,respectively.The results showed that the root length of the overexpression lines of Cd ZAT8 and CdbZIP23 were significantly longer than those of the wild type,indicating that overexpression of Cd ZAT8 and CdbZIP23 enhanced tolerance to ABA and Na Cl in Arabidopsis.The CdbZIP23 transgenic and wild type plants were treated with high temperature at 42°C.The data revealed that the leaves of the three transgenic lines were healthier than the wild type in terms of leaf yellowing,wilting and withering.The chlorosisnecrosis severity indices,electrolyte leakage and the content of malondialdehyde were significantly lower than those of wild type,and the content of soluble sugar was higher than that of wild type.These results suggested that overexpression of CdbZIP23improved heat tolerance in Arabidopsis.4.CdbZIP23 might regulate Cd ZAT8 in bermudagrass in response to abiotic stresses.To explore the regulatory network between Cd ZAT8 and CdbZIP23,yeast one-hybrid experiments and dual-luciferase experiments were carried out based on the results of promoter cis-acting elements analysis.The preliminary results showed that CdbZIP23may improve tolerance of bermudagrass by binding the ABRE element in the promoter region of Cd ZAT8 to regulate the expression of downsteeam genes and enhance stress tolerance in bermudagrass.The results above indicated that Cd ZAT8 and CdbZIP23 in bermudagrass were induced by various abiotic stresses.Cd ZAT8 and CdbZIP23 localize in the nucleus.CdbZIP23 might regulate Cd ZAT8 expression in bermudagrass to improve tolerance to ABA and Na Cl in transgenic plant.These results provided a theoretical basis for illustrating the molecular mechanism of bermudagrass under abiotic stress condition.