Molecular Mechanism Of TaWAK20 In Triticum Aestivum In Response To Cadmium Stress

Wheat(Triticum aestivum L.)it is one of the main food crops in the world.Its growth,development,yield and quality are affected by abiotic stresses such as heavy metals,drought,low temperature and salinity.With the development of industrialization,the pollution of heavy metals to environment is becoming more and more serious,especially the pollution of cadmium(Cd).Cd is a non-essential element in plants.Cd stress causes plant nutritional deficiency,decreased Photosynthetic capacity,oxidative stress and other reactions.Cd is highly toxic and bioavailable,and is easily transferred and accumulated by plant roots,thus endangering human health through the food chain.Therefore,reducing the uptake of Cd by plant roots is an important method to reduce the accumulation of Cd in plants.It is important to reveal the molecular mechanism of Cd tolerance in wheat for improving yield and quality.When plants are subjected to abiotic stress,the stress signal is transduced through receptor perception and a series of stress-related genes are activated to alleviate abiotic stress damage to plants.By sensing environmental stress signals,plants activate different protein Phosphorylation pathways in their cells,and then regulate the transcription and expression of stress-tolerant genes downstream,initiating the corresponding Physiological and biochemical processes of stress tolerance,it becomes an important part of plant stress-tolerance response mechanism.Plant receptor-like kinases(rlks)are one of the most important protein kinases in plants,which include extracellular receptor domain,Transmembrane domain domain and intracellular kinase domain,as a component of upstream signaling pathways,it often plays a key role as a "Switch".Plant-like receptor kinases play an important role in abiotic stresses in plants.In our previous studies,the receptor-like kinase gene Ta WAK20 in response to Cd was screened in wheat by high-throughput sequencing and transcriptome analysis,in this study,we used real-time quantitative PCR to detect the expression of Ta WAK20 gene in wheat under different time treatment,and analyzed the changes of Physiological and biochemical indexes and PHenotypes.Tawak20-regulated upstream target genes were screened by Y1 H assay,and then verified by gel migration assay(EMSA)and transient expression assay.Subsequently,the interacting proteins downstream of Ta WAK20 were screened by Y2 H assay,co-immunoprecipitation(Co-IP),pull-down,Bi FC and Phosphorylation in vitro were used to determine the interaction.Ta WAK20 gene regulation mode was revealed through the above experiments.Therefore,the study was carried out and the following results were obtained:1.Ta WAK20 gene was mainly expressed in root by real-time fluorescence quantitative PCR analysis and reached the highest level at12 h.The responses of Ta WAK20 transgenic wheat plants to Cd stress were analyzed.The results showed that the activities of SOD,CAT and POD and the content of H2O2 in the transgenic wheat plants were increased.These results indicated that Ta WAK20 transgenic plants showed increased tolerance to Cd stress and were positive regulators of Cd stress.2.In this study,Ta WAK20 gene was cloned from wheat,and then Ta WAK20 was transformed into yeast Natural competence by Y1 H system,the resulting library plasmid sequences were then found to have the highest similarity to Tab HLH35 by screening,which we subsequently validated by methods such as gel migration experiments(EMSA)and transient expression;The results showed that Tab HLH35 was the transcription factor that could interact with the core sequence of Ta WAK20 promoter.3.The protein interacting with Ta WAK20 in wheat c DNA library was screened by Y2 H screening.The positive protein was identified,sequenced and analyzed as Ta SPL5.In order to further study the mechanism of Ta WAK20,the interaction between Ta WAK20 and Ta SPL5 was confirmed by pull-down test,co-immunoprecipitation(Co-IP),Bi FC and Phosphorylation in vitro,and Phosphorylated Ta SPL5.4.Ta WAK20 was found to Phosphorylate Thr-55 and Thr-174 in Ta SPL5 amino acid domain by in vitro Phosphorylation experiments and mass spectrometry,these two sites are the key auto Phosphorylation sites regulating the activity of Ta WAK20 protein kinase.The results showed that Ta WAK20 was tissue-specific and responsive to both Tab HLH35 gene and TASPL5 protein.In addition,the construction of wheat c DNA library and the summary of the problems in the experiment were summarized,it will lay a foundation for further optimizing the experiment and screening out the transcription factors regulating TAWAK20 mgene...