Preliminarily Study On The Function And Regulation Mechanism Of WRKY Transcription Factor OsHsHS In Rice

Transcription regulation is the most important in regulation network,and gene transcription is mainly regulated by transcription factors.WRKY transcription factors belong to a large group of plant transcription factors,which usually regulate biotic and abiotic stress responses.Our previous study found a WRKY transcription factor that displayed hypersensitivity to heat shock,and was named OsHsHS(Oryza sativa Hypersensitive to Heat Shock).OsHsHS located in the nucleus like other WRKY transcription factors.In this study,the function of OsHsHS gene was studied by changing the express-ion of OsHsHS gene.We established overexpression,RNAi and CRISPR-CAS9 con-struct,and then transformed them into MH86 or Zhong hua 11.We obtained two transgenic plants that were overexpressed,and one transgenic plant that was repressed after checking with RT-PCR and Northern Blot.We have already got a few transgenic plants with CRISPR/Cas9.To further reveal the function of OSHsHS gene,we detected the expressions of OsHsHS gene in wild type seedling under different treatments.Result showed that the expression of leaf and leaf sheath was higher than that of lower part,and OsHsHS gene could be induced by JA,ABA,high temperature,low temperature,drought and high salt.We also examined the expression of OsHsfA2d and OsPr1a after heat treatment.Interestingly,the expression of OsHsfA2d gene was inhibited,whereas OsPr1a gene expression increased rapidly and then decreased.In order to reveal the interaction protein with OsHsHS,the yeast two-hybrid library was screened,and no protein was found to interact with OsHsHS.We noticed that some WRKY transcription factors in Arabidopsis can interact with VQ proteins,and VQ8,VQ9,VQ15 and VQ33 were discovered to interact with OsHsHS with different strengths.OsHsHS gene could bind to W-box,and EMSA(electrostatic mobility shift assay)results demonstrated that OsHsHS weakly bind to W-box.Previous result showed that OsHsHS could regulate the expression of ROF gene.Sequence analysis showed that the promoter region of the gene contained W-box sequence.EMSA detection also revealed that OsHsHS could bind to W-box in ROF gene promoter,but the binding was weak.The result implied that there may be other sequences that affect the OsHsHS protein binding to the promoter.We used SELEX(Systematic Evolution of Ligands by EXponential enrichment)technique to select DNA fragments that could bind to OsHsHS.We also used this method to detect WRKY45 and Myb42 for finding their binding motif.Myb42 could bind to GATHY motif,but we didn't find WRKY45 and OsHsHS binding motif.ChIP(Chromatin Immunoprecipitation)can be used to detect the interaction between transcription factor and promoter in vivo.The ChIP result can be amplified and recovered,and then the product was sequenced with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins.In this experiment,antibody is an important factor,which was used to pull-down the DNA-associated protein.We try to prepare the antibodies required for ChIP experiments.Western Blot result show that our antibody can effectively and specifically bind OsHsHS and should be used for ChIP assay.Summarizing upper results indicated that OsHsHS should involve in a variety response reactions,and is a multifunctional protein.OsHsHS can interact with several VQ proteins.To further reveal the OsHsHS regulatory network,we will investigate the VQ proteins functions.