Identification And Expression Analysis Of OsHDT703 In Rice

Plant-specific HD2 family proteins are an important histone deacetylase?HDAC?,plays an important role in regulating the level of histone acetylation in eukaryotes and plays an important role in plant growth and development and stress adaptation.There are three HD2 family proteins in rice,among which the biological functions of OsHDT701 and OsHDT702 have been reported,but the biological functions of OsHDT703 have not been reported.In this study,on the basis of analyzing the characteristics of OsHDT703 gene and its encoded proteins,the possible alternative splicing bodies of the gene were further identified,and the expression patterns of OsHDT703 in different tissue parts of rice under normal and stress conditions were revealed.The expression site of OsHDT703 protein in cells and its histone deacetylase activity were preliminarily determined.The transgenic rice materials with CRISPR knockout expression,over expression of OsHDT703 and its fusion expression with FLAG were created.It lays a foundation for further study on the biological function of OsHDT703 gene.Main knot The results are as follows:1.Bioinformatics Analysis of OsHDT703 and its coding proteinOsHDT703 is located on chromosome 1 of rice.The promoter sequence contains many elements related to hormone and stress response,such as drought-induced MYB binding site MBS,ABA response element,gibberellin response element and so on.There are five alternative splicing in this gene,all of which have conserved NPL domain of HD2 family proteins.Phylogenetic tree analysis showed that the evolutionary relationship between OsHDT703 protein and OsHDT702 protein was closer than that with OsHDT701 protein.2.Subcellular localization and biochemical function of OsHDT703 proteinBy constructing OsHDT703-GFP fusion expression vector and transforming Arabidopsis protoplasts into Arabidopsis thaliana protoplasts,subcellular localization analysis showed that OsHDT703 protein was mainly expressed in the nucleus of Arabidopsis thaliana.Western blot analysis showed that the acetylation level of H4histone in over-expressed plants showed a tendency to decrease.Therefore,OsHDT703 is a histone deacetylase,which can affect the deacetylation level of histone H4.3.Tissue expression characteristics of OsHDT703 in Rice under normal and stress conditionsThe results of quantitative PCR showed that the OsHDT703 gene was expressed in different tissues of rice,and the highest expression was the stem,followed by the young spike,and then the root and the leaf,and the amount of the expression in the seed was the lowest.the OsHDT703 gene has a uniform response to the low temperature,the drought and the high salt stress,but the mode of response is different;under the cold stress,the expression amount of the OsHDT703 gene in the roots,the stems and the leaves is obviously up-regulated;and after the drought treatment,the expression amount of the gene in the roots and the stems is obviously up-regulated,The expression level of the leaves is up-regulated;after high-salt treatment,the OsHDT703 The expression of the gene was up-regulated in roots and stems,but not in leaves.4.The CRISPR knockout homozygous mutant of OsHDT703 was obtained.Using CRISPR gene editing technique,24 transgenic T₀ plants were obtained,20 of them were mutated at the target site,11 of the 20 positive plants were homozygous mutations,and 45.83%of the 20 positive plants were homozygous,and the rate of homozygous mutation was 45.83%.There are four types of homozygous mutations,in which the insertion mutation rate of A base is 12.50%,T base insertion mutation rate is 16.67%,C base insertion mutation rate is 4.17%,C base deletion mutation rate is 12.50%.These four types of homozygous knockout mutations all result in the amino acid sequence inversion of the OsHDT703 gene.The translation is terminated in advance.5.The overexpression of OsHDT703 and its fusion with FLAG in transgenic rice were established.The overexpression vector of OsHDT703 and the OsHDT703-FLAG fusion expression vector were constructed,and the indica rice was transformed by Agrobacterium-mediated transformation.At present,four families of OsHDT703gene over-expressed T₂ homozygous plants,OsHDT703-FLAG fusion expression of transgenic positive T₀ plants 6 strains,laid the foundation for further study of the biological function of OsHDT703 gene...