Transformation Of Wheat 5-Methylcytosine DNA Glycosylase Silencer And Identification Of HD2 Gene Family

Epigenetic modification is one of the most important regulation methods in organisms.Methylation and acetylation modification are two important epigenetic modifications,which participate in many regulatory processes.Wheat is one of the most important food crops in the world.Since the genome of wheat is huge and complex,there are still many unknows in the study of epigenetic modification in wheat.5-methylcytosine DNA glycosylase?DME?is an important demethylated enzyme,which is involved in many important biological processes in plants.The HD2 family is one of the subfamilies of HDACs that specifically exist in plants.It is involved in the development of multiple tissues in plants,and also plays an crucial role in abiotic stress responses of plant.In this study,the plasmid Ubi-DMEhp was constructed and transformed into the wheat plant.Silencing the expression of DME gene in wheat lays the foundation for the further study of DNA methylation/demethylation in wheat.HD2 genes of wheat have been identified.Meanwhile,their genomic organization,phylogenetic relationship and expression profiles have been analyzed by using bioinformatics methods.Moreover,the quantitative real-time PCR was performed to investigate the expression of wheat HD2 genes under heat stress.Overexpression vectors were constructed to provide basis for further study of the function of wheat HD2 genes and there use to improve wheat quality.The main results of the study are as follows:1.Based on the pHMW-DMEhp plasmid and pGEM-Ubi-GFP-nos,a one-step cloning method was used to construct plasmid pUbi-DMEhp.2.The pUbi-DMEhp was transformed into wheat immature embryos using the high-efficiency gene gun transformation method in our laboratory.The transgenic T₀generation wheat were obtained and 5 positive plants were screened out by PCR.The T₁generation were continued to grow and 35 positive plants were screened out by PCR.3.Using the methods of bioinformatics such as HMMER and BLAST,12 wheat HD2genes were identified and their isoelectric point,subcellular localization and relative molecular mass of protein were predicted.According to whether or not the C-terminus of the protein has a zinc finger structure,the HD2 genes were divided into Group 1 and Group 2.These genes were named according to their chromosomal location information.4.The WheatExp website was used to analyze the expression level of wheat HD2 gene in different tissues at different times.According to the results,log 2 was used to plot the heat map.The RNA-seq data in NCBI-SRA database was used to analyze the expression levels of wheat HD2 gene under heat stress,drought stress,and heat stress plus drought stress.According to the results,log 2 was used to plot the heat map.5.Specific primers were designed.Wheat leaves,that treated with 35?/1h or 42?/1h,of12 different development stages,three days of seedlings,seven days of seedlings,tillering stage,vernalization period,standing stage,jointing stage,flag leaf stage,heading date,blooming stage,early filling stage,late filling stage and maturation period of wheat,were selected to detect the expressions of HD2 genes.Most of HD2 genes were highly expressed in flag leaf stage and heading date under heat stress treatment.It suggests that the HD2 genes may play roles in the development of wheat in leaf stage and heading date in response to heat stress.