Methylation Varation Of HMW-GS Gene Promoters And Their Influence On Related Gene Expression During Wheat Endosperm Development

Wheat is one of the world’s most widely distributed, top three planting area and output, and the most abundant processing products, whose processing quality is mainly decided by glutenin and gliadin in the flour. Previous researches show that the regulation of gluten expression is mainly in the transcription level, but some data shows that epigenetic modifications such as DNA methylation also play an important role on it. In this paper, firstly, we used 5 - azaC as a demethylating agent to treat Chinese Spring young ear. It was found that the accumulation of grain glutenin increased by about 21%. Then we analyzed the DNA methylation status in the promoter of HMW - GS genes (1 Bx7,1 By8, and 1 Dx2) in different development stages of wheat endosperm (10 DPA,15 DPA,20 DPA,25 DPA and 30 DPA). It was showed that the DNA methylation level of HMW-GS promoter in the endosperm is significantly lower than that of in the flag leaf. We also found that 10 DPA possessed the highest DNA methylation level compared to other development stage of endosperm, and its methylation level reduced obviously after 5-azaC treatment in the promoter regions Bx7 -561～-90 and By8 -789～-364 which possessed higher DNA methylation than other regions. Intrestingly,5-azaC treatment does not make the methylation degree lower in the low methylation region of promoter in this period. In order to investigate the influence of DNA methylation changes on the HMW-GS gene expression, we also analysed HMW-GS gene expression during the endosperm development. The results was shown that the HMW-GS gene expression as a whole was increased with the DNA methylation level decreased under the 50 μM 5-azaC treatment, especially for the Bx7 and By8 whose gene expression sharply increased with promoter methylation level significantly decreased. It is suggested that there were correlation between DNA methylation level and HMW-GS gene expression. We also analyzed the expression of major endosperm TFs TaSPA, TaPBF and TaGAMYB. The results showed that the expression of HMW-GS is consistent with the expression of related transcription factors in 15 DPA,20 DPA and 25 DPA under 50 μM 5 - azaC treatment. Correlation analysis suggested that the expression of transcription factor was significantly positive correlation with the expression of HMW-GS. For example, the expression of transcription factors TaPBF-B, TaPBF-D and that of By8, Dx2, Dyl2 showed extremely significant correlation. It was suggested that endosperm related TFs can paly important role on the expressional regulation of HMW-GS genes. To further study the effect of endosperm related TFs on DNA methylation of HMW-GS gene promoter, eukaryotic expression vectors of TaSPA, TaPBF, TaGAMYB were constructed and then transformed spring wheat. One T1 positive transgenic strain with TaSPA and three To strains with TaPBF were obtained. In addition, we also built prokaryotic expression vectors of these three TFs, and purified the protein of TaSPA and TaPBF. This built a foundation for deeper study of the relationship between the three endosperm related TFs and their corresponding cis-element with different methylation level.