Study Of Sss?a?sbe?a Promoter Methylation Changes And Starch Content Of Wheat In Response To High Temperature After Anthesis

Objective: Wheat(Triticum aestivum L.)is one of the most important grain crops in the world.The most important component of wheat grain is starch,and there are many enzymes involved in the starch synthesis pathway.The promoter is located in the upstream region of the gene,which contains regulatory elements that play an important role in the transcription level,and studies the promoter of the key enzyme gene of starch synthesis.It is helpful to understand the transcriptional level of the key enzyme gene of starch synthesis from the source.The methylation level of sssIIa and sbeIIa promoter regions in the key enzymes of starch synthesis at different developmental stages were studied.The changes of methylation in promoter region were analyzed to elucidate the molecular mechanism of wheat starch synthesis response to post anthesis high temperature.Methods: The sss?a and sbe?b promoter were cloned from genomic DNA of Wheat(Triticum aestivum L.)Variety Xinchun 11.The promoter of the clone was linked to the plant expression vector PBI221-GFP,which removed the 35 S promoter,and its activity was identified in protoplasts of maize leaves.High temperature treatment(HT)and post 5-azaC treatment before flowering and then high temperature treatment(5azaC-HT)were carried out on spring wheat 11.The methylation level of sss?a and sbe?b promoter region was detected by MethylTarget multiple target region methylation and sequencing,and the relative expression of sss?a and sbe?b was detected by real time fluorescence.Results:(1)The sss?a promoter 1862 bp fragment and the sbe?b promoter 1864 bp fragment were cloned from the genomic DNA of the wheat variety Xinchun 11.The sss?a promoter was fused into the plant expression vector containing GFP gene and expressed instantaneously in the protoplast of maize leaves.The fluorescence was observed under confocal laser microscope,and the promoter activity of the cloned sss?a promoter was found.(2)The promoter region of sss?a and sbe?b were detected on 410 methylation sites,131 of CHG type methylation,116 for CHH type methylation and 163 for CG type methylation.(3)The level of methylation in the promoter region of sss?a and sbe?b was significantly changed after HT treatment.After 5azaC-HT treatment,there were 14 significant changes in the loci.There were 17 differences between the two different treatments of HT and 5azaC-HT.(4)After HT treatment,the relative expression of sssIIa and sbeIIa was higher at 15 DAA and 20 DAA.After 5azaC-HT treatment,the relative expression of sssIIa and sbeIIa was higher in 10 DAA,the improvement of starch content is not obvious.(5)The total starch content of wheat was lower than that of CK at all times by HT treatment.5azaC-HT treatment increased total wheat starch content at 10 DAA and 15 DAA,higher than CK and HT.But the starch content of wheat was lower than that of CK and HT in the later period.Conclusion:The methylation level of wheat grains is different at different developmental stages.When the starch synthesis of wheat responds to high temperature after anthesis,the level of the promoter methylation site increases or decreases at high temperature.The level of methylation increased,the binding capacity of the transcription factors decreased,the level of methylation decreased,the binding capacity of the transcription factors increased,and the expression of the regulatory genes affected the starch content.The 5-azaC methylation inhibitor changed the site of methylation of the promoter region at high temperatures.