Study On The Relationship Between α-amylase 1,2 From Hard Wheat “Chara” And PHS Or LMA

Wheat starch is an important storage material,accounting for more than 70% of dry weight.At the early stage of wheat development,starch as the main energy material is hydrolyzed by various enzymes to provide the metabolic source and energy supply for plant growth.The hydrolysis of starch requires the participation of many enzymes,among which α-amylase is the most important,which exists widely in the plant,animal,and microbial kingdom.Wheat α-amylase has many subtypes,including Ta Amy1,Ta Amy2,Ta Amy3,and Ta Amy4.According to different isoelectric points,it can be divided into high electric point α-amylase and low electric point α-amylase.These twoα-amylases are related to the quality deterioration in wheat,pre-harvest sprouting(PHS),and late maturity α-amylase(LMA).During germination,the increasing expression levels of Ta Amy1 and Ta Amy2 was increased in wheat grains promotes starch to hydrolyze into soluble sugar to provide energy and a carbon source for plant growth.So far,the mechanism of α-amylase affecting PHS is unclear,and the mechanism of starch hydrolysis by Ta AMY1 and Ta AMY2 needs to be studied further.In this study,Ta Amy1 and Ta Amy2 were cloned from hard wheat Chara.Ta Amy1 and Ta Amy2 were overexpressed in Fielder by Bx17 and Ubiquitin promoters,respectively.Overexpression of Ta Amy1 and Ta Amy2 driven by Bx17 and Ubiquitin promoters,respectively,parsing the difference between Bx17A1 OEs and LMA and the relation between α-amylase and PHS.The differences between Bx17A1 OEs and LMA wheat and the relationship between α-amylase,and PHS were analyzed.The main results were described as follows:Fifty-one Amy1 genes were isolated from 12 species of Gramineae and a Ta Amy1 gene was cloned from Chara.Phylogenetic analysis showed that there were some differences between Ta Amy1-Chara and 11 Ta Amy1 genes of CS,but the identity of amino acid sequence between Ta AMY1-Chara and Ta AMY1-A3 was 97.42%.Ta Amy1-Chara was overexpressed in wheat cultivar Fielder driven by endosperm specific promoter Bx17 and obtained five different transgenic lines.The α-amylase activity in the grains of Bx17A1 OEs was increased by 20.8~229.9 times,significantly improving the damaged starch percentage.The high α-amylase activity in leaves of Bx17A1OE1.2,Bx17A1OE2.2,and Bx17A1OE3.1 had no effect on starch and sugar.According to the comparison between Bx17A1 OEs and LMA wheat,the α-amylase in Bx17A1 OEs mainly accumulated in the endosperm,while the α-amylase in LMA wheat mainly concentrated in the aleurone layer.The high α-amylase activity in the endosperm of Bx17A1 OEs increased the content of soluble sugar(sucrose,α-gluco-oligosaccharide and glucose)in grains,while the high α-amylase activity in the endosperm of LMA wheat did not affect the content of soluble sugar in grains.High α-amylase activity in Bx17A1 OEs and LMA wheat significantly reduced RVA(Rapid Visco Analyser)value but had no effect on starch thermochemical properties and amylose.The high soluble sugar content in seeds of Bx17A1 OEs reduced the grain dormancy and enhanced the insensitivity to ABA.During germination,when α-amylase is inhibited,starch degradation can be degraded through other pathways.39 Amy2 genes were isolated from 12 species of Gramineae.One Ta Amy2 was isolated from Chara.Phylogenetic analysis showed that there were some differences between Ta Amy2-Chara and nine Ta Amy2 genes,but the identity of amino acid sequence between Ta Amy2-Chara and Ta Amy2-D1 was 99.08%.Gene expression analysis showed that the Ta Amy2 gene was mainly expressed by Ta Amy2-A1/Ta Amy2-B1/Ta Amy2-D1 during seed development and germination,while the other six Ta Amy2 genes were almost not expressed.Ta Amy2-Chara was overexpressed by maize Ubiquitin promoter in Fielder,and three different transgenic lines were obtained.During the stage of grain development,the expression level of Ta Amy2-Chara was increasing by more than one hundred times higher compared to NC.In mature seeds,α-amylase activity increased by 116.7~437.6 times.High α-amylase activity in grains resulted in decreased starch content,changed starch viscosity,and significantly increased soluble sugar content(mainly sucrose and α-glucose oligosaccharides,Germination experiments showed that high soluble sugar content in seeds reduced grain dormancy and enhanced insensitivity to ABA(Abscisic acid).During germination,the inhibition of α-amylase resulted in the increase of α-gluco-oligosaccharide content but did not affect seed germination.The increase of α-amylase activity in leaves had no effect on the mechanism of starch and soluble sugar.