Analysis Of Important Functional Enzymes Involved In Starch Synthesis And Isolation Of Transcription Factors Regulating Expression Of TaAGPS1 In Bread Wheat

Wheat is one of the most important food crops in the world,and starch as the main storage form of carbohydrates is the major component of gramineous crops,which provides energy and nutrients for humans and animals.The wheat filling period is an essential period for the synthesis of grain starch.Due to the rapid temperature changes in the wheat-producing regions of China,the wheat filling period is difficult to extend.Therefore,increasing the filling rate is the most important way to stabilize and increase the wheat grain weight.As the main carbonhydrate compound in the endosperm of cereal crops,starch accounts for more than 70% of grain weight,which plays an important role in stabilizing and increasing wheat grain weight.There are five main types of enzymes involved in the catalytic process of wheat starch biosynthesis.ADP-glucose pyrophosphorylase(AGPase)is a key enzyme in starch biosynthesis,which participates in the initial steps of starch synthesis.AGPase is a heterotetramer composed of two pairs of large subunits(AGPL)and two pairs of small subunits(AGPS),which is divided into cytoplasmic and plastid types.Our previous research proved that the important role of TaAGPS1 gene in starch synthesis by using fluorescent quantitative PCR method and the overexpression of the TaAGPS1 gene in transgenic plants.In this study,differentially expressed proteins,especially related enzymes involved in starch synthesis at the filling stage were identified and then the yeast one-hybrid technique was used to perform the yeast one-hybridization of the TaAGPS1 promoter and the wheat grain c DNA library to screen out the upstream regulatory factor of TaAGPS1 gene expression,so as to explore the molecular mechanism of TaAGPS1 gene expression.The main results are as follows:(1)The Label-Free proteomics technology was used to identify differentially expressed proteins in wheat after anthesis and the relevant enzymes involved in starch synthesis are11 isozymes including TaAGPL1,TaAGPS1,Ta SSI,Ta SSII,Ta BEI,Ta BEIIa,Ta BEIIb,Ta PUL and Ta DPE1.The trend of these isozymes’ expression abundance in each period of grain filling stage was analyzed,so as to reveal the molecular mechanism of wheat starch synthesis.At the same time,the proteomic results were compared with the results ofthe previous transcriptome analysis in the laboratory.9 isozymes related with starch synthesis were identified from both transcriptional and proteomic analysis,which is conducive to further reveal the mechanism of wheat starch synthesis.(2)The TaAP2 transcription factor was screened out by yeast one-hybrid technique and the c DNA sequence of TaAP2 gene was isolated.As a large family of transcription factors,AP2 is divided into three subfamilies: AP2,RAV and ERF.According to the analysis fo phylogenetic tree,TaAP2 including 2 AP2 domains belongs to AP2 subfamily.It laid the foundation for the next step to verify the interaction between the transcription factor TaAP2 and TaAGPS1 gene and whether the transcription factor regulating the expression of other starch synthesis functional genes.(3)BSMV-VIGS gene silencing technique was used to vertify the function of transcription factor TaAP2.The results showed that expression level of gene TaAP2 in BSMV-VIGS-TaAP2 silencing plants was notably reduced at 17 d after inoculation.Grain length,eidth and starch content on mature grains of TaAP2 silencing plants were decreased by 10%,which suggested that TaAP2 may participate in the starch synthase of bread wheat.