Study Of Transcription Factors ZmEREB192 And ZmEREB25 Involved In Starch Synthesis In Maize Endosperm

Starch is a kind of polymer compounds grouped by single molecule glucose,and it is applied in many fields such as food and industry.As the world's largest food crop,about 70%of storage is starch in maize(Z.Mays)seed.Maize starch is one of the starchs which have best chemical ingredients,and its biosynthetic path is a system network that involves many enzymes.There are five key enzymes:ADP-glucose pyrophosphorylase(AGPascs),starch synthases(SSs),starch branching enzymes(SBEs),starch-debranching enzymes(DBE)and starch phosphorylascs(PHOs).They are encoded by different genes.At present,the exploration for enzymatic properties of the key enzymes have been relatively thorough in biology field.But the research on the regulatory mechanism of the key genes is relatively limited.Transcriptional regulation is a very important way in the many pathways of starch biosynthesis,and it is usually associated with signaling molecules.There have been few reports about transcription factors can affect grain starch content and crop seed yield by regulating the expression of several key genes in the process of starch biosynthesis.In this study,maize master inbred lines 18-599 were used as experimental materials.We selected the TFs called ZmEREB 192 and ZmEREB25 which belong to the AP2/ERENP family as the candidate transcription factors according to the results of co-corrclation analysis and the RNA-seq of maize endosperm deal with sucrose,ABA.Our aim is to analyze the transcription mechanism involved in maize starch biosynthesis of two candidate TFs.The expression patterns of candidate genes were analyzed by semi-quantitative RT-PCR and real-time PCR.The structural characteristics of candidate TFs were verified by subcellular localization and activation experiment in yeast.The expression of key genes in starch synthesis was regulated by gene gun-mediated transient overexpression of endosperm and yeast single hybridization.Analyzing the effects of candidate TF ZmEREB 192 on endosperm starch synthesis at the genetic phenotype level by identifying the TF ZmEREB 192 Mu insert mutant.The yeast two-hybrid library was used to construct the vector with ZmEREB25 as a bait gene to verify whether it was combined with other proteins.Testing the TF ZmEREB25 combined with other proteins or not by yeast two-hybrid experiment.The results are as follows:1.The candidate genes ZmEREB192 and ZmEREB25 were successfully cloned from the endosperm of maize.The gene coding region of ZmEREB192 is 990bp and the protein encoded by it contains 329 amino acids.The gene coding region of ZmEREB25 is 879bp and the protein encoded by it contains 292 amino acids.Analyzing the amino acid sequence of candidate genes,we found they both contain an AP2 conserved domain.The two candiate TFs are belong to different subfamily in the AP2 transcription factor family.2.The expression patterns of candidate transcription factors were analyzed by semi-quantitative RT-PCR and real-time quantitative PCR.The results showed that the candidate genes ZmEREB192 and ZmEREB25 were expressed in multiple tissues respectively,and they were relatively high expressed at different stages of endosperm development.The expression level of ZmEREB192 and ZmEREB25 was the highest at 12DAP-15DAP.This period is a critical period of starch synthesis in maize.The real-time quantitative PCR was used to determine the expression of candidate genes under the conditions processing of sucrose,ABA,sucrose and ABA.The results showed that sucrose can significantly up-regulate the expression of ZmEREB192,and extreme significantly up-regulated the expression of ZmEREB25.ABA can significantly inhibit the expression of ZmEREB192,but significantly increased the expression of ZmEREB25;Sucrose and ABA co-treatment can significantly up-regulate the expression of two candidate genes.This conclusion is consistent with the results of sequencing of the RNA-seq.3.The candidate TFs ZmEREB192 and ZmEREB25 were verified to locate in the nucleus of onion epidermal cells by subcellular localization experiment.The transcriptional activity of the proteins encoded by candidate genes were verified by yeast activation experiments.The results showed that the candidate TF ZmEREB192 having transcriptional activity,but ZmEREB25 don't have transcriptional activity.So the TF ZmEREB25 was used as the bait protein for the yeast two-hybrid library,and a transcription factor ARF27 was screened to respond to auxin.But whether there is interaction between the two proteins need further verification.Constructing the constructions of the candidate gene promoters linked the reporter gene Gus,then The introduced them into the 15DAP maize endosperm.The GUS staining showed that the two candidate genes were expressed in the starch synthesis area.4.The results of overexpression experiment and yeast single hybrid experiment showed that the TF ZmEREB192 inhibited the activity of pSu2,pSul and pSh2 by directly binding to them,and significantly reduced the activity of pAe,pDul,pBtl by indirect effect.The TF ZmEREB25 promoted he activity of pSh2,pSSl,pBtl by indirect effect,and increased the activity of pDul,pBt2 by directly binding to them.5.The mutant material of the TF ZmEREB192 was found from the Mu mutant library by sequence alignment based on the gene registration number.The PCR results showed that there were 6 homozygous plants and 15 heterozygous plants.The total starch content,amylose content and amylopectin content in the mutant material and wild type W22 were measured respectively.The results showed that the total starch content in the mutant material was 10.4233 mg/g higher than that in the wild type material(Mass ratio),and is mainly caused by an increase in amylopectin content.