The Regulation Mechanism Of Opaque2 On Starch Synthesis Genes In Maize Endosperm

Maize(Zea mays L.)is an important crop,food and feed or industrial raw material in the world.Maize kernels can be divided into three main parts: maternal tissue,endosperm and embryo.The endosperm occupies most volume of the seed and storges most nutrients,including carbohydrate,fat,protein,hemicellulose and oil.Besides,the starch is the basic form of carbonhydrates,which accounts for 60% to 80% of the endosperm.The synthesis of starch involves many enzymes,including sucrose synthase(SUS),ADPG pyrophosphorylase(AGPase),starch synthase(SS),starch branching enzyme(SBE)and starch debranching enzyme(DBE).The synergistic and interactive effects of these enzymes enable the synthesis of different structure and amount of starch.Opaque 2(O2)plays an important regulatory role in the growth and metabolic pathway of maize endosperm.O2 protein is involved in the synthesis of starch in endosperm.We produced a pair of near-isogenic lines of o2 mutation which were constructed by MAS.Then the regulation mechanism of O2 protein in the starch synthesis pathway was explored by qRT-PCR,transient expression and yeast-one hybrid.The present study can provide theoretical support for the application of o2 to cultivate high quality protein maize(QPM)in breeding practice.The main research contents of this article are as follows:1.The genes were screened according to differently protein spots in the two-dimensional electrophoresis and RNA-Seq analysis in previous study.Then fluorescence quantification was used to analyse their expression level in the endosperms of CAL58 and CAL58/o2 near-isogenic lines at 10 DAP,14 DAP,18 DAP,22 DAP and 26 DAP.The results showed that in CAL58,the expressions of Sh1,Sh2 increased at 10 DAP to 18 DAP and the expression of SSIV increased at 10 DAP to 22 DAP,reaching a peak then decreased.18 DAP~22 DAP is the key stage of starch synthesis in endosperm and most of the starch granules are synthesized during this period,indicating these three genes play an important role in starch synthesis.2.Promoter sequences of Sh1,Sh2 and SSIV genes were used to construct reporter vectors,and PRI101-AN-O2 plasmid was constructed as an expression vector.These vectors were used for transient expression in Arabidopsis protoplasts.The fluorescent signal of the reporter vectors can reflect the transcriptional activity of promoters,which were regulated by the transcription factor O2.According to the analysis of fluorescent signal,it was speculated that the O2 transcription factor can activate transcriptional activation of the Sh1 gene and inhibit transcriptional activation of the Sh2 and SSIV genes.However,due to the low activity of the promoter of Sh2 and SSIV gene,the detection of O2 protein regulating these two genes is difficult,and further research is needed.3.Futhermore,we analyzed the promoters of Sh1,Sh2 and SSIV genes and selected target sites which contain O2 box(ACGT)as the core sequences to construct reporter vectors.Meanwhile,the PGADT7-O2 was constructed as an expression vector to carry out yeast one-hybrid experiments.The current experiment results showed that the O2 can bind to the 724 bp~694 bp in front of ATG of the Sh1 gene promoter,indicating that the O2 protein can regulate the transcriptional activity of Sh1 gene by binding this fragment.The study showed that the expression levels of Sh1? Sh2 and SSIV genes were changed significantly because of o2 mutation during the critical period of grain development.We found that O2 protein can bind to the Sh1 promoter directly to activate its transcriptional expression,while O2 inhibits the transcription expression of Sh2 and SSIV.Thus,O2 is involved in the starch synthesis pathway.This study is helpful to understand the function of O2 and is of great significance for perfecting O2 protein regulatory network.