Effects On Starch Synthesis Affected By OPAQUE2 Protein In Maize Endosperm

Starch is the main component of corn kernels.The total amount of starch and the ratio of amylose to amylopectin are closely related to the yield,food quality and industrial quality of maize.Starch synthesis in kernel requires the synergistic action of enzymes such as APGase,starch synthase(SSs),starch branching enzyme(SBE)and starch debranching enzyme(DBE).OPAQUE2 protein(O2)plays an important role in the metabolic pathway of corn endosperm.The O2 was found to participate in the starch synthesis of maize endosperm in the previous work.This experiment used CAL58 and the constructed o2 near-isogenic lines CAL58/o2 as materials.A better understanding of the regulatory role of O2 in the starch synthesis pathway will facilitate the development of more valuable maize in the future.The main research results of this article are as follows:1.The phenotypic analysis of CAL58 and CAL58/o2.Compared with the wild-type full hard endosperm,the o2 mutant exhibit a soft endosperm,with a high percentage of embryos and a significant decrease in 100-kernel weight.Ultrastructure images revealed that the size,shape,distribution,and the combination of starch and protein granules were significantly different.The content of nutrient component analysis showed that it changed significantly in o2 mutant,and the change of crude starch content was the most significant.Analysis of the zein by SDS-PAGE showed that the content of ? and ?-zein was significantly reduced in the o2 mutant,and the content of ?-zein was increased compared to the wild type.2.The total of 3051 differentially expressed genes(DEGs)were screened using RNA sequencing(RNA-Seq)between wild-type and o2 maize endosperm at 18 DAP.Through GO and KEGG functional analysis,the DEGs were divided into 65 significant GO terms and 123 Pathways,covering transmembrane transport,biosynthesis of secondary metabolites,plant hormone signal transduction,lipid metabolism,nitrogen metabolism,etc.The Q value of sucrose metabolism,pentose phosphate pathway,and glycolysis/gluconeogenesis pathways closely related to starch synthesis and starch metabolism were less than 0.05,and the expression levels of some genes rlated in starch synthesis were significantly up-regulated in o2 mutant.Therefore,it is speculated that O2 may affect the starch synthesis in endosperm by regulating the expression of starch synthesis-related genes.3.Endosperms of CAL58 and CAL58/o2 seeds were harvested at 10,14,18,22 and 26 DAP,and O2 and some genes related in starch synthesis(Sh2,Bt2,SSI,SSIIIa,SSIV,ISA and SBEIIb)were quantified by fluorescence quantitative analysis.The expression pattern of O2 in CAL58 was similar to that of Sh2,Bt2,SSI,SSIIIa and SSIV.The expression level increased gradually at 10-22 DAP,reached the peak at 22 DAP,and then decreased.The expression levels of SBEIIb,Sh2,SSI and SSIV genes were significantly higher than those of wild type.It is speculated that O2 has inhibitory effects on the expression of these genes.4.The promoter vectors of Sh2,SBEIIb,SSIV,SSI,SSIIIa,Waxy and ISAI were selected to constructreport vectors,and p B42AD-O2 plasmid was used as the expression vector to carry out yeast onehybrid experiment.It was found that the O2 protein could be combined with the promoter fragments of the Sh2(1904 bp),SSI(848 bp)and SSIV(2051 bp)genes.The promoter segments of Sh2 and SSIV genes were segmented and amplified according to the location of O2 box,and the reporter vectors were constructed for Yeast one-hybrid experiments.The results showed that O2 protein interacted with the first 343 bp of Sh2 initial codon(ATG)and the 199 bp of SSIV initiator codon.