Functional Analysis Of Maize ZmVPP5,Opaque7 And Shrunken4 Genes

Corn is an important food crops.High quality,high yield and the resistance to environment stress are the aim of maize breeding improvement.The study of the related functional genes is significant for maize production and breeding.Three important genes were introduced in this research.They are salinity tolerance related gene Zm VPP5,quality related gene Opaque7,and yield related gene Shrunken4.Vacuole H+-Pyrophosphatase(VPPs)are important proton pumps,which are encoded by multiple genes and related with salinity tolerance.ZmVPP5 was a truncated VPPs(vacuolar H+-Pyrophosphatase)protein,lacks the complete structure of the conserved proton transport and the inorganic pyrophosphatase-related domain,but the polymerization structure of VPP1.So Zm VPP5 may be a regulation gene of VPP1.ZmVPP5 was detected in the plasma membrane,vacuole membrane and nucleus of maize cells.Transgenic maize lines and yeast cells with overexpressed Zm VPP5 exhibited the salt hypersensitivity phenotype.A yeast two-hybrid analysis identified the ZmBag6-like protein as a putative ZmVPP5-interacting protein.The results of subcellular co-localization and a LCI assay indicated an interaction between ZmBag6-like protein and ZmVPP5 in cell.This study suggests that ZmVPP5 might act as a VPP antagonist and participated in the cellular response to salt stress.O ur study of Zm VPP5 has expanded the understanding of the biological functions of plant VPPs family.opaque7(o7)is a classic maize kernel mutant with high quality.The grains of o7 have soft,starchy and opaque phenotype.Studies have shown that,o7 mutant can significantly improve the lysine content.The O7 gene used in this study was cloned by map-based cloning in our lab.Phylogenetic analysis showed that the gene encodes a protein called acyl-CoA synthetase.The enzyme activity assay of purified O7 protein showed that oxalate is the specific substrate.O7 localized in the cytoplasm by subcellular localization analysis.The contents of TCA compositions in O7 and o7 kernels were determined by HPLC-MS.The synthesis of OAA and ?-KG has been disturbed by opaque7.That is to say,the amino acid contents were influenced by the synthetic precursor reducing.Therefore,the functional defection of Opaque7 res ults the reduction of zein and then opaque phenotype.shrunken4 is a classic maize kernel mutant with grain weight decreased.The kernel of sh4 has the collapse of the crown and sides,with significant decrease of starch and protein contents.The mutant kernel shows underfill storage material kernel,and abnormal endosperm cell.The starch granules and protein body were sharply reduced in sh4 kernels under scanning electron microscope(TEM).The biochemical component measurement results showed the significantly decreased of the protein content,starch contents,fat contents,free amino acids,glucose and the other basic metabolites.The genes expression were changed between Wild-type and mutant kernel by RNA-seq analysis.Serious PCD phenotype were observed by living cell staining.Also we could see the antophagy cell in sh4 endosperms under TEM.At last,the sh4 gene was located within a 43 Kb genomic region by fine mapping.Now we analysis the two candidate genes by transgene.The research of functional genes above improved our understanding on maize high quality,high yield and the resistance to environment stress mechanisms.These research also provided the knowledge and clues to cultivate maize lines with high quality,high yield and the stress tolerance.