Research Of Transcriptional Regulation Of Opaque2Gene And DNA Methylation Of19-kDa α-zeins In Maize

Maize (Zea mays) is one of the most important cereals in the world, and seed is an important organ of nutrient storage, where seed storage proteins account about70%of the total proteins. The prolamins, also known as zeins, are the major seed storage proteins in maize, representing about70%of the seed storage proteins. However, the nutrient value of corn was restricted because a-zeins contain poor essential amino acids. The Opaque2(02) is a regulator of a-zeins through binding the conserved TCCACGTAGA motif in the promoters of22-kDa a-zeins, which mutent will down-regulating the expression of22-kDa a-zeins, increasing essential amino content, improving the nutritional value of corn. In addition,02can also regulate the ribosome-inactivating proteins (RIP) genes by binding the GATGAPyPuTGPu motif. In this research, bioinformatics was used to search the potential genes regulated by02gene in maize, combined two-dimensional electrophoresis (2-DE) and mass spectrometry techniques to analyze the differential proteins in W64A inbred line and its mutant W64Ao2; and the DNA methylation status of19-kDa a-zeins genes were analyzed on their promoters and gene bodies, to explore the expression and regulation mechanisms of19-kDa and22-kDa a-zeins. The main results are as follows:1. Thirty-one genes were obtained in the maize genome whose promoters contain TCCACGTAGA motif, of which8are22-kDa a-zeins genes. GO analysis showed that the822-kDa a-zein genes are involved in nutrient reservoir activity, while other genes are mainly involved in the catalytic activity. Total of683genes were obtained with GATGAPyPuTGPu motif in their promoters, of which197genes were functional annotated. GO analysis revealed that most of these genes are involved in binding, catalytic activity and cellular components, and in metabolism catalyzed, protein, amino acid and carbohydrate reaction, and some genes encode transcription factors. The tissue expression analysis showed that12genes expressed in the endosperm specifically, of which8are22-kDa a-zein genes, and others have the GATGAPyPuTGPu motif, one is the RIP gene that is regulated by02directly.2. Total of19differential proteins were identified by two-dimensional electrophoresis (2-DE) and mass spectrometry techniques. GO analysis revealed that most of these proteins are enzymes mainly involved in catalysis and metabolism process, as well as metabolic process and response to stimulus. Tissue expression analysis indicated that6genes high expressed specifically in seeds, include the seed storage protein, protein disulfide isomerase (PDI) and RIP protein, which were regulated by02directly. Five differential expression genes were detected by RT-PCR.3. DNA methylation analysis on19-kDa a-zeins genes revealed that hypermethylation on promoter will prevents gene expression, while tissue culture altered the DNA methylation status and expression levels. What’s more, this change differs from genes, showing site-specific. DNA methylation level of promoters has negative relationship with gene expression; while gene body methylation plays less effect on gene expression. Further analysis showed that expressed gene has medium DNA methylation on their bodies, while hypermethylation or hypomethylation will restrict expression.