Preliminarily Functional Analysis Of OsMBDl In Rice

Epigenetic refers to heritable changes in gene expression owing to various modifications of nucleotide sequence,but not to the variation of DNA sequence.DNA methylation,one of the key epigenetic regulatory mechanisms of epigenetics,can not only alter the expression of genes,but also participate in the genetic defense process.Methyl-binding domain protein(MBD)binds to methylated DNA,and regulates the expression of genes.In the previous study,we have identified a functional unknown methyl binding protein gene OsMBD1 in rice.In this study,we performed functional analysis of this OsMBD1 gene.The results are as follows:(1)Expression pattern analysis of OsMBD1:qRT-PCR analysis revealed constitutive expression of OsMBD1 in rice root,stem,leaf,seed and young panicle,whereas the expression level in leaf showed higher than in other tissues.To further analyse the expression pattern of OsMBD1,an expression vector containing the OsMBD1 promoter fused with a GUS report gene was constructed and pOsMBDl-GUS transgenic rice plants were developed.The transgenic plants therefore provided important material for future analysis of OsMBDl promoter expression pattern.(2)Functional analysis of OsMBD1:An overexpression vector pCXUN-OsMBDl and a RNAi vector pCXSN-MBD1-RNAi were constructed and transformed into rice Nipponbare calli to produce transgenic plants.Investigation of the T0 and T1 transgenic plants showed that overexpression of OsMBD1 led to delayed heading,enhanced plant height,reduced tillering,dispersed plant type.In contrast,no significant difference exhibited in RNAi lines,compared with the wild type.(3)Subcellular localization of OsMBD1 protein:a fusion expression vector pC-NGFP-OsMBD 1 harboring gfp-OsMBD1 was constructed and transiently expressed in both tobacco and rice cell.The results showed that OsMBD1 was localized in the nucleus,consistent with the functional prediction of binding to methylated DNA for OsMBD1.(4)Prokaryotic expression and purification of OsMBDl protein:a prokaryotic expression vector pMAL-c2X-OsMBD1 harboring a maltose binding protein(MBP)tag sequence fused with OsMBDl was constructed.The fusion MBP-OsMBD1 protein was successfully expressed in E.coli,and soluble recombinant MBP-OsMBD1 were purified,providing an important material for future biochemical research.Our results showed that OsMBD1 plays an important role in rice growth and development.The results in this research establish a certain foundation for deeply understanding the mechanism of OsMBD1.