Preliminary Functional Study of Rice OsLIR1 Gene
|Keywords||Oryza sativa OsLIR1 vector construction genetic transformation|
LIR1(light–induced, rice) is a kind of protein family regulated by light and the biological clock. The level of the LIR1 mRNA is different in different light/dark condition, performancing for movement cycle of approximate nature diurnal variation of rhythmic. LIR1 proteins from different plants have high homologous fragments, and play a major role in the light reaction and biological rhythm. There is a detailed report that rice LIR1 protein in different lights shows up rhythm following different time, but what is the regulation mechanism of rhythm and how to participate in the regulation of light system still remain unknown. Therefore, knowledge about the structure and the function of OsLIR1 protein is of great significance to fully understand LIR1 protein family on molecular mechanism.The length of the OsLIR1 gene was cloned by RT-PCR, sequence analysis showed that it is a unstable hydrophilic protein, mainly composited by ruleless curl and alpha helix, containing N-chloroplasts transshipment peptide, existing glycosyl melt, the phosphorylation and nutmeg acylation modification site. In addition, the protein includes a spiral acrossing the membrane between 3-19 amino acids, a cracking site in the KG-R of the 21-22 amino acids, which participates in the energy metabolization. The tissure-spccific expression analysis of OsLIR1 in root, leaf sheath and leaf was carried out by RT-PCR and the results demonstrated that the expression level of OsLIR1 is much higher in leave than that in leaf sheath and root.A series of expression vectors were constructed and then transferred into agrobacterium. Transgenic tobacco and rice seedlings was achieved and recognized by PCR and GUS dyeing. The green root of transgenic rice with Ubi::OsLIR1 was observated by the transmission electron microscopy(TEM), and the results showed that the chloroplasts majority have larger starch grains. The transcriptional levels of chloroplast development related gene such as Rbc L, Rbc S in the transgenic rice were different from these in the wild rice by RT-PCR.The OsLIR1 gene promoter was amplified according to the upstream sequence of OsLIR1. the cis-regulatory regulation componets analysis of the OsLIR1 gene promoter throughing the Plantcare showed that a significant amount of light regulation related components such as AE-box, DNA conservative components Box4 motif and ATCT-box, light response related components G-box, partial light response components GAG-motif, GATA-motif, I-box, Sp1 motif and TCT motif, etc. And the GUS dyeing of transgenic tobacco by OsLIR1::GUS also suggested that OsLIR1 mainly in leaf, followed by stem, and in roots almost invisible. The expression of GUS in transgenic tobacco leaf is very low by RT-PCR.In the transgenic tobacco transformed by OsLIR1-GFP, the result showed that OsLIR1 localizes in the chloroplasts of the cytoplasm.