Isolation And Expression Of Rice OsbolA1 Gene

Organisms can sense disadvantageous environment signals and trigger a network of signal transductions, which can finally lead to changes of gene expression patterns to allow the adaptation of organisms to the disadvantageous external environment. Transcription level regulation of genes plays a key role in the process of the organism response to the environment factors. Transcription level of gene is regulated mainly through transcription factor, which act as trans-factor to interact with the cis-element locating in the corresponding promoter region or with other trans-factors to exert the regulation functions in the responses to stresses.To date, researchers have isolated and identified a large number of transcription factors in animals, plants, and microorganisms. BolA-like protein family is one of the transcription factor families discovered in the recent years. The preliminary studies in the prokaryote indicate that BolA-like proteins are related to the regulation of biosynthetic enzymes in cell wall, the biofilm forms, and the permeability of the cell membrane, but the BolA-like protein family was seldom studied in eukaryotic organisms.In this study, a bolA-like gene OsbolA1 was isolated from rice. The gene was studied for its expression levels under various stress conditions and in different tissues by RT-PCR. The OsBolA1 protein was expressed in Escherichia coli for functional complementation and in rice callus cells for checking the subcellurlar localization of this protein. The main results are as following:1. By using RT-PCR, the full-length cDNA of OsboIA1 was isolated from rice. The homologous genes of OsbolA1 in rice and Arabidopsis thaliana genomes were retrived and analyzed for their sequence similarity with reported prokaryotic bolA genes. There are 3 and 4 homologous bolA genes in rice and Arabidopsis thaliana respectively, and genes encoding proteins with a SufE motif and a BolA motif are existed in both rice and Arabidopsis. Rice OsbolA1 gene encodes a predicted protein with 167 amino acids (AA) showing 52% sequence identity with Escherichia coli BolA protein. 2. RT-PCR method was used to study the expression level of OsbolA1 in eight different rice tissues including root, stem, flag leaf, callus. The result indicated this gene expressed in all the tissues investigated, and the expression level was highest in leaves. This result was consistent with the expression pattern of homologous gene AtbolA1(AC:EMB1374) identified in Arabidopsis, which may suggest that OsbolA1 may have the similar functions with.the homologous gene in Arabidopsis.3. By using RT-PCR, the expression level of OsbolA1 was investigated in rice plants under drought, salt, and cold stresses, and different treatments of phytohormones. The results showed that the OsbolA1 mRNA was down-regulated by drought, salt, but not affected by cold stresses and abscisic acid (ABA), kinetin (KT), or gibberellin (GA), indicating that OsbolA1 gene maybe involved in the the response to abiotic stresses. Howaver, the homologous genes in prokaryote was up-regulated by the stress signals. This difference reflects a diversification of expression pattern of the homologous boIA genes between prokaryote and eukaryote.4. The experimental result of subcellular localization suggested that OsBolA1 and GFP fussion protein mainly located in the nucleus of rice cells. This result highlights the possibility of OsBolA1 as a transcription factor in eukaryote.5. A mutant Escherichia coli strain in which the bolA gene was knocked out was generated by homozygous recombination method. The OsBolA1 fusion protein was expressed in the Escherichia coli. Function complementation in E. coli showed that rice OsBolA1 protein could partially complementate the function of E..coli BolA protein. Considering the sequence similarity of OsBolA1 with prokaryotic bolA proteins, this data indicate that rice OsBolA1 may have the conserved function as E. coli BolA has in prokaryoteS. All this information has provided enlightenments for further studies of the function of BolA-like protein family in eukaryotes.