The Functional Analysis Of The Unknown Gene AtBRU-X In Arabidopsis Thaliana

In this paper,the expression pattern of unkown AtBRU-X (Brassinosteroid-Upregulated-X) gene under various treatments was investigated by semi-quantitative RT-PCR in Arabidopsis.Meanwhile,the interaction between BR and NO in the process of expression and regulation of AtBRU-X gene was studied,and the preliminary analysis and prediction of the function of AtBRU-X protein were performed.This paper aims to preliminarily explore on the function of unkown AtBRU-X gene of Arabidopsis.The summary is as follows:1.AtBRU-X gene is 1219 bp in length with an ORF of 263 amino acids, corresponding to a protein of molecular mass 29.3 KD and an isoelectric point of 10.06. The hydropathy plot analysis of AtBRU-X sequence showed most amino acids to be hydrophilic.There exists an OprD conserbed domain within the amino acids between 255 to 262,which is an outer membrane porin.2.RT-PCR analysis showed that expression of AtBRU-X gene could be detected in roots,stems,leaves and flower at a certain level without tissue-specific.3.AtBRU-X gene could be induced by brassinolide to a great extent in Col-0, which was much more significant in the det2 mutant.However,while NO scavenger PTIO had been added,the transcript abundance of AtBRU-X gene was no longer induced by BL.This indicates that endogenous NO accumulation plays an important role in the inducing AtBRU-X gene expression by BL.AtBRU-X expression is also sensitive to the signal molecular NO and H₂O₂ at different degrees.The RT-PCR analysis in BR mutants suggests that the expression and regulation of AtBRU-X gene by NO does not depend on BR signals.These observations indicate that the regutory site of the target gene which BR reacts in may locate downstream of where NO does. BR signaling pathway may have a feedback regulation on NO response of AtBRU-X gene.4.We found that expression of the AtBRU-X gene significantly increased in green tissues and roots after exposed to 4℃,150 mM NaCl and 300 mM mannitol for 12h, which decreased after treatment with 38℃for 3h.These results indicate that AtBRU-X gene is involved in the responses to cold stress,salt stress and osmotic stress of Arabidopsis.The various stress treatments in det2 mutant were also carried out,which showed that salt and water stress induction of AtBRU-X gene depended on the BR, implying that the process of stress response of AtBRU-X gene closely relates to BR.5.Also two pairs of specific primers carrying restriction sites were designed according to the published sequence of AtBRU-X gene cDNA.We obtained the AtBRU-X gene CDS region At(BRU-X)1 and At(BRU-X)2 by PCR.The prokaryotic expression vector pGEX-At(BRU-X)1 and eukaryotic expression vector pCAMBIA1300-At(BRU-X)2-GFP were successfully constructed.And the preliminary analysis of prokaryotic expression in Rosetta(DE3),protoplast transient expression of AtBRU-X protein were carried out.These will be helpful for further study of AtBRU-X gene.6.Besides,We obtained the homozygous mutant of the AtBRU-X gene with T-DNA insertion ordered from SIAGnAL by using PCR.This provides the experimental material for the further study of the gene function.