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Expression Influence Of Ribosomal Protein From The Mutation Of Arabidopsis Ribosomal Protein Gene RPL36B Upstream Motifs

Posted on:2008-09-19Degree:MasterType:Thesis
Country:ChinaCandidate:Q LiuFull Text:PDF
GTID:2250360242470464Subject:Biochemistry and Molecular Biology
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There is an elaborate network of gene expression regulation in cell. Especially in the eukaryote, the special and temporal-specific gene expressions can respond to the changes of internal and external environment, meeting the demands of life activities such as growth and development. Eukaryote Ribosome consists of 4 ribosomal RNAs and 80 ribosomal proteins. It is one of the most important organelle for its unique role in protein biosynthesis and critical manipulating cell growth in different conditions such as environmental stresses, nutrients variation, growth factors and development stages. There are over 200 ribosomal protein genes in Arabidopsis. Our previous work had found the expression of most of these ribosomal protein genes are tightly coordinated controlled. Bioinformatics analyzing have found two conserved motifs display in most ribosomal protein gene promoters in Arabidopsis. To further analyze these two motifs for their role in regulating ribosomal protein gene expression, we have mutated the conserved position in these two motifs in the promoter of ribosomal protein gene RPL36B respectively, and transformed these mutations into yeast, carrot and tobacco respectively. We further using Northern Blotting to analyze the expression of these mutations to character the role of these two motifs in regulating ribosomal protein genes expression.The experiment is operated following these steps. At first, the gene RPL36B encoding Arabidopsis ribosomal protein was legated to pBluescript SK(-) vector with 3xHA/3xflag markers to be cloned. And then in the mutation PCR reactions, we designed suitable primers to bring mutation in the three motifs found in the upstream conservative sequences of the Arabidopsis RPL36B promoter. After the transformation of mutants into the Saccharomyces cerevisiae, we can differentiate the endogenous RPL36B’s transcription with the help of HA markers. We can detect the difference of transcription level by Northern Blotting of the RNAs drawn from mutants, by contrast of endogenous RPL36B.
Keywords/Search Tags:RPL36B, motif, promoter
PDF Full Text Request
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