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Expression Of Arabidopsis Ribosomal Protein Gene RPL36B In Carrot And Yeast

Posted on:2007-09-24Degree:MasterType:Thesis
Country:ChinaCandidate:C XuFull Text:PDF
GTID:2120360185961012Subject:Biochemistry and Molecular Biology
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Regulation of gene expression can occur at different levels, for instance, initiation of transcription, splicing, stablity of RNA, translation and modification of protein and so on. Every level is very important to the normal growth of cell. 5' TOP (terminal oligopyrimidine) mRNA species contain a stretch of oligopyrimidines at its very 5' end; predominantly encode for translation factors and ribosomal protein subunits. There has been extensive study in the regulation of 5' TOP mRNA in vertebrate, but there hasn't been any report about similar translational control on plant ribosomal protein mRNAs.In the carrot genome, there are three different copies of RPL36 genes encoding an identical polypeptide. It will be difficult to distinguish them. Therefore, we choose Arabidopsis ribosomal protein gene RPL36B to be expressed in carrot as an example to investigate the phenomenon and mechanism of translational control on plant ribosomal protein genes. By introducing Arabidopsis ribosomal protein gene RPL36B into carrot to generate transgenic cell lines, it is possible to study the mechanisms of translational regulation of transgenic Arabidopsis ribosomal protein gene RPL36B in comparison with the endogenous carrot RPL36 mRNAs in developing carrot embryos.This thesis focuses on the procedures of transformation of Arabidopsis RPL36B gene into carrot and yeast, detection of the transformed genes in the host cells, and examination of the expression of the transfected genes in different systems. From this work, a standard protocol of making transgenic plant and detection of the existence and expression of the transgene were established. Transformation of carrot was mediated by Agrobacterium tumefaciens GV3101 and transformation of yeast was achieved by chemical method. By using X-Gluc staining, PCR, and RT-PCR, we were able to confirm expression of the Arabidopsis RPL36B gene in transgenic carrot and in yeast. Interestingly, the results indicate that Arabidopsis RPL36B gene is differentially expressed in carrot and in yeast.Besides, we performed 5'RACE combined with DNA sequencing to confirm the transcription initiation site of Arabidopsis RPL36B gene and the complete 5' untranslated region of its mRNA. This work lays a foundation for future study of the mechanisms of...
Keywords/Search Tags:TOP mRNA, transformation, RPL36B, gene expression
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