Molecular cloning and characterization of chloroplast phage-type RNA polymerase gene.
| Autor: | Yih-Lin Chen, 陳奕霖 |
|---|---|
| Rok vydání: | 1999 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 87 Two types of rpoZ mRNAs, one without exon-10 and one with all exons, were observed in Arabidopsis cells. Clones containing complementary DNAs synthesized from these two types of rpoZ mRNAs by RT-PCR were constructed and sequenced, respectively. The exon-10 deleted rpoZ mRNA, presumably due to exon skipping, were detected not only in green leaves, but also in non-green tissues, calli. In order to quantify the amount of both types of rpoZ mRNAs in different cell types, a quantitative RT-PCR method using competitor RNA was performed. Two DNA clones, DC344 and FLC350, were constructed to serve as templates (each contains a cDNA fragment shortened about 100 bp of its original cDNA) for the production of competitor RNA. Results of quantitative RT-PCR revealed that exon-10 skipped transcript represents approximately 17% or 2% of the total rpoZ mRNA in Arabidopsis leaves or calli, respectively. This observation suggests that exon-10 skipping may play a role in regulation of rpoZ gene expression and exhibits different sensitivity in different cell type. In this study, Pichia pastoris expression system was chosen for the production of rpoZ protein. This system provides better expression of rpoZ gene in terms of codon usage and glycosylation. Both the intact and exon-10 deleted rpoZ cDNA were fused to gst gene or 6xhis tag respectively in the Pichia expression vectors which were used to transform P. pastoris. The P. pastoris transformants were selected and confirmed by PCR and their ability to produce rpoZ fusion proteins were identified through GST activity and Western blot analyses. Only three out of ten P. pastoris recombinants expressed efficiently the GST-RPOZ fusion proteins for each type of rpoZ cDNA. Back extraction using ammonium sulfate indicated that most GST-RPOZ fusing proteins were located in 20~40% fraction. Further purification using GST sepharose 4B affinity column did not get good yield of these fusion proteins. In order to get enough amount of rpoZ protein for activity assay, methods that can improve the purification efficiency will be tested in the future study. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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