碩士 / 國立中興大學 / 生物科技學研究所 / 97 / Chlorella sp. is an unicellular green algae with spherical morphology, which can be cultured inexpensively on a large scale. These characteristics provide rationale for using Chlorella as a new system for foreign protein expression. Different methods have been developed for the nuclear transformation of Chlorella sp., such as polyethylene glycol-mediated transformation, particle bombardment and electroporation. However the transformation frequencies of these methods are very low. In this study we established a nuclear transformation system of Chlorella sp. by Agrobacterium tumefaciens C58C1. Bamboo mosaic virus (BaMV) viral vector
carrying the green florescent protein (GFP) gene constructed in a plant binary vector (pCAMBG) was used as a reporter system. The highest nuclear transformation frequency, 92.5 ± 6.4 cells/108 cells, was achieved when Chlorella cells were co- cultivated with A. tumefaciens in dark for 48 hours, in the presence of 100 μM Acetosyringone (AS). Polymerase chain reaction (PCR) results showed a precise amplification of a 1 kb gene fragment from Agrobacterium transformed Chlorella. Southern blot analysis confirmed the integration of transgene fragment in transformed Chlorella. In the transformed cells, hygromycin resistance gene, BaMV CP and GFP
can be detected by RT-PCR. BaMV coat protein and GFP accumulation in
transformed Chlorella sp. was also confirmed by western blot analysis. In addition, the transformed Chlorella showed the expression of GFP as detected by confocal laser scanning microscope. To confirm the replication of BaMV viral vector in Chlorella sp. cells. Mutant pCAMBGdGDD was constructed with a deletion of GDD motif to disrupt the replicase activity as a negative control. To compare with pCAMBG for its BaMV CP accumulation and GFP expression, we found pCAMBGdGDD transgenic lines lack of BaMV CP accumulation and GFP expression. Moreover, transformation of Chlorella cells with the plasmid
pCAM2pBG, which carries duplicate 35S promoter of Cauliflower mosaic virus, as compared to pCAMBG for efficiency in expressing BaMV CP and GFP. Here, we report the first evidence of the successful transformation of Chlorella sp. by A. tumefaciens harboring the pCAMBG plasmid. We had established a system that can be applied for foreign protein expression with plant viral vector in green algae. This work is a first demonstration that a higher plant virus replicates in green algae.
| Identifer | oai:union.ndltd.org:TW/097NCHU5111021 |
| Creators | Chung-Chan Lu, 呂權蓁 |
| Contributors | 徐堯煇 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 68 |
Page generated in 0.0131 seconds