碩士 / 國立臺灣大學 / 漁業科學研究所 / 88 / Abstract
Microtubules is found in all eukaryotic cells which is majorly formed by polymerization of -andβ-tubulin (tub) subunits. β-tubulin, a multifunctional filament, is abundent in organelles and subcellular structures. β-tubulin gene is highly conserved among eukaryotes. Despite the nucleotide and aminio acid sequences of tub from higher eukaryotes is well studied, few studies have been involved the marine invetebrate tub gene families, including arthropod.
This study is to understand the organization structures ofβ-tubulin genes of giant freshwater prawns, Macrobrachium rosenbergii. Primers were designed and used for the β-tubulin genomic sequence PCR amplification. Five clones containing β-tubulin sequence of M. rosenbergii were isolated and sequenced. Results revealed that β-tubulin gene family may comprise at least five members (Type 1.2.3.4.5). We also used extender PCR (Extender Polymerase Chain Reaction) method to complete the 5’ and 3’end of tub genomic sequence. Two types of M. rosenbergii tub genomic clones were isolated. Type 1 (1359 bp) and Type 5 (1368 bp). Type 1 and Type 5 tub encoded for 452 and 455 amino acid squences, respectively. They exhited 81% and 71% similarities between nucleotide and amino acid sequences, respectively. But there existed high divergency at 3’ region. Phylogenetic analysis of M. rosenbergii β-tubulin genes by neighbor-joining method analysis suggest that M. rosenbergii β-tubulin gene is closely related to American lobster (Homarus americanus).
We microinjection two DNA constracts into the fertilized egg of medaka： pEFGPSβ0.4 (Upstream —102 bp to +322 bp) and pEFGPSβ0.1(only contains upstream —102 bp). Data showed that reporter gene (EGFP cDNA) could be driven by the promoter sequence we cloned at the medaka embryos. The GFP expression of embryos injected with pEFGPSβ0.4 was stronger than that of pEFGPSβ0.1.
PCR-based strategy with one set of specific primers： Tu-f6 and Tu-r7. The predicted 426 bp fragment was always amplified from 32 different invertebrte species. This primer pair may be widely applicable for amplifying invertebrate β-tubulin genes and may also provide a useful tool for genome analyses of β-tubulin in other species of invetebrate.
| Identifer | oai:union.ndltd.org:TW/088NTU00451002 |
| Date | January 2000 |
| Creators | Ping-Hong Chen, 陳炳宏 |
| Contributors | Huai-Jen Tsai, 蔡懷楨 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 70 |
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