碩士 / 國立海洋大學 / 海洋生物研究所 / 92 / Abstrat
We tested the accuracy of two techniques that were used to detect gene expression in phytoplankton. These techniques were quantitative reverse transcription -polymerase chain reaction（Q-RT-PCR）and dot blotting. Items tested included specificity of primer pairs and target dots, consistency of results when RNA was extracted from pure and mixed algal cultures, and differential gene expression in low-phosphate and normal growth conditions. In addition, several field samples were tested and how experiences gained in laboratory tests can help to explain the field data was discussed. The genes and algal species used in the tests were rbcL、PHO and MCM of Skeletonema costatum, rbcL and PHO of Tetraselmis chui, and rbcL of Isochrysis sp. Fragments of these genes were cloned as part of this thesis except MCM of S. costatum and PHO of T. chui. Results of Q-RT-PCR indicated that all primers designed showed high degrees of specificity. Primers designed for one algal class did not generate any signal amplification from phytoplankton of different classes. Even for species of the same class, amplification strength of target alga are 67 fold higher than that of a non-target alga. The technique of dot blotting have the same level of specificity. For algae grown at different physiological situations, only the mRNA expression of T. chui PHO showed statistically significant between low-phosphate and. Using dot blotting mRNA expressions of algae grown in low-phosphate and nutrient replete conditions were indistiguishable. Whether the total RNA was extracted from pure or mixed cultures, Q-RT-PCR detected similar mRNA content for S. costatum MCM , Isochrysis sp. rbcL, and T. chui PHO, but detected inconsistent results for others. In field test, both Q-RT-PCR and dot blotting detected gene expression of diatoms at Sta. 11 in the East China Sea, but failed to obtain signals at others stations for other phytoplankton. In conclusion, our results indicated that the sensitivity of real-time Q-RT-PCR is higher than that of dot blotting. Q-RT-PCR can detect in RNA with very low content in cells. Although the sensitivity and quantification power of dot blotting can not match that of Q-RT-PCR, expressions of multiple genes in natural phytoplankton can be detected at the same time. Dot blotting is suitable for preliminary screening of gene expressions, and identification of interested genes for further quantifications.
| Identifer | oai:union.ndltd.org:TW/092NTOU0270001 |
| Date | January 2004 |
| Creators | 慮永修 |
| Contributors | , 張正, 黃生蘋 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 63 |
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