Cloning of constantly-expression genes in a diatom Skeletonema costatum and its application to thedetermination of cell replication activities

• Main Authors: Lin Hsin-I, 林欣誼
• Other Authors: Chang, Jeng
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/96674834697421285165

碩士 === 國立海洋大學 === 海洋生物研究所 === 89 === The technique of quantitative reverse transcription-polymerase chain reaction ( Q-RT-PCR) was used to detect the cellular content of glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) mRNA、18S rRNA and proliferating cell nuclear antigen ( PCNA) DNA in a marine diatom, Skeletonema costatam. The main purpose is to defind the relation ships between the level of these molecules and the population growth rate, and to evaluate if they are good biomass standards. We also measured the amount of PCNA mRNA, a gene product related to DNA replication, and for diatom the species in the natural environment the ratios between PCNA mRNA and the mRNA level of those biomass standards were used to form the basis of comparison. Our results indicated that PCNA mRNA possesseda very clear pattern of diel change. The average level of PCNA mRNA ranged from 0.8 to 0.01 molecules cell-1. PCNA DNA also has a mild diel change with the average level between 0.8 and 1.2 molecules cell-1. In contrast, GAPDH and 18S rRNA did not show diel changes. The average content of GAPDH was around 0.01 molecules cell-1, and the average content of 18S rRNA was around 2 x 104 molecules cell-1. However, none of the 4 molecules showed a clear correlation between their cellular content and the population growth rate. Compared to the trend of PCNA expression on a per cell basis, both the PCNA mRNA / GAPDH mRNA ratio and the PCNA mRNA / 18S rRNA showed a similar pattem. For the reason, GAPDH mRNA and 18S rRNA were regared as acceptalde biomass standards. When the same technique was applied to nature marine environment, the primers for 18S rRNA performed the best in specificity with 77﹪amplified sequences identical to the 18S rRNA sequence from cultured Skeletonema costatam. The primers for PCNA mRNA was less satifactory, and the primers for GAPDH failed to produce a single band in several cases. The amount of PCNA mRNA、GAPDH mRNA and 18S rRNA decreased when Skeletonema costatam became less abundant. On the molecules per cell basis, the amount of these 3 molecules measured in field samples was more than 100 time higher than data measured in cultured Skeletonema costatam. These observations suggest that 18S rRNA is the most useful standard in field applications.