The Study Of The Relationship Between The Proliferating Cell Nuclear Antigen Gene Expression Amount And The Growth Rate Of Skeletonema Costatum

The mensuration of the physiological and ecological parameters such as the in situ growth rate is the basic of understanding the regulation of phytoplankton dynamics, constructing the ecological dynamic models and even forecasting the happening of red tide. While a correctly estimating method of the in situ growth rate is still lack. In recent years, the international research on the molecular markers of the cell cycle has shown that the proliferating cell nuclear antigen (PCNA) is of encouraging potential to study the phytoplankton growth rate, which provids a new visual angle of study the algal growth rate.In this study, Skeletonema costatum was taken as the object, which was one dominant specie of red tide algae in Chinese coast. An effective RNA extraction method for S. costatum was developed. The partical sequence of S. costatum PCNA gene was obtained by RT-PCR and RACE technique. A 714bp cytochrome b (Cyt b) gene fragment of S. costatum was cloned for the first time. The phylogenetic trees of PCNA and Cyt b were constructed respectively. Furthermore, the primers and the Taqman probes were designed for RFQ-PCR based on the S. costatum PCNA and Cyt b sequences. The equation for detecting S. costatum PCNA was defined as y=-3.055x+41.093 (r=0.999), where x was the logarithm of the plasmid copy number, and y was the CT values. The equations for Cyt b was defined as y=-4.0x+44.96 (r=0.997). The species-specificity of the RFQ-PCR primers for amplifying PCNA was validated by the other 16 algal species,. The accuracy of the standard curves were verified by detecting the plasmid samples with the known concentration. The above methods were applied to the study of the relationship between the S. costatum growth rateμ(d-1) and the expression amount of PCNA gene. The expression amount of PCNA gene had large variation in different culture stages, and the trend had good consistent with the growth rateμ(d-1), which suggested that the expression amount of PCNA gene correlated well with the cell division, and the PCNA maybe was a promising indicator for the S. costatum cell proliferation.While the expression amount of Cyt b had no obviously variation during different culture stages, which suggested that the Cyt b was a good potential house-keeping gene. A new formula for enumerating S. costatum growth rate in the lab situation was established between the growth rateμ(d-1) and the relative expression amount of PCNA gene. In which, the PCNA gene was used as the objective gene, the Cyt b gene was used as the house-keeping gene, and the relative expression amount of PCNA gene(REQ) was used as an indicator to the S. costatum growth rate. This research paved a way for using molecular biological methods to enumerating the S. costatum in situ growth rate accurately, and which could provide an effective approach to study the red tide algae.