摘要
对中国近海常见的赤潮藻-中肋骨条藻(Skeletonema costatum)生长相关的分子标记进行了研究,通过RT-PCR和RACE技术获得了其PCNA基因的部分序列,首次克隆测序得到了714bp的中肋骨条藻细胞色素b(Cytochrom eb,Cytb)基因序列。根据得到的PCNA与Cytb基因序列,分别建立了检测中肋骨条藻PCNA与Cytb基因的实时荧光定量PCR的标准曲线,并通过对已知浓度样品的检测证明了所建立标准曲线的准确性。以上述建立的方法为基础,研究了中肋骨条藻生长率与增殖细胞核抗原基因表达量之间的关系。结果发现,平均单细胞中PCNA表达量在培养的不同阶段变化很大,且变化趋势与生长率表现出良好的一致性,说明PCNA表达量与细胞分裂密切相关,体现了其作为细胞增殖指标的潜能;而Cytb基因表达稳定,表达量变化很小,说明Cytb基因是一个潜在的良好的内参基因。基于此,提出以PCNA为目的基因、Cytb为内参基因,将PCNA基因的相对表达量(REQ)作为中肋骨条藻生长率的一个指标,并建立了实验室条件下PCNA相对表达量与中肋骨条藻生长率之间的关系式。
The mensuration of the physiological and ecological parameters such as the in situ growth rate is the basic of un- derstanding 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 in- ternational 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 provides a new visual angle of study the algal growth rate. In this study, Skeletonema costatum was taken as the object,which, was a familiar red tide alga in Chinese coast, and its molecular markers about growth rate were studied in this paper. The partial sequence of S. costatum proliferating cell nuclear antigen （PCNA） gene was obtained by RT-PCR and 3＇-RACE techniques, and a 714bp cytochrome b （ Cyt b） gene fragment of S. costatum was cloned for the first time. Based on the cloned sequences, a RFQ-PCR method was developed to detect the S. costatum PCNA and Cyt b gene respectively and the accuracy of the standard curves was verified. 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 above method was applied to study the relationship between the S. costatum growth rateμ （/d） and the average expression quantity of PCNA gene in a single cell. In about 3 weeks＇ culture, the PCNA gene expression showed distinct changes in the different growth phases and was consistent to the growth rate. The PCNA expression was the highest in the exponential growth phase （46. 200 copies/cell, μ = 0. 742）. The PCNA expression in the stationary phase was lower （0. 950 copies/cell,μ = 0. 002） , and reached to the lowest level （0. 040 copies/cell,μ = -0. 104） in the de- cline phase. And in group 1 and 2, the expression amount of PCNA gene also had large variation in different culture phases, and the trend was well consistent with the growth rate, all of which suggested that the expression amount of PCNA gene correlated well with the cell division, and the PCNA might be a promising indicator for the S. costatum cell proliferation. While the expression amount of Cyt b gene had no obvious variation during different culture phases, which indicated 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） 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 expres- sion amount of PCNA gene （REQ） was used as an indicator to the S. costatum growth rate, and the formula REQ = lg （ PCNA expression quantity/Cyt b expression quantity） ± 10 was used to calculate the PCNA REQ. The results of group 1 and group 2 suggested that the PCNA REQ was well consistent with the grow rate, and their correlation was 0.90 in group 1 and 0.97 in group 2. So the PCNA REQ might be a good method to estimate the growth rate of S. costatum. How- ever, more experiment was necessary to show on the different illuminations, temperatures and nutrition conditions and even the spot samples, whether there were good relationship between them all the same. Furthermore, a growth rate （/x） esti- mation method by PCNA REQ could be established. 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.
出处
《水生生物学报》
CAS
CSCD
北大核心
2009年第1期103-112,共10页
Acta Hydrobiologica Sinica
基金
国家自然科学基金项目40406028
国家高技术研究发展计划项目(863)2001AA635090资助
