增殖细胞核抗原基因表达量与中肋骨条藻生长的关系
RELATI ONSHIP BETWEEN PROLIFERATI NG CELL NUCLEAR ANTI GEN GENE EXPRESSION AMOUNT AND GROWTH RATE OF SKELETONEMA COSTATUM
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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.Abstract: Themensuration of the physiological and ecological parameters such as the in situ growth rate is the basic of un-derstanding the regulation of phytoplankton dyna m ics, constructing the ecological dynamicmodels and even forecasting the happening of red tide. W hile a correctly estm i ating method of the in situ growth rate is still lack. In recent years, the in-ternational research on themolecularmarkers of the cell cycle has shown that the proliferating cell nuclear antigen (PC-NA) is of encouraging potential to study the phytoplankton growth rate, which provides a ne w visual angle of study the a-l gal growth rate. In this study, Skeletone ma costatum was taken as the ob ject, wh ich, was a familiar red tide alga in Chinese coast, and itsmolecularmarkers about growth ratewere studied in this paper. The partial sequence ofS. costatum prolif erating cell nuclear antigen (PCNA) genewas obtained by RT-PCR and 3c-RACE techn iques, and a 714bp cytochrome b (Cyt b) gene fragment ofS. costatum was cloned for the first tm i e. 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 detectingS. costatum PCNA was defined as y=23.055x+ 41.093 (r=0.999), wherex was the logarithm of the p lasmid copy number, and y was theCTvalues. The equations forCyt bwas defined as y=24. 0x+44.96 (r=0.997). The abovemethod was applied to study the relationship bet ween theS. costatum growth rate L (/d) and the average expression quantity of PCNA gene in a single cel.l In about 3 weekspcu lture, the PCNA gene expression showed d istinct changes in the d ifferent growth phases andwas consistent to the growth rate. ThePCNA expressionwas the h ighest in the exponential growth phase (46.200 copies/cel, l L=0.742). The PCNA expression in the stationary phase was lower (0. 950 copies/cel, l L=0.002), and reached to the lowest level (0.040 copies/cel, l L=20. 104) in the de-cline phase. And in group 1 and 2, the expression a mount ofPCNA gene also had large variation in different culture phases, and the trend waswell consistent with the growth rate, all ofwh ich suggested that the expression a mount of PCNA gene correlated wellwith the cell division, and the PCNA m ight be a pro m ising indicator for theS. costatum cell prolifera-tion. W hile the expression a mount ofCyt b gene had no obvious variation during different culture phases, which ind icated that theCyt b was a good potential house-keeping gene. A new for mula for enumeratingS. costatum growth rate in the lab situation was established between the growth rate L (/d) and the relative expression amount ofPCNA gene. In which, the PCNA genewas used as the ob jective gene, the Cyt b genewas used as the house-keeping gene, and the relative expres-sion a mount of PCNA gene (REQ) was used as an indicator to theS. costatum growth rate, and the for mula REQ=lg (PCNA expression quantity/Cyt b expression quantity) + 10 was used to calculate the PCNA REQ. The results of group 1 and group-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. So thePCNA REQ m ight be a goodmethod to estmiate the growth rate ofS. costatum. How-ever, more experm i entwas necessary to show on the different illum inations, temperatures and nutrition conditions and even the spot samples, whether therewere good relationship bet ween them all the same. Further more, a growth rate (L) est-i mation method by PCNA REQ could be established. This research paved away for using molecu lar biologicalmethods to enumerating theS. costatum in situ growth rate accurately, and which could provide an effective approach to study the red tide algae.
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Keywords:
- Skeletonema costatum /
- Growth rate /
- Fluorescent quantitative PCR /
- PCNA /
- Cytb
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