GLOBEC: Physiological Status of Zooplankton: Utilizing PCR to Rapidly Assess Species-Specific Enzyme Expression

该项目为识别、分类和评估浮游动物的生理状态提供了一个独特的解决方案。分子技术将被开发，可以快速识别和量化在桡足动物，Calanus finmarchicus生理上重要的基因表达水平。这些技术利用聚合酶链反应（PCR）的特异性和敏感性，定量扩增编码调节代谢通量的酶的物种特异性mrna。为了设计PCR所需的引物，将利用这些酶的系统发育保守的一级序列克隆这些酶的编码cdna，包括己糖激酶、磷酸果糖激酶、磷酸葡萄糖异构酶和柠檬酸合成酶。根据这些cDNA序列设计物种特异性引物，定量扩增各酶mRNA，以确定环境参数对酶表达水平的影响。通过同时测量一个生理上不变的基因（如肌动蛋白）的特定浓度，可以使在分类上复杂的浮游动物组合中存在的C. finmarchicus数量的差异归一化。结果将相当于C. finmarchicus每重量的酶，一个可与更传统的生理研究相媲美的单位。

This project offers a unique solution to the problems associated with identifying, sorting and assessing the physiological status of zooplankton. Molecular techniques will be developed that can rapidly identify and quantify the level of expression of physiologically significant genes in the copepod, Calanus finmarchicus. These techniques take advantage of the specificity and sensitivity of the polymerase chain reaction (PCR) to quantitatively amplify species-specific mRNAs that encode enzymes that regulate metabolic flux. In order to design the necessary primers for PCR, the cDNAs that encode for the enzymes, hexokinase, phosphofructokinase, phosphoglucose isomerase and citrate synthetase will be cloned utilizing the phylogenetically conserved primary sequences of these enzymes. Species-specific primers will be designed from these cDNA sequences and used to quantitatively amplify each enzyme's mRNA only from C. finmarchicus to ascertain the affect of environmental parameters on the level of enzyme expression. Differences in the quantity of C. finmarchicus present in a taxonomically complex assemblage of zooplankton will be normalized by simultaneously measuring the specific concentration of a physiologically invariant gene, such as actin. The results will be equivalent to enzyme per weight of C. finmarchicus, a unit comparable to more traditional physiological studies.