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

该项目为确定、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。分子技术将被开发出来，可以快速识别和量化桡足类动物Calanus finmarchicus中具有生理意义的基因的表达水平。这些技术利用聚合酶链式反应(PCR)的特异性和敏感性来定量扩增物种特有的mRNAs，这些mRNAs编码调节代谢流量的酶。为了设计用于聚合酶链式反应的必需的引物，将利用这些酶的系统发育保守的初级序列来克隆编码己糖激酶、磷酸果糖激酶、磷酸葡萄糖异构酶和柠檬酸合成酶的cDNA。根据这些cDNAs序列设计出物种特异性的引物，并仅用于定量扩增来自Finmarchicus的每个酶的mRNA，以确定环境参数对酶表达水平的影响。通过同时测量生理不变基因的特定浓度，如肌动蛋白，浮游动物分类复杂的组合中存在的Finmarchicus数量的差异将被归一化。这一结果将相当于Finmarchicus每重量的酶，这是一个与更传统的生理学研究相当的单位。

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.