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

GLOBEC：浮游动物的生理状态：利用 PCR 快速评估物种特异性酶表达

基本信息

批准号：
9121744
负责人：
Lewis Incze
金额：
$ 9.25万
依托单位：
Bigelow Laboratory for Ocean Sciences
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
1992
资助国家：
美国
起止时间：
1992-01-15 至 1995-06-30
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=9121744&HistoricalAwards=false
关键词：
GLOBEC Physiological Status Zooplankton Utilizing

项目摘要

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.

该项目为与识别，分类和评估浮游动物的生理状况相关的问题提供了独特的解决方案。将开发可以快速识别和量化生理意义基因表达水平的分子技术。这些技术利用了聚合酶链反应（PCR）的特异性和灵敏度，以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的必要引物，使用这些酶的系统发育的主要序列，将克隆用于克隆这些酶的己糖酶，己糖酶，磷酸果糖异构酶和柠檬酸合成酶。物种特异性引物将是通过这些cDNA序列设计的，并仅用于从芬马那氏梭菌进行定量扩增每个酶的mRNA，以确定环境参数对酶表达水平的影响。浮游动物的分类学复杂组合中存在的Finmarchicus量的差异将通过同时测量生理上不变基因的特异性浓度（例如肌动蛋白）来归一化。结果将等效于每重量芬马那菌的重量，这是一个与更传统的生理研究相当的单位。