Cloning And Expression Analysis Of The CRH-BP And EcR Gene Of Eurytemora Pacifica Under Environmental Stress

As an important part of the marine plankton,copepods play a pivotal role in maintaining the balance of global marine ecosystem.At the same time,copepods are susceptible to environmental changes,which makes them a good model organism in the field of environmental toxicology.Different species of marine copepods require specific environmental conditions and food.As the important part for the life in the seas of the world,Copepods play an important role in the monitoring and control of the global marine ecological environment.The study on the change of marine environment under the influence of copepod biological characteristics will help understand the current changes in the marine environment caused by pollution,and it can provide a reference for the development of the marine ecological environmental protection standards.In this paper we choose the change of salinity,temperature and pH as the stress condition,and use the copepod Eurytemora pacifica(E.pacifica)that is a common planktonic copepod in the East China Sea in winter and spring as the research object in this study.We study the expression changes of of CRH-BP and EcR under the condition of different salinity,temperature and pH,and discusses the toxic effects of three environmental factors stress on Eurytemora Pacifica.It will provide a scientific basis for the biological monitoring.This paper is divided into 3 chapters:The first chapter is summary of research.This chapter mainly discusses the overview of copepods and the application of CRH-BP and EcR in toxicology analysis.It mainly expounds the ecological status of copepods in the nature,the research value and significance of the study on copepods in ecotoxicology,and an overview of the copepods E.pacifica;the present situation and characteristics of environmental factors in marine,the molecular mechanism of biological toxicity of environmental factors in marine.At the same time,this paper discusses the application prospect and research value of copepods as the indicator organism.The second chapter is the full length cDNA cloning of CRH-BP and the analysis of mRNA expression level.According to CRH-BP gene sequence in Genbank conservation region of primer design,amplification of Ep.CRH-BP gene sequence of parts.After cloning sequencing in the NCBI gene identified as purpose,design on the known sequence 3'and 5' specific primers,using the end of the RACE gene cloning method quickly out of the E.pacifica 3'and 5' end,CRH-BP length gene cDNA sequence is obtained by joining together.Gene identified for the purpose,then designed according to known sequence specific primers and 3'and 5' end,respectively,and 3'and 5' RACE cloning,finally get the Ep.CRH-BP length gene cDNA sequence.Results show that the CRH-BP cDNA gene length is 1950 bp,open reading frame of 1245 bp,encoding 414 amino acids(AA).According to the online software to predict Ep.CRH-BP gene of relative molecular weight of 45.816KDa;theory of pI value is 5.89.Software to predict Ep.CRH-BP for hydrophilic protein.The amino acid sequence homology analysis showed that the CRH-BP have high homology with Eurytemor affinis.The phylogetical tree shows that,Eurytemora pacifica and Eurytemor affinis and Tigriopus japonicus clustered into one branch.According to the cloning of CRH-BP genetic design qRT-PCR primers,we analyzed the time-depended and dose-depended expression features of CRH-BP of E.pacifica under different salinity,temperature and pH by the method of qRT-PCR.The results showed that the gene expression showed a certain concentration and time effect,and the gene expression increased significantly after a period of salinity or temperature stimulation.We found it's more sensitive to acidification stress.The increase of CRH-BP gene expression can relieve the stress response of animals.The third chapter is the full length cDNA cloning of EcR and the analysis of mRNA expression level.According to ECR gene sequence in Genbank conservation region of primer design,amplification of Ep.ECR gene sequence of parts.After cloning sequencing in the NCBI gene identified as purpose,design on the known sequence 3'and 5' specific primers,using the end of the RACE gene cloning method quickly out of the E.pacifica 3'and 5' end,ECR length gene cDNA sequence is obtained by joining together.Gene identified for the purpose,then designed according to known sequence specific primers and 3'and 5' end,respectively,and 3'and 5' RACE cloning,finally get the Ep.ECR length gene cDNA sequence.Results show that the ECR cDNA gene length is 2782 bp,open reading frame of 1575 bp,encoding 524 amino acids(AA).According to the online software to predict Ep.ECR gene of relative molecular weight of 53.798KDa;theory of pI value is 8.09.Software to predict Ep.ECR for hydrophilic protein.The amino acid sequence homology analysis showed that the ECR have high homology with Eurytemor affinis.The phylogetical tree shows that Eurytemora pacifica and Eurytemor affinis and Tigriopus japonicus clustered into one branch.According to the cloning of ECR genetic design qRT-PCR primers,we analyzed the time-depended and dose-depended expression features of ECR of E.pacifica under different salinity,temperature and pH by the method of qRT-PCR.The results showed that the response to temperature stress is not obvious in a short time,and it is more sensitive to high salinity or seawater acidification stress.Analyzing the expression characteristics of Ep.ECR gene under acute environmental stress,it can be further concluded that ECR is an important regulatory factor affecting molting under salinity,temperature,and seawater acidification stress.