Development of microarray techniques for the study of gene expression in the European eel (Anguilla anguilla) during silvering and migration to seawater

DNA microarrays;Anguilla anguilla--Genetics;Anguilla anguilla--Development;Osmoregulation;Gene expression

McWilliam, Iain Stuart ; Cramb, Gordon ; Cramb, Gordon

University:University of St Andrews

Department:Biology (School of)

关键词: DNA microarrays;    Anguilla anguilla--Genetics;    Anguilla anguilla--Development;    Osmoregulation;    Gene expression;   

Others  :  https://research-repository.st-andrews.ac.uk/bitstream/handle/10023/502/Iain%20McWilliam%20PhD%20thesis.pdf?sequence=11&isAllowed=y

来源: DR-NTU

【 摘 要 】

The European eel, Anguilla anguilla, has a complex life-cycle involvingmigrations between the Sargasso Sea and the river systems of Europe andNorth Africa. The requirement to move across large salinity gradientspresents a significant physiological challenge and the developmental stagesof the eel are closely linked to these migrations. Microarrays were created toelucidate gene expression changes occurring during;i. The transition from juvenile yellow to the adult sexuallymaturing, migrating silver eel and;ii. Salinity adaptation during the migration from freshwater toseawater.Groups (n = 6) of freshwater-acclimated yellow or silver eels weretransferred to seawater for between 6 hours and 5 months andcomplementary control groups were transferred to freshwater. Brain, kidney,intestine and gill cDNA libraries were constructed using suppressionsubtractive hybridisation (SSH) techniques and a novel protocol based onInvitrogen's Gateway cloning system. The latter technique produced a lowredundancy (~4 %) EST bank with a wide range of insert sizes (0.5 – 10 kb).Two microarray types were produced; one comprised 5760 clones from thetwo brain libraries whilst the other was a multi-tissue microarray incorporating6144 clones from the SSH libraries. Pooled RNA samples were probedagainst the microarrays to highlight differentially expressed genes. Real-timequantitative PCR (QPCR) was used to validate the observed expressionchanges of selected genes in the tissues of individual fish. Following yellowto silver transformation of freshwater-adapted eels, the expression of tyrosine3-mono-oxygenase/tryptophan 5-mono-oxygenase activation protein (14-3-3)and vaccinia related kinase 3 was shown to be consistently elevated.Prolactin expression increased in the brains of silver eels following two-dayseawater-acclimation but QPCR analysis revealed high variation amongstfreshwater-adapted eels. This is the first eel microarray study and theexpression profiles highlighted herein will provide new avenues for researchinto the sexual development and salinity acclimation of A. anguilla