Date of Completion

5-24-2018

Embargo Period

5-24-2018

Keywords

Onchorhynchus mykiss, cecropins, antimicrobial peptide, RNA-Seq, RT-qPCR array

Major Advisor

Thomas T. Chen, PhD

Associate Advisor

Johann P. Gogarten, PhD

Associate Advisor

Joerg Graf, PhD

Associate Advisor

Charles Giardina, PhD

Associate Advisor

Ping Zhang, PhD

Field of Study

Molecular and Cell Biology

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Introduction of antimicrobial peptide (AMP) genes into fish genome by the transgenic technology provided a promising solution to control fish disease. Cecropin P1, a known porcine originated AMP, had been introduced into the rainbow trout genome for production of transgenic fish by Chiou et al. (2014) in Chen’s laboratory. Repeated, in vivo, challenge studies demonstrated that these transgenic fish exhibited resistant characteristic to infection by microbial pathogens. Here, we hypothesized that cecropin P1 transgene product may not only directly eliminate microbial pathogens, but also indirectly exert immunomodulatory activity in the transgenic hosts to elevate their disease resistance. To address this hypothesis, I have employed the technologies of mRNA deep sequencing (mRNA-Seq) and reverse transcription quantitative real-time PCR (RT-qPCR) array. In a preliminary cDNA microarray analysis, Lo et al. (2014) reported functional perturbations of the immune relevant pathways in three immune competent tissues (namely, the spleen, kidney and liver) of the cecropin P1 transgenic rainbow trout. To overcome the technical limitations of the cDNA microarray, the differentially expressed genes (DEGs) of immune relevant pathways in the spleen, kidney and liver of two families of transgenic rainbow trout was analyzed by de novo mRNA-Seq. By sorting of Reads Per Kilobase of transcript per million mapped reads, the DEGs were determined. From the GeneCodis enrichment analysis, functional alterations of biological processes with identifiers of Gene Ontology: Biological Process and Kyoto Encyclopedia of Genes and Genomes databases were observed. Via pathway analyses, immune relevant processes in the spleen and kidney, and energy metabolism relevant processes in the liver were significantly perturbed. By employing a custom-layout RT-qPCR array with pre-defined immune relevant DEGs, the expression patterns in three remaining transgenic families were profiled. The results of mRNA-Seq and RT-qPCR array analyses supported the hypothesis that the combined effects of transgenic cecropin P1 elevate the host disease resistance. Furthermore, the RT-qPCR array with pre-defined DEGs as bio-markers promises a fast, effective and inexpensive technique for screening of disease resistant fish strains for aquaculture.

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