Differentiated Expressed Genes Of Turbot Following Challenge With Vibrio Harveyi

Turbot (Scophthalmus maximus) is an economically important marine fish species, and is valued for its rapid growth and good taste. However, the impact of diseases is still a limiting factor to aquaculture. In particular, Vibrio harveyi is a bacterial pathogen responsible for serious disease outbreaks in numerous species of cultured fish including turbotIn order to solve the disease problems in aquaculture and to improve the resistance of fish against varies pathogens, it is important to illustrate the immune-related genes of turbot and its resistance to bacterial pathogens. However, most of the immune-related genes are not constitutive expressed but inducible expressed. Therefore, if we can analyse the differences in gene expression of turbot following challenge with pathogens, we can identify new immune-related genes and illustrate the relationship between immune-related genes and pathogens.Suppression subtractive hybridization (SSH) technology was established on the basis of mRNA differential display technology, in order to select differentially expressed genes. It is mainly based on suppression PCR and connected with normalization and subtractive hybridization technology. It overcomes shortcomings of the high false positive in mRNA differential display and more rounds in representational difference analysis. It is particular used to analyse the differences in gene expression and it has become the most potential techniques for selection of differentially expressed genes.In this thesis, the differences in gene expression in the unchallenged and V. harveyi challenged turbot were compared by SSH. Trizol reagent was used to extract total RNA from kidney and spleen of experimental and control group, and mRNA was then separated and purified. The SSH library was contructed and differentiated expressed genes were cloned. A total of 49 ESTs (expressed sequence tags) were obtained from subtractive libraries, and sequenced. After blasting in GenBank, it was found that most of the differentiated expressed genes were immune-related genes including major histocompatibility complex (MHC) class Ia gene, Hsp70 gene and some signaling molecules genes such as src-family tyrosine kinase SCK, sgk-1 serine-threonine protein kinase and amyloid precursor-like protein 2 and so on. One EST was identified as MHC classⅠa gene and the full-length cDNA were obtained by Race. The gene was sequenced and the character of encoding protein were analysed. The full-length cDNA clone of the turbot MHC class Ia (1466 bp) contained a single open reading frame (ORF) of 1062 bp. The 5'untranslated region (UTR) and the longer 3'UTR were 127 bp and 277 bp, respectively. Sequence analysis identified a possible polyadenylation signal (ATTAA) in the 3'UTR at position 1185 bp. The 1062 bp encoding region was found to code for a protein with 354 amino acid residues. The putative amino acid sequence encoded by the turbot MHC class Ia was aligned with amino acid sequences from human (Accession no. U21053), mouse (Accession no. XM974660), chicken (Accession no. NM001031338), rainbow trout (Accession no. AY278455), bastard halibut (Accession no. AB126921), medaka (Accession no. AB026978), Atlantic cod (Accession no. AF414203) and tiger puffer (Accession no. AF001216 ). Overall, the deduced amino acid sequence of turbo MHC class Ia had 68%, 54%, 51%, 52%, 57%, 33%, 29% and 29% identities with those of bastard halibut, medaka, rainbow trout, Atlantic cod, tiger puffer, chicken, mouse and human, respectively.Realtime PCR is a technology that adds fluorescence to PCR reaction system, which is based on the detection of a fluorescent reporter molecule that increases as PCR product accumulates with each cycle of amplification. It exceeds the limitations of traditional end-point PCR methods by allowing either absolute or relative quantification of PCR product at the end of each cycle, and avoiding the pollution in traditional PCR. In this thesis, the differences in MHC class Ia gene expression in the unchallenged and V. harveyi challenged turbot were compared by 2 Standard Curves Relative Quantitation, and the houskeeping geneβ-actin was used as control. The results showed that the expression level of the MHC class Ia gene in V. harveyi challenged turbot increased to 4-fold of the controls. It domenstrated that MHC class Ia gene plays a role during immune response of turbot.