Transcriptional Expression Of Immune-related Genes And Heat Shock Protein Genes Of Japanese Flounder(Paralichthys Olivaceus)

Japanese flounder(Paralichthys olivaceus) is one of economically important marine fish in C hina. However, diseases of Japanese flounder have become increasingly prominent, and there were few disease-resistant species, which restricted sustainable development of Japanese flounder aquiculture. However, immune system and defend mechanism to pathogen were scarce, the comprehension of immune-related genes and signaling pathways of Japanese flounder from the point of view of the transcriptome level will contribute to deeply understand immune system and defend mechanism of fish. In this study, we conducted transcriptome sequencing of Japanese flounder spleen and discovered immune-related genes and signaling pathways. Moreover, we further studied the gene expression changes of heat shock proteins of Japanese flounder after temperature stimulation, pathogen infection and vaccine immunity. This study will have a very important significance to deeply understand of fish immune system and disease control of Japanese flounder.Main results of the study were as follows: 1. We studied the spleen transcriptome of Japanese flounder after infected with Vibrio anguillarum. Two c DNA libraries(the spleen of Japanese flounder before and after infected with V. anguillarum) were successfully constructed. 314,377 transcripts were obtained from Illumina Mi-seq sequencing. Using Trinity software, a total of 96,627 unigenes were assembled, which were annotated using the Nr databases. There were a total of 21,392(22.14%) unigenes with significant blast hits to known proteins. Approximately 12,503(58.45%) unigenes were categorized into three Gene Ontology groups, 19,547(91.38 %) were classified into 26 C luster of Orthologous Groups, and 10,649(49.78%) were assigned to six Kyoto Encyclopedia of Genes and Genomes pathways.The Japanese flounder assembled unigene sequences were compared to unigenes from zebrafish(Danio rerio) and known flounder EST and nucleotide sequences in the NCBI database. 11,799 of the unigenes could be matched in the EST sequences, 4,442 sequences were shared between the flounder unigenes and zebrafish unigenes.Among the species distribution of BLAST hits, 18,627(87.1%) originated from fish species, 778(3.6%) from other vertebrates, including mammals, birds, amphibians, and reptiles, and 1,987(9.3%) from other species, mainly plant and microorganisms.We identified 1,563 putative immune-associated unigenes that mapped to 15 immune signaling pathways. Many genes, such as TOLLIP, IRAK1/4, TRAF3/6, C2, C6, C8 g, C5AR1 were even first found to express in Japanese flounder.A total of 47,362 putative SNPs were identified from the 314,377 transcripts, including 28,142 transitions and 19,220 transversions. Moreover, a total of 40,928 putative SSR loci were discovered in 21,392 unigenes; Among the 1,563 immune unigenes, a total of putative 1,579 SNPs were detected, which were distributed in 339 unigene sequences.A total of 189 differentially expressed genes(DEGs) were detected between the spleen transcriptomes of the infected and control group. 38 genes were up-regulated while 151 were down-regulated in the two transcriptomes. 2. Using qRT-PCR detection, we initially constructed a n expression pattern of Hsp10, Hsp40A4, Hsp60, Hsp70 and Hsp110 of Japanese flounder after temperature stimulation, pathogen infection and vaccine immunity.The expression pattern of HSP genes of healthy Japanese flounder in spleen was compared. The highest expression quantities was Hsp90β, followed by Hsp90α å'ŒHsp70, and the lowest expression was Hsp10 and Hsp110. HSP genes of healthy Japanese flounder were expressed in different degrees in 12 different tissues. The expression difference of HSP genes between 12 tissues was large. The expression quantity in the midintestine, liver and muscle were the highest.The expression changes of HSP genes were gr eat after 1 hour at 5℃, 10℃, 16℃, 22℃, 28℃ and 32℃. Hsp40 and Hsp70 were not sensitive to cold stress, but sensitive to heat shock, the expression quantitiy of the two genes increased abundantly. The expression level of Hsp10 genes was lower under cold stress and 32℃ heat shock than that at normal conditions. And the expression level of Hsp10 increased abundantly at 28℃ heat shock. Hsp60 was not sensitive to cold stress and heat shock. Hsp110 was sensitive to cold stress, and the expression level increased abundantly at 28℃ heat shock.QRT-PCR was performed to detect the expression pattern of HSP genes of Japanese flounder spleen after cold stress(10℃) and heat shock(28℃) 1h, 3h, 5h, 8h. The mRNA expression level of Hsp10 and Hsp70 decreased after cold stress. Otherwise, the expression level of Hsp40, Hsp60 and Hsp110 were similar, expression quantity increased over time and were the highest at 5 hour after cold stress. Heat shock protein genes were sensitive to heat shock. The mRNA expression level of HSP increased significantly and was the highest at 3 hour after heat shock, in addition to the expression amount of Hsp60 did not change significantly. Especially the expression quantity of Hsp70 and Hsp40 increased rapidly after heat shock(28℃).QRT-PCR was performed to detect the expression pattern of HSP genes in the spleen of Japanese flounder after infected with V. anguillarum and immuned by inactvated V. anguillarum. Different heat shock protein genes seemed to show different expression changes at 6 h, 12 h, 24 h and 72 h after infected with V. anguillarum. The expression of most of HSP genes was down-regulated, but the expression of Hsp60 was up-regulated. By contrast, the expression of most of HSP genes was up-regulated at 1 day after vaccine immunity, subsequently down-regulated at 3 day. Hsp60 was up-regulated at 3 day after vaccine immunity. The expression level of HSP genes were back to normal levels at 7 and 14 day, which were not differ with the control group significantly.