Cloning And Expression Analysis Of Heat Shock Protein And Hypoxia Responsive Genes In Mandarin Fish (Siniperca Chuatsi)

The mandarin fish, Siniperca chuatsi, is an economically important fish and has been widely cultured in China. With the development of intensive production in the aquaculture industry, various environmental stresses, such as high temperature, hypoxia and pathogenic infection and etc, have frequently threatened the health of this fish, causing serious economic losses. Heat shock proteins (HSPs) are ubiquitous essential molecular chaperones that maintain cellular protein homeostasis under normal conditions. HSPs can protect organism or cell against damages caused by environmental stresses, however, this function of protection varied among environmental stressors as well as the fish species. In order to better understand the relationship between environmental stresses and HSPs in S. chuatsi, multiple HSPs were isolated and characterized from this fish and their expression in tissues, during embryonic development and under different environmental stresses was investigated in this study. On the other hand, the stress response is controlled by multiple genes. Beside the HSPs, there are various hypoxia responsive genes (HRGs) in the process of response to hypoxia. Therefore, the RNA-seq technique was used to analysis the liver transcriptome of S. chuatsi on exposure to hypoxia. Further, complete cDNAs of some HRGs were cloned and their expression profiles in S. chuatsi under hypoxia were analyzed by real-time RT-PCR. The main topics of this paper are as follows:1. Using technique of rapid amplification of cDNA ends (RACE), three S. chuatsi HSP family cDNAs and gDNAs were cloned.(1) The HSP90family contains ScHSP90α and ScHSP90β, their complete cDNA sequences were2638and2722bp in length, with2193and2178bp open reading frame (ORF) encoding730and725amino acids, respectively. ScHSP90α gDNA containes10exons and9introns in its coding region, while the coding region of ScHSP90β gDNA is composed of11exons and10introns.(2) The HSP70family is devided into HSC70subfamily and HSP70subfamily. The S. chuatsi HSC70subfamily contains two members, ScHSC70-1and ScHSC70-2. The full lengths of ScHSC70-1and ScHSC70-2cDNAs are2344and2468bp, respectively, with ORFs of1950bp encoding649amino acids. The coding regions of both ScHSC70gDNAs are composed of8exons and7introns. In the HSP70subfamily，two isoforms, ScHSP70a and ScHSP70b, were identified from S. chuatsi. The full lengths of ScHSP70a and ScHSP70b cDNAs are2245and2371bp, respectively, with ORFs of1920bp encoding639amino acids. There are no introns in the coding regions of gDNAs of both ScHSP70isoforms.(3) The complete ScHSP60cDNA was2505bp in length with an ORF of1731bp encoding576amino acid residues. The coding region of ScHSP70gDNA contains10exons and7introns.2. During embryonic development, S. chuatsi HSPs transcripts were maternal in origin and characterized by phase-specific expression. ScHSP90α was constitutively expressed in the embryonic development. The expression levels of ScHSP90β and ScHSP60increased from the stage of closure of blastopore, and had the highest levels at the metameres appearance stage. ScHSC70-1was elevated from the gastrula stage, and peaked at the muscle burl stage. ScHSC70-2was increased from the metameres appearance stage and peaked at the crystal stage. In contrast, the expression levels of ScHSP70a and ScHSP70b significantly decreased from the gastrula stage to a low level, however, during the muscle burl stage and crystal stage, ScHSP70a was significantly increased. The different expression patterns of S. chuatsi HSPs suggested that they may participate in morphogenesis of some tissues or organs, such as the somite development, nervous system development or lens formation.3. Tissue distribution showed that the expression of S. chuatsi HSPs (except for ScHSC70-2) in ovary were much higher than that in other tissues, suggesting these HSPs are involved in the ovarian mature. In the non-gonad tissues, ScHSP90α and ScHSC70-1were constitutively expressied in all tissues tested. ScHSP90α, ScHSC70-2and ScHSP60had the highest expression level in the brain, heart and opisthonephros, respectively. Both ScHSP70a and ScHSP70b were expressed ubiquitously and at a low level in tissues under non-stressed conditions. The expression of HSPs varied among tissues indicated that they are engaged in different biological functions in normal physical conditions.4. Acute heat shock strongly induced the expression of S. chuatsi HSPs, and the induction were tissue-specific and in time-dependent manners. The induction of ScHSP90β in heart and liver were positively correlated with exposure time of heat shock, while the expression of ScHSP90β in head kidney maintained at a constant level after heat shock of2h. Acute heat shock could not induce the expression of ScHSC70-1in the liver, but could slightly increase its expression in the heart and head kidney. During the12-h heat shock, ScHSP90α, ScHSC70-2, ScHSP70a, ScHSP70b and ScHSP60showed the expression trend of sharply induction at first and then gradually returned to the normal levels. Under gradual heat shock regimes, all HSPs expression levels in S. chuatsi were significantly higher than that in the hybrid (S. chuatsi♀×S. scherzeri♂) at the sublethal temperature, which was in accordance with the fact that the S. chuatsi had a higher tolerable temperature than that of the hybrid, indicating that the accumulation of HSPs is positively related with the hyperthermal tolerance of S. chuatsi.5. Acute cold shock also slightly induced the expression of ScHSP90β, ScHSC70-2, ScHSP70a, ScHSP70b and ScHSP60in the heart, as well as ScHSP90β, ScHSC70-1, ScHSP70a and ScHSP60in the gill. However, the expression of ScHSC70-2and ScHSP70b in the muscle was down-regulated under cold shock, indicating they were regulated in a different way compared with the HSPs up-regulated in the liver and gill.6. During the72-h of Aeromonas hydrophila infection，the expression of ScHSP90α, ScHSP70a, ScHSP70b and ScHSP60in the head kidney and spleen were markedly augmented at early infective stages，and then gradually returned to the control levels. The expression of ScHSP90β in the spleen at6h post infection was also significantly increased, but that in the head kidney was decreased at first and then recovered. The expression of ScHSC70-1in head kidney and spleen were also significantly increased at the early infective stages, however, it was significantly decreased in head kidney at24h post infection. The bacterial infection induced the ScHSC70-2expression in the head kidney but did not alter its expression in the spleen. These results suggested that different S. chuatsi HSPs participate in the fish immune response in time-dependent and tissue-specific manners during the bacterial infection.7. Using RNA-seq technology, the S. chuatsi liver transcriptome between under normoxic and hypoxic conditions were analyzed. A total of84,773Unigenes were obtained, in which there were5,088different expression Unigenes (DEUs), including4,139up-regulated ones and949down-regulated ones. GO analysis of DEUs showed that914DEUs were annotated to the biological process including metabolism, biological regulation, stress response and cell death, and831DEUs were annotated to molecular function, such as binding, nucleic acid combining-transcription factors activity, catalytic activity, etc. There were1,393DEUs were annotated to the KEGG database, and significantly enriched in49pathways, such as metabolism, MAPK signaling pathway, VEGF signaling pathway, cytokine-cytokine receptor interaction, protein processing in endoplasmic reticulum, apoptosis, etc.8. Complete cDNAs of six HRGs in S. chuatsi (ScHIF-1α, ScHIF-2α, ScIGFBP-1, ScTf, ScHO-1a and ScHO-1b) were obtained by RACE. Hypoxia significantly induced the expression of ScHIF-1α, and the induction levels were positively correlated with the hypoxic duration. After24-h reoxygenation, the expression of ScHIF-1α returned to the control level. The level of ScHIF-2α was down-regulated post6h of hypoxic exposure. ScIGFBP-1presented the trend of decreasing at first and increasing then, followed by recovering. The expression of ScTf was down-regulated at first, and then elevated to the control level. Two isoforms of HO-1, ScHO-1a and ScHO-1b had the adverse expression profiles under hypoxia: ScHO-1a was up-regulated, while ScHO-1b was down-regulated. Hypoxia strongly induced the expression of ScHSP90α, ScHSP70a, ScHSP70b and ScHSP60in a time-dependent manner, and after the reoxygenation their expression returned to the control level. These results were consistent with that of transcriptome sequencing, meanwhile, indicated these genes contribute differently to protection against damage from hypoxia.