| Most fish have great economic importance and become main aquaculture speciesin China and other countries. However, aggravated climate change and fluctuating lowtemperature may cause increased costs and fish mortality, which severely hampered thehealthy development of fish farming industry. Therefore, establishment of coldtolerance fish model and elucidation of gene regulatory networks during temperatureacclimation may help to solve this problem. In this study, three cold tolerance relatedgenes including common carp (Cyprinus carpio) muscle form Ⅲ of creatine kinase(m3ck), zebrafish (Danio rerio) glutathione s-transferases M (GSTM) and oliveflounder (Paralichthys olivaceus) type-IV antifreeze protein (AFP-IV), were analyzed.The m3ck was firstly used to construct a germline transgenic zebrafish (Danio rerio),which was used to investigate the possible molecular regulatory networks at lowtemperatures. GSTM had performed gene polymorphism analysis and a SNP molecularmarker had association with low temperature tolerance was detected. The flounderAFP-IV was cloned and its recombinant protein was expressed in E. coli BL21(DE3).The detailed results are shown as follows:1. Two transgenic plasmids were constructed and the corresponding tissue-specfictransgenic zebrafish, Tg (smyd1: m3ck) and Tg (cmlc: m3ck) were established by micro-injection. Tg (smyd1: m3ck) fish could express carp m3ck directed by zebrafish muscle-specific smyd1promoter and Tg (cmlc: m3ck) fish could express carp m3ck directed byzebrafish cardiac-specific cmlc promoter. Both transgenic line were raised to F2generation and the positive rate was34.90%(22/63) and58.75%(47/80), respectively.The behavioral observations after cold treatment (13°C) showed that the percentagesof adult Tg (smyd1: m3ck) possessed better swimming ability, occasional body swingand response to external stimuli were increased significantly than wild type (P <0.05).The percentages of Tg (smyd1: m3ck) completely still, inverted and dead were reduced significantly than wild type (P <0.05). In Tg (cmlc: m3ck), only the percentage ofinverted was significantly reduced than wild type (P <0.05). In addition, energyproperties (ATP concentration) were recorded. In Tg (smyd1: m3ck) fish, ATPconcentration in muscle was significantly higher than wild type both under28°C and13°C (P <0.05). There was no statistically significant difference in heart ATPconcentration between Tg (cmlc: m3ck) and wild type.2. The germline transgenic zebrafish Tg (smyd1: m3ck) was used as a model toinvestigate the gene expression profile changes at room temperature and lowtemperature. Microarray analysis showed that a total of1249transcripts were affectedin Tg (smyd1: m3ck) fish during cold stress, compared with wild-type fish. Biologicalprocesses up-regulated in Tg (smyd1: m3ck) fish at low temperature included steroidbiosynthesis and phosphoinositide pathway, which participated in environmentaladaptation, signal transduction, cell membranes construction and other biologicalprocesses. Pathways such as MAPK signaling pathway, NOD receptor signalingpathway, Toll-like receptor signaling pathway and conversion of pentose andglucuronide were cold-induced in wild type. Pathways such as PPAR signaling pathway,cytokine receptor interaction were found down-regulated in transgenic fish. In wild type,primary bile acid biosynthesis, pyruvate metabolism, ether lipid metabolism and TCAcycle, which were mainly involved in energy metabolism were down-regulated. Thesefindings gave new interesting clues for the elucidation of mechanisms underlying thetemperature acclimation in fish.3. The germline transgenic zebrafish Tg (cmlc: m3ck) was used as a model toanalyze the impact of energy on the heart. Heart rates analysis showed that in larvalwild type fish (48hpf), the heart rate was152.88±4.01beats/min under28°C andsignificantly decreased to41.20±4.13beats/min under13°C (P <0.01). In48hpfTg (cmlc: m3ck) fish, heart rate was187.50±7.84beats/min under28°C and87.82±9.71beats/min under13°C, which were all increased significantly than wild type (P <0.05). And transgenic zebrafish heart rate under low temperature could reach to57.44%of wild zebrafish under nomal temperature. In addition, in situ hybridization analysisshowed that Tg (cmlc: m3ck) fish has an advanced developmental pattern than wild typeduring heart development.4. PCR-SSCP was performed to explore the correlation between GSTM gene andcold tolerance traits in zebrafish. In intron1of the GSTM gene, three genotypes(denoted DD, DE and EE) were found, and there were D and E alleles. The observedheterozygosity and expected heterozygosity of this locus were0.408and0.477,respectively. The polymorphism information content was0.362and the genotypesdistribution fitted to the Hardy-Weinberg equilibrium. Correlation analysis amongdifferent genotypes and cold-tolerance traits of zebrafish was studied by the Pearson’schi-square test. The results indicated that the genotypes of intron1were significantlyassociated with the cold-tolerance traits (χ2=8.498，P <0.05). Among them, the DDgenotype was dominant in the cold-tolerance group (50.00%) and performed as aprotective factor for zebrafish under cold stress (OR=0.520,95%CI=0.255-1.061).The DE genotype was dominant in the cold-sensitive group (51.31%) and acted as arisk factor for zebrafish enduring cold stress (OR=3.012,95%CI=1.413-6.419).These results could provide a basis for a further research on GSTM gene in associationwith cold tolerance in fish.5. The type IV antifreeze protein (AFP-IV) homologous gene, AFP-IV-1and AFP-IV-2, were screened in olive flounder cDNA library. The full-length cDNA of AFP-IV-1was652bp, containing a375bp ORF which encoding124amino acids. The full-length cDNA of AFP-IV-2was781bp, containing a465bp ORF which encoding154amino acids. In order to investigate the anti-freeze activity of these two genes, theywere cloned into expression vector pET30a and expressed in E. coli BL21(DE3). Therecombinant AFP-IV-1(19.36kDa) was successfully induced by IPTG and the fusionprotein was mainly expressed in the form of soluble protein, and little fusion protein expressed in the form of inclusion bodies. The fusion AFP-IV-1protein had the highestexpression after4h induction with0.6mM IPTG. The results will provide the basis forfurther study on the application of recombinant flounder AFP-IV-1.In summary, this study reveals a large number of cold-torlerance related candidategenes and regulatory networks, screened a molecular marker associated with coldtolerance traits in fish and purified a recombinant protein possessing potential antifreezeactivity. This work could not only help to understand the fish cold tolerance generegulatory networks and mechanism, but also provide a reference for the MarkerAssistant Selection Breeding in commercial fish culture. |
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