2.1. Fish and sample collections
Loaches were obtained from Baishazhou fish market (Wuhan, China). All loaches were subjected to a ploidy analyzer (Partec, Germany) to determine their ploidy level. Diploid loaches were used and divided into four groups. The first group (15g ± 0.2) was used for cloning chop. Three female and three male adult loaches of the second group (15g ± 0.2) were used for determining expression levels of chop in ten tissues including liver, gill, brain, spleen, heart, intestine, muscle, gonad, kidney and skin, and each tissue served as an experimental unit (n = 3). A pair of parents from the third group (20g ± 0.2) was used for reproduction and embryos were collected at different early life stages, including oosperm, 2-cell stage, 8-cell stage, 32-cell stage, blastula stage, gastrula stage, neurula stage, tail-bud forming stage, muscle effect stage, heart-beating stage, and hatching stage. All samples were frozen at -80°C prior to RNA extraction. The fourth group (5g ± 0.2) contained 270 individuals, was used for H2O2 stress experiment.
2.2. Cloning the full-length cDNA of chop in loach
Total RNA was isolated from the liver tissues by using RNA isoPlus (TaKaRa, Japan). Quantities and qualities of isolated RNAs were measured by electrophoresis and spectrophotometry (Nanodrop 2000, Thermo Scientific, USA). Following the method of the SMART™ RACE cDNA amplification kit (Clontech, USA), the RNA obtained was completely reverse-transcribed into cDNA, which was next used to clone the gene.
A pair of universal amplified primers was designed (Table S1), depending on the multiple sequences alignments of numerous species including zebrafish (Barchydanio rerio var), common carp (Cyprinus carpio), roughskin sculpin (Trachidermus fasciatus), yellow cartfish (Pseudobagrus fulvidraco), etc. The PCR program was set as follow: initial denaturing for 5 min at 95°C, 35 cycles of 30 s at 95°C, 30 s at 55°C and 18 s at 72°C, and extra elongation for 5 min at 72°C. The PCR products were collected with 2% agarose (Sangon, China) and then purified with a TaKaRa Agarose Gel DNA Purification Kit Ver.2.0 (TaKaRa). After purification, the DNA fragments were ligated into PMD19 (TaKaRa) and the randomly selected positive transformants were opted and sequenced (Invitrogen, China).
2.3. Sequence and phylogenetic analysis
Similarity analysis of all the sequences was conducted by BLAST program at the National Center of Biotechnology Information (NCBI) (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequences of loach chop were translated into amino acid sequences by ORF Finder (http://www.ncbi.nlm.nih.gov/projects/gorf/). Multiple alignments of the deduced amino acid sequences were conducted by BioEdit software. Other vertebrate chop amino acid sequences for alignments and constructing phylogenetic tree were obtained from NCBI and the identities of these sequences were blasted by Blastp (NCBI). The phylogenetic tree of amino acid sequence of chop was constructed by MEGA6.0 program using the neighbor-joining method (NJ, bootstrap method: 1000 replications, Arizona State University, USA). The evolutionary distance between loach chop and other chop sequences was calculated by using p-distance and gaps were removed by pairwise detection, using default parameters.
2.4. H2O2 stress experiment
270 loaches were stocked in nine 30 L white tanks (10 L water volume and 25 ± 0.5°C water temperature) at a stocking density of 30 fishes/tank. Loaches were respectively treated with 0 µM, 50 µM and 100 µM H2O2 with triplicate for 24 h. During the experiment, there was no feeding and human interference to prevent introducing other stressors. The number of dead individuals in each tank was recorded every 6 hours. The data were input into GraphPad Prism 6.0 and the survival rate curves were generated.
Gill, liver and spleen tissues were collected from three loaches per tank at 6-, 12-, 18-, and 24-hour H2O2 stress exposure. All tissues were frozen in liquid nitrogen and stored at -80°C for determining catalase (CAT), glutathione peroxidase (GPX) and superoxide dismutase (SOD) activities. In addition, gills were also used for detecting expression levels of chop and its related genes (atf4, bcl2 and bax).
2.5. Real-time quantitative PCR (qPCR)
qPCR was performed using a Mini opticon real-time detector (BIO-RAD, Hercules, CA USA). Specific primers of the target genes and reference genes (β-actin and gapdh (glyceraldehyde-3-phosphate dehydrogenase)) are summarized in Table S1. The relative expressions of the target genes were calculated with the comparative Ct method (2 (−ΔΔCt)). All the procedures were based on the methods from our laboratory described by Cui et al. (2018).
2.6. Determination of antioxidant enzyme activities
The activities of SOD, GPX and CAT were estimated by using the commercially available kits (Nanjing Jiancheng Bioengineering Institute, China). The examinations of the three antioxidant enzyme activities were carried out according to the manuals of the kits.
2.7. Statistical analysis
The data were expressed as the means ± SD (standard deviation) or SE (standard error). One-way analysis of variance (ANOVA) was conducted by using Tukey’s post hoc test in SPSS statistical package version 25.0 (SPSS Inc., USA). P < 0.05 was significantly different (*P < 0.05, **P < 0.01, ***P < 0.001).