Trophotometry (absorbance at 260 nm) and agarose gel electrophoresis, respectively.RTqPCR analysisMethodsFish
Trophotometry (absorbance at 260 nm) and agarose gel electrophoresis, respectively.RTqPCR analysisMethodsFish PubMed ID: samplingIn 2015, from March to August, healthy fish (n = 98) from six populations of C. nasus, were collected at seven time points from their major regional habitats in the Yangtze River during their migration upstream (Fig. 1). The collection sites were at: Anqing (AQ); Dangtu (DT); Zhenjiang (ZJ); Jingjiang (JJ); Nantong (NT), and Chongming (CM). Fish were kept on dry ice immediately after collection, and were transferred to the laboratory in the dry ice boxes.Analysis of development stage and tissue collectionAfter measuring the body weights (BW ?0.01 g wet weight; WW) of the fish their gonads were dissected andTo analyze the mRNA transcript expression patterns at each fish developmental PubMed ID: stage, total RNA (about 2 g) that had been isolated from the brains and ovary tissues was reverse transcribed into cDNA using the SMARTTM cDNA kit (Clonetech, USA) and RTqPCR analysis was performed, using the PrimeScript Real-time PCR Kit (TaKaRa, Japan). Target fragments of cDNA that encode GnRH-R2 were chosen basing on the constructed transcriptome library using BLAST tool [35]. First-strand cDNA was prepared as described above; the genespecific primer pair (GnRH-R2-F and GnRH-R2-R; Table 1) were designed based on the cDNA sequences (GenBank accession numbers KU861569) to produce 387 bp amplicons. The PCR reaction conditions were as per the qPCR Kit protocol. Samples were run in triplicate using pooled RNA (as described above) at the same concentration, and normalized to the selected control gene18sRNA; the primer pair 18sRNA-R and 18sRNA-F (Table 1) were designed based on the C. nasus 18sRNA and to amplify a fragment of 232 bp. The gene expression levels were calculated using the 2-Ct comparative CT method [36]. Mean and standard deviation valuesDuan et al. BMC Developmental Biology (2016) 16:Page 3 ofFig. 1 Sampling distribution locations for C. nasus in the Yangze River. Black dot display the sampling distribution locations. AQ: Anqing; DT: Dangtu; ZJ: Zhenjiang; JJ: Jingjiang; NT: Nantong; CM: Chongming, TH: Taihu lake; HZH: hongzehu lake; BYH: Boyanghu lakewere calculated from the triplicate runs, and presented as fold differences in expression, relative to 18 s RNA expression. Data were analyzed using CFX ManagerTM software (version 1.0).GnRH-R2 antiserum preparingAn increase in antibody titers against the peptide was verified by enzyme-linked immuno sorbent assay (ELISA).ELISAGnRH-R2 antiserum was produced commercially by Hua-an Biol. Co., Ltd. (Hua-an, Hangzhou, China). get Vercirnon Briefly, a synthetic signature peptide (LVVVSLDRH) for GnRH-R2, conjugated with the keyhole limpet hemocyanin, was emulsified with complete (for the first immunization) and incomplete (for the second to fourth immunizations) Freund’s adjuvant, and injected into a New Zealand rabbit at intervals of 2 to 3 weeks. Before immunization and after the third and fourth injections, the rabbit was bled and serum samples were collected.Table 1 Sequences of primers used in the present studyPrimer Name DNA-Sequence 5-3 F–Forward/ R–Reverse Gene-specific Primer pairs for RT-qPCR GnRH-R2-F GnRH-R2-R 5-CGTGCGGTGAAGG CGAAGGGGGTGG-3 5-ACACAACCCCAAC TAAGCAAGCATA-3 60.7 64.7 387 Annealing Fragment Temperature ( ) Size (bp)18sRNA primers 18sRNA-R 18sRNA-F 5- TGATTGGGACTGG GGATTGAA-3 5- TAGCGACGGGC GGTGTGT-3 59.2 62.4ELISAs were used to measure the GnRH-R2 concentrations in the fish ser.

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