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| cDNA-AFLP Analysis of Differentially Expressed Genes Related to Hypoxia Stress in Bighead Carp (Hypophthalmichthys nobilis) |
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Abstract Abstract Hypoxia is one of the major stresses in aquaculture animals. Bighead carp (Hypophthalmichthys nobilis) is one of the most important aquaculture fish in China, which is sensitive to hypoxia stress, and the related genetic research is rarely reported. In this study, six hypoxia-sensitive samples (S group) and six hoypoxia-tolerant samples (T group) from a hypoxia stress experiment of bighead carp were selected, and differentially expressed genes between the two groups were analyzed using cDNA-amplified fragment length polymorphism (cDNA-AFLP) technique. A total of 10 571 transcript-derived fragments (TDFs) were obtained from 256 primer combinations of the EcoRⅠand MseⅠ AFLP system, and the number of TDFs produced by a single primer combination ranged from 21 to 63, with an average of 41. Four types of differential straps were screened in the hypoxia stress experiment of bighead carp, including A (up-regulated band), B (down-regulated band), C (the fragment only expressed in one sample) and D (the fragment expressed in all samples), and bands of types A and B were chosen as target bands. Among those 221 differentially expressed TDFs between two groups, 137 (62%) were up-regulated, and 84 (38%) were down-regulated. 70 TDFs were successfully cloned and sequenced, and the length of fragments ranged from 96 to 402 bp. The results of sequence blast against public databases revealed that 29 TDFs (TDF42~TDF70) had no hits to any known homologous sequences, 9 TDFs (TDF33~TDF41) were unknown functional sequences, and 32 TDFs (TDF1~TDF32) showed homology to genes of known functions. Among the 32 differentially expressed TDFs, TDF29 and TDF32 sequences were different but both encoded the same protein (S- adenosylmethionine synthase). These functional genes participated in such physiological pathways as transcriptional regulation (16%), stress and signal transduction (9%), energy metabolism (7%), protein synthesis (4%), immune defense (4%) and cell proliferation (6%). Four TDFs with the genes of known functions (ANXA6, TRIM25, Tnp and RBAT) were randomly selected for verification by qRT-PCR analysis. For ANXA6, TRIM25 and Tnp, the expression patterns from qRT-PCR were consistent with those from AFLP analysis, while the expression pattern of RBAT was different in two methods. The results of this study would facilitate revealing molecular mechanism of genetic regulation responsible to hypoxia stress in bighead carp, and they would also provide useful information for potential gene (marker)-assisted selection towards hypoxia-tolerant variety in aquaculture of this species.
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Received: 17 April 2017
Published: 20 July 2017
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