Identification and characterization of apoptosis regulator Bax involved in air-exposure stress of the mud crab, Scylla Paramamosain Estampador, 1949

in Crustaceana
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The mud crab, Scylla paramamosain Estampador, 1949, is a widely farmed commercial species in the South-East coastal areas of China. The crabs are captured and placed in a water-free container for further transportation. The long-time air exposure makes the crabs suffer from oxidative stress, triggering cell apoptosis and leading to the crab’s death. In this study, the homologue of the apoptosis regulator BAX was firstly identified in S. paramamosain and named as SpBAX. The coding region of SpBAX yielded a polypeptide of 278 amino acids, consisting of the defining motif of the BAXs family including Bcl-2 homologous BH1, BH2, BH3 regions and a transmembrane (TM) domain. Subcellular prediction suggested that SpBAX was located in the cytoplasm. The three-dimensional structure showed that SpBAX contained 8 helical regions. Two central α-helices (α5 and α6) flanked on one side by α3 and α4, and on the other side by α1, α2 and α7, which showed the same structure as BAXs in mammals. The highest expression level of SpBAX was detected in hepatopancreas tissue. The expression level of SpBAX was up-regulated in 12 h in hepatopancreas and in 24 h in haemocytes after air exposure. Meanwhile, a flow cytometry assay revealed that the proportion of apoptotic haemocytes exceeded 65% after air exposure for 36 h, showing a significant difference with the control group. These results indicated that the cloned SpBAX might be involved in the response to air exposure stress by causing cell apoptosis. This study may thus be helpful to clarify the mechanisms of the air-exposure stress response in the mud crab.

Identification and characterization of apoptosis regulator Bax involved in air-exposure stress of the mud crab, Scylla Paramamosain Estampador, 1949

in Crustaceana



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    Nucleotide sequence of the SpBAX cDNA (KY348742.1) and its predicted amino-acid sequence. Start and stop codons (*) are in boldface. The putative conserved domains of BH1-3 and TM are underlined. The poly(A) signal (AATAAA) is highlighted with a box.

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    Amino-acid alignment of BAX from Scylla paramamosain Estampador, 1949 with other species. GenBank accession numbers are listed as follows: Scylla paramamosain (AQS99485.1), Lepeophtheirus salmonis (Krøyer, 1837) (ADD24081.1), Plutella xylostella (Linnaeus, 1758) (AHG99281.1), Daphnia magna Straus, 1820 (KZS14244.1), Xenopus laevis (Daudin, 1802) (NP_001079104), Sus scrofa Linnaeus, 1758 (XP_005664767), Mus musculus Linnaeus, 1758 (NP_031553), Homo sapiens Linnaeus, 1758 (CAD10744), Danio rerio (Hamilton-Buchanan, 1822) (NP_571637), Crassostrea gigas (Thunberg, 1793) (EKC42310.1). The secondary structural elements, such as helix and coils, are highlighted with a wavy line.

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    Phylogenetic analysis of the amino-acid sequence of BAX. Amino acid sequences of BAX from various species were obtained from NCBI protein database. The number at the forks indicated the bootstrap value. The confidence values of the branching pattern were tested on 1000 bootstrap replicates. The scale bar indicates the number of 10 residue substitutions per site. The protein sequences used for phylogenetic analysis were: Ovis aries Linnaeus, 1758 (XP_004015412), Canis lupus familiaris Linnaeus, 1758 (NP_001003011), Panthera tigris altaica Temminck, 1844 (XP_007074210), Sus scrofa Linnaeus, 1758 (XP_005664767), Mus musculus Linnaeus, 1758 (NP_031553), Homo sapiens Linnaeus, 1758 (CAD10744), Rattus norvegicus (Berkenhout, 1769) (Q63690), Chrysochloris asiatica (Linnaeus, 1758) (XP_006868239), Python bivittatus Kuhl, 1820 (XP_007435463), Anolis carolinensis (Voigt, 1832) (XP_003226241), Xenopus laevis (Daudin, 1802) (NP_001079104), Danio rerio (Hamilton-Buchanan, 1822) (NP_571637), Salmo salar Linnaeus, 1758 (XP_013981944.1), Capitella teleta Blake, Grassle & Eckelbarger, 2009 (ELU02334), Aplysia californica (James Graham Cooper, 1863) (XP_005104447), Mytilus galloprovincialis Lamarck, 1819 (AGK88247), Crassostrea hongkongensis Lam & Morton, 2003 (KM262836), Stegodyphus mimosarum Pavesi, 1883 (KFM65908.1), Daphnia magna Straus, 1820 (KZS14244.1), Lepeophtheirus salmonis (Krøyer, 1837) (ADD24081.1) and Scylla paramamosain Estampador, 1949 (AQS99485.1).

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    Predicted three-dimensional structure of SpBAX protein. BH1 is situated in the α4 and α5 helical region (green). BH2 is situated in the α7 helical region (yellow). BH3 is situated in the α2 helix region (sea blue).

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    Gene expression stability of three reference genes calculated by Bestkeeper in a, different tissues, b, hepatopancreas after air exposure treatment and c, haemocytes after air exposure treatment. The y-axis represents the standard deviation (SD) of the Ct values.

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    Relative mRNA expression level of SpBAX in different tissues, including stomach (Sto), haemocytes (Hae), heart (Hea), muscle (Mus), ganglion (Gan), intestine (Int), gill (Gil), epidermis (Epi) and hepatopancreas (Hep). Relative gene expression was calculated using the 2ΔΔCt method with initial normalization of SpBAX against EIF within each sample. The expression level of SpBAX in the stomach was arbitrarily set at 1.0 and the level of other tissues were adjusted correspondingly. Vertical bars represent the mean ± SEM (n=7). Different superscript letters indicate significant differences (P<0.05).

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    The relative expression level of SpBAX mRNA in haemocytes (a) and hepatopancreas (b) at 0, 6, 12, 24 and 36 h post air exposure. Relative gene expression was calculated using the 2ΔΔCt method with initial normalization of SpBAX against EIF within each sample. The expression level of SpBAX in the 0 h group was arbitrarily set at 1.0 and the level of other groups were adjusted correspondingly. Vertical bars represent the mean ± SEM (n=7). Different superscript letters indicate significant differences (P<0.05).

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    Apoptotic rate of haemocytes detection after air-exposure treatment for 36 h by flow cytometry. a, Apoptotic rate of haemocytes in the control group. b, Apoptotic rate of haemocytes under the air exposure treatment for 36 h. c, Percentage of apoptotic rate in the control group and air exposure group. Vertical bars represent the mean ± SEM (n=5). Asterisks indicate significant differences (**P<0.01) compared with values of the control group.

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