The metallothionein gene from the Oriental river prawn Macrobrachium nipponense (De Haan, 1849): characterization and expression in response to hypoxia and reoxygenation

in Crustaceana
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Since mammals make use of metallothioneins (MTs) to protect against hypoxia-generated reactive oxygen species (ROS), here we investigate whether MTs have the same effect in the Oriental river prawn Macrobrachium nipponense (De Haan, 1849). Levels of M. nipponense MT (MnMT) mRNA in different tissues were analysed using a real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). We found that MnMT is differentially expressed in the haemocytes, intestine, gills, heart, hepatopancreas and muscle. The highest levels of expression occurred in the hepatopancreas and heart. The results of the qRT-PCR and Western blot indicated that MnMT mRNA and protein expression in the hepatopancreas increased significantly in response to hypoxia 24 h and followed by 1 h reoxygenation compared to normoxia. Similarly, there were also significant increases in respiratory bursts and ROS production in the hepatopancreas. The MT protein possibly has a protective effect against ROS generated during hypoxia and reoxygenation stimuli.

The metallothionein gene from the Oriental river prawn Macrobrachium nipponense (De Haan, 1849): characterization and expression in response to hypoxia and reoxygenation

in Crustaceana

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Figures

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    (A) Nucleotide and deduced amino-acid sequences of MnMT gene. The deduced amino acid sequence is shown as a capital letter under each codon of the coding sequence. The characteristic 18 cysteine residues MTs are shown in bold; (B) Clustal alignment of MT amino acid sequences from various organisms. The crustacean MT N-terminus motifs P-[GD]-P-CC-X-(3,4)-C-X-C are shown in the red box. The cysteine residues characteristic of metallothioneins are shown in the last line. This figure is published in colour in the online edition of this journal, which can be accessed via http://booksandjournals.brillonline.com/content/journals/15685403.

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    The phylogenetic tree based on the alignment of known amino acid sequences of MnMT proteins. The percentages of replicate trees in which the associated taxa are clustered together in the bootstrap test (1000 replicates) are shown next to the branches. GenBank accession numbers are provided next to each species name.

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    Tissue-specific mRNA expression of MnMT determined using quantitative real-time PCR. Vertical bars represent the mean ± SE values for triplicate samples.

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    Quantitative real-time PCR analysis of MnMT expression in the hepatopancreas of the Oriental river prawn exposed to normoxia (Control) and 24 h hypoxia (H24); and after recovery of 1 h (R1), 2 h (R2) and 4 h (R4) in normoxic water. Significant differences (P<0.05) in MnMT expression between the challenge and control groups are indicated with asterisks. Values are mean ± SE for triplicate samples.

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    Expression of MnMT in the hepatopancreas of the Oriental river prawn exposed to normoxia (Control); and after recovery of 1 h (R1), 2 h (R2) and 4 h (R4) in normoxic water. β-Actin was used as the loading control. This figure is published in colour in the online edition of this journal, which can be accessed via http://booksandjournals.brillonline.com/content/journals/15685403.

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    Respiratory burst activity (A) and ROS generation capacity (B) in the hepatopancreas of Macrobrachium nipponense at different time intervals after hypoxia and reoxygenation. Asterisks indicate a significant difference (P<0.05) between the treatment and control groups. Values are mean ± SE for triplicate samples.

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