Characterization and expression analysis of PP2C from Penaeus monodon under acute low salt and ammonia nitrogen stress
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摘要: PP2C家族蛋白 (Protein phosphatase 2C family protein) 是一类在抗逆过程中具有重要作用的蛋白磷酸酶,但其在甲壳动物中研究较少。通过RACE (Rapid amplification of cDNA ends) 方法克隆并获得了斑节对虾 (Penaeus monodon) 具有c型结构域的蛋白磷酸酶2C的cDNA全长 (PmPP2C)。该基因cDNA开放阅读框 (ORF) 全长2 079 bp,可编码692个氨基酸。实时荧光定量结果显示,PmPP2C基因在所有检测的组织中均有表达,在肝胰腺和鳃组织中的表达量最高,其次是胸神经、精巢、肌肉等组织。96 h急性低盐胁迫过程中,肝胰腺和鳃组织PmPP2C表达量先下调后上调。96 h急性氨氮胁迫中肝胰腺和鳃组织PmPP2C表达量整体呈下调-上调-下调趋势。结果表明,PmPP2C基因可能参与了斑节对虾急性低盐和氨氮胁迫响应过程,表明其可能在斑节对虾抗环境胁迫的免疫防御过程中发挥重要作用。Abstract: Protein phosphatase 2C family protein is a class of protein phosphatases that plays an important role in stress resistance. However, there are few studies in crustaceans. In this study, we cloned and obtained the full-length cDNA of protein phosphatase 2C (PmPP2C) with c-type domain in Penaeus monodon by rapid amplification of cDNA ends (RACE). The full length of ORF was 2 079 bp, encoding 692 amino acids. The real-time fluorescence quantitative results show that PmPP2C was expressed in all the tested tissues. The expression was the highest in hepatopancreas and gill tissues, followed by thoracic nerve, testis and muscle. The expression of PmPP2C in hepatopancreas and gill was first down-regulated and then up-regulated under 96 h acute low salt stress. The expression of PmPP2C in hepatopancreas and gill showed a downward-upward-downward trend under 96 h acute ammonia nitrogen stress. The results indicate that PmPP2C can be involved in the response of P. monodon to acute low salt and ammonia nitrogen stress, suggesting that it may play an important role in the immune defense against environmental stress.
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Key words:
- Penaeus monodon /
- PmPP2C /
- Molecular cloning /
- Low salt /
- Ammonia nitrogen
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表 1 实验中所用引物序列
Table 1. Oligonucleotide primers used in experiment
引物
Primer引物序列 (5'—3')
Primer Sequence (5'—3')退火温度
Annealing temperature/℃用途
FunctionPP2C-F1 AGACGAACTCAGCAGGGAA 55.1 cDNA序列验证 PP2C-R1 CAATAAGCGTTGGTCTACAGG 55.5 PP2C-F2 TCAAAGTGGTAGGCGTAATGG 58.0 PP2C-R1 ATAAGCGTTGGTCTACAGGGT 55.9 PP2C-3'GSP1 CAAGACGAACTCAGCAGGGAA 60.3 3' RACE PP2C-3'GSP2 GAGGCAAGCAGCCCGTAACCAA 62.2 PP2C-5'GSP1 TCCCTGCTGAGTTCGTCTTG 58.4 5' RACE PP2C-5'GSP2 TTGGAAGGCATTTGTCGG 57.2 PP2C-qF GCAAGCAGCCCGTAACCAAT 62.5 qPCR PP2C-qR ACCATCTCCAAGCACAACCC 59.4 EF-1α-qF AAGCCAGGTATGGTTGTCAACTTT 57.3 EF-1α-qR CGTGGTGCATCTCCACAGACT 59.2 -
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