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，而在甲壳动物中研究较少。该研究通过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，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。实验结果表明，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 immune defense against environmental stress.
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Key words:
- Penaeus monodon /
- PmPP2C /
- Molecular cloning /
- Low salt /
- Ammonia nitrogen
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图 1 PmPP2C 核酸序列及氨基酸序列展示
起始密码子 (ATG)、终止密码子 (TAA) 用灰色阴影显示；糖基化位点用黑色圆圈显示；黑色画线部分为PmPP2C c型结构域。
Figure 1. Nucleotide and deduced amino acid sequence of PmPP2C
The start code (ATG) and the termination code (TAA) are highlighted by grey shading; glycosylation sites are highlighted by blank circle; PmPP2C c domain is highlighted by black underline.
图 2 PmPP2C c亚型与其他物种氨基酸序列比对
Figure 2. Multiple alignment of PmPP2C c subtype amino acid sequences from P. monodon and other species
图 3 运用Clustal和MEGA 6.06软件基于NJ法构建PmPP2C c亚型与其他物种的系统进化树
Figure 3. NJ phylogenetic tree of PmPP2C c subtype with other species by Clustal and MEGA 6.06
图 4 SOPMA软件对PmPP2C蛋白二级结构预测结果
蓝色为α-螺旋；绿色为β-转角；紫色为无规则卷曲；红色为延伸链。
Figure 4. Secondary structure of PmPP2C protein predicted by SOPMA
Blue. α-helix; Green. β-turn; Purple. Random coli; Red. Extended strand.
图 6 PmPP2C在各组织的相对表达量
图中数值为“平均值±标准差”(N=3)，不同小写字母表示组间差异显著 (P<0.05)；后图同此。
Figure 6. The expression of PmPP2C in different tissues
The values are $\bar x$±SD (N=3). Bars with different lowercase letters indicate significant differences (P<0.05); the same case in the following figures.
图 7 急性低盐胁迫下斑节对虾PmPP2C在肝胰腺和鳃组织中的相对表达水平
Figure 7. mRNA relative expression of PmPP2C in hepatopancreas and gill under acute low salt stress
图 8 急性氨氮胁迫下斑节对虾PmPP2C在肝胰腺和鳃组织中的相对表达水平
Figure 8. mRNA relative expression of PmPP2C in hepatopancreas and gill under acute ammonia nitrogen stress
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退火温度
Tm/℃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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[1] TONG Y, QUIRION R, SHEN S H. Cloning and characterization of a novel mammalian PP2C isozyme[J]. J Biol Chem, 1998, 273(52): 35282-35290. doi: 10.1074/jbc.273.52.35282 [2] TAYEBEH O B, KEIVAN M A, REZVAN E. Wip1: a candidate phosphatase for cancer diagnosis and treatment[J]. DNA Repair, 2017, 54: 63-66. doi: 10.1016/j.dnarep.2017.03.004 [3] LU X, AN H, JIN R, et al. PPM1A is a RelA phosphatase with tumor suppressor-like activity[J]. Oncogene, 2014, 33(22): 2918-2927. doi: 10.1038/onc.2013.246 [4] LIU T, LIU Y, CAO J, et al. ILKAP binding to and dephosphorylating HIF-1α is essential for apoptosis induced by severe hypoxia[J]. Cell Physiol Biochem, 2018, 46(6): 2500-2507. doi: 10.1159/000489656 [5] TANG Y T, PAN B, ZHOU X, et al. Wip1-dependent modulation of macrophage migration and phagocytosis[J]. Redox Biol, 2017, 13: 665-673. doi: 10.1016/j.redox.2017.08.006 [6] MATHUR A, PANDEY V K, KAKKAR P. PHLPP: a putative cellular target during insulin resistance and type 2 diabetes[J]. J Endocrinol, 2017, 233(3): 185-198. doi: 10.1530/JOE-17-0081 [7] AMARJEET S, AMITA P, ASHISH K. S, et al Plant protein phosphatases 2C: from genomic diversity to functional multiplicity and importance in stress management[J]. Crit Rev Biotechnol, 2016, 36(6): 1023-1035. doi: 10.3109/07388551.2015.1083941 [8] FAN K, CHEN Y, MAO Z, et al. Pervasive duplication, biased molecular evolution and comprehensive functional analysis of the PP2C family in Glycine max[J]. BMC Genomics, 2020, 21(1): 465-481. doi: 10.1186/s12864-020-06877-4 [9] 齐阳, 许维恒, 张俊平, 等. PP2C蛋白磷酸酶调控的细胞信号通路研究进展[J]. 药学实践杂志, 2018, 36(5): 385-388,456. doi: 10.3969/j.issn.1006-0111.2018.05.001 [10] JAKKULA P, RAHILA Q, ATIF I, et al. Leishmania donovani PP2C: kinetics, structural attributes and in vitro immune response[J]. Mol Biochem Parasit, 2018, 223: 37-49. doi: 10.1016/j.molbiopara.2018.06.005 [11] HAIDER M S, KURJOGI M M, KHALIL-UR-REHMAN M, et al. Grapevine immune signaling network in response to drought stress as revealed by transcriptomic analysis[J]. Plant Physiol Biochem, 2017, 121: 187-195. doi: 10.1016/j.plaphy.2017.10.026 [12] 胡秋涛, 侯丹, 赵钟毓, 等. 毛竹PP2C基因家族鉴定与表达分析[J]. 农业生物技术学报, 2020, 28(10): 1776-1787. [13] 范红弟, 李运东, 杨其彬, 等. 斑节对虾MKK7基因的克隆及在不同胁迫条件下的表达分析[J]. 中国水产科学, 2020, 27(7): 748-758. [14] LIU G, HU X, SUN B, et al. Phosphatase Wip1 negatively regulates neutrophil development through p38 MAPK-STAT1[J]. Blood, 2013, 121(3): 519-529. doi: 10.1182/blood-2012-05-432674 [15] BOLLMANN P, WERNER F, JARON M, et al. Initial characterization of stressed transgenic mice with cardiomyocyte-specific overexpression of protein phosphatase 2C[J]. Front Pharmacol, 2021, 11: 591773-591789. doi: 10.3389/fphar.2020.591773 [16] 杨其彬, 叶乐, 温为庚, 等. 盐度对斑节对虾蜕壳, 存活, 生长和饲料转化率的影响[J]. 南方水产科学, 2008, 4(1): 16-21. doi: 10.3969/j.issn.2095-0780.2008.01.003 [17] JOSEPH A, PHILIP R. Immunocompetence of Penaeus monodon under acute salinity stress and pathogenicity of Vibrio harveyi with respect to ambient salinity[J]. Fish Shellfish Immunol, 2020, 106: 555-562. doi: 10.1016/j.fsi.2020.07.067 [18] LI Y D, ZHOU F L, HUANG J H, et al. Transcriptome reveals involvement of immune defense, oxidative imbalance, and apoptosis in ammonia-stress response of the black tiger shrimp (Penaeus monodon)[J]. Fish Shellfish Immunol, 2018, 83: 162-170. doi: 10.1016/j.fsi.2018.09.026 [19] 陈劲松, 江世贵, 黄建华, 等. 斑节对虾天门冬氨酸转氨酶基因的克隆及氨氮胁迫条件下的表达分析[J]. 南方水产科学, 2017, 13(3): 73-82. doi: 10.3969/j.issn.2095-0780.2017.03.010 [20] DOHONEY K M, GUILLERM C, WHITEFORD C, et al. Phosphorylation of p53 at serine 37 is important for transcriptional activity and regulation in response to DNA damage[J]. Oncogene, 2004, 23(1): 49-57. doi: 10.1038/sj.onc.1207005 [21] LI D W C, LIU J P, SCHMID P C, et al. Protein serine/threonine phosphatase-1 dephosphorylates p53 at Ser-15 and Ser-37 to modulate its transcriptional and apoptotic activities[J]. Oncogene, 2006, 25(21): 3006-3022. doi: 10.1038/sj.onc.1209334 [22] LI D W C, FASS U, HUIZAR I, et al. Okadaic acid-induced lens epithelial cell apoptosis requires inhibition of phosphatase-1 and is associated with induction of gene expression including p53 and bax[J]. Eur J Biochem, 1998, 257(2): 351-361. doi: 10.1046/j.1432-1327.1998.2570351.x [23] OLSEN J V, BLAGOEV B, GNAD F, et al. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks[J]. Cell, 2006, 127(3): 635-648. doi: 10.1016/j.cell.2006.09.026 [24] ZHAI Y F, HE P, SHI D J, et al. iTRAQ-based proteomic analysis of the hepatopancreas from Litopenaeus vannamei after trans-vp28 gene Synechocystis sp. PCC6803 immunization[J]. Fish Shellfish Immunol, 2020, 104: 686-692. doi: 10.1016/j.fsi.2020.05.078 [25] DAVIE E, FORTE G A, PETERSEN J. Nitrogen regulates AMPK to control TORC1 signaling[J]. Curr Biol, 2015, 25(4): 445-454. doi: 10.1016/j.cub.2014.12.034 [26] PAN L Q, SI L J, LIU S N, et al. Levels of metabolic enzymes and nitrogenous compounds in the swimming crab Portunus trituberculatus exposed to elevated ambient ammonia-N[J]. J Ocean Univ China, 2018, 17(4): 957-966. doi: 10.1007/s11802-018-3574-y [27] LIU L, HU X, SONG J, et al. Over-expression of a Zea mays L. protein phosphatase 2C gene (ZmPP2C) in Arabidopsis thaliana decreases tolerance to salt and drought[J]. J Plant Physiol, 2009, 166(5): 531-542. doi: 10.1016/j.jplph.2008.07.008 [28] UMEZAWA T, NAKASHIMA K, MIYAKAWA T, et al. Molecular basis of the core regulatory network in ABA responses: sensing, signaling and transport[J]. Plant Cell Physiol, 2010, 51(11): 1821-1839. doi: 10.1093/pcp/pcq156 [29] MCNAMARA J C, FARIA S C. Evolution of osmoregulatory patterns and gill ion transport mechanisms in the decapod Crustacea: a review[J]. J Comp Physiol B, 2012, 182(8): 997-1014. doi: 10.1007/s00360-012-0665-8 [30] PENG T, WANG W N, GU M M, et al. Essential roles of Cdc42 and MAPK in cadmium-induced apoptosis in Litopenaeus vannamei[J]. Aquat Toxicol, 2015, 163: 89-96. doi: 10.1016/j.aquatox.2015.03.023 [31] LOTZ J, ANTON L S, SOTO M. Effect of chronic Taura syndrome virus infection on salinity tolerance of Litopenaeus vannamei[J]. Dis Aquat Organ, 2005, 65(1): 75-78. [32] FAN H D, LI Y D, YANG Q B, et al. Isolation and characterization of a MAPKK gene from Penaeus monodon in response to bacterial infection and low-salinity challenge[J]. Aquacult Rep, 2021, 20: 671-681. [33] 姚万龙, 何玉英, 刘萍, 等. 中国明对虾MKK3基因cDNA克隆及其在氨氮胁迫下的表达[J]. 中国水产科学, 2016, 23(1): 34-43. [34] 姚万龙, 何玉英, 刘萍, 等. 中国明对虾MKK4基因克隆及其在氨氮胁迫下的表达分析[J]. 水产学报, 2015, 39(6): 779-789. [35] YU Y H, LI J X, YU W, et al. GADD45α induction by nickel negatively regulates JNKs/p38 activation via promoting PP2Cα expression[J]. PloS One, 2013, 8(3): 185-193. [36] LUO L Y, JIANG S S, HUANG D Q, et al. MLK3 phophorylates AMPK independently of LKB1[J]. PLoS ONE, 2015, 10(4): 927-935. -
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