斑节对虾<i>PP</i>2<i>C</i>基因的克隆及其在急性低盐和氨氮胁迫下表达模式的相关研究

司梦茹; 李运东; 杨其彬; 姜松; 杨丽诗; 黄建华; 江世贵; 周发林

doi:10.12131/20210193

斑节对虾PP2C基因的克隆及其在急性低盐和氨氮胁迫下表达模式的相关研究

doi: 10.12131/20210193

司梦茹^{1, 2,},
李运东^{2, 3},
杨其彬³,
姜松²,
杨丽诗²,
黄建华²,
江世贵^{2, 3},
周发林^{2, 3, ,}

1.
上海海洋大学水产与生命学院 201306
2.
中国水产科学研究院南海水产研究所/农业农村部南海渔业资源开发利用重点实验室，广东广州 510300
3.
中国水产科学研究院南海水产研究所热带水产研究开发中心，海南三亚 572018

基金项目: 财政部和农业农村部国家现代农业产业技术体系资助 (CARS-48)；中国水产科学研究院中央级公益性科研院基本科研业务费专项资金资助 (2020TD30)；中国水产科学研究院南海水产研究所中央级公益性科研院所基本科研业务费专项资金资助 (2021SD13)；农业农村部财政专项 (NHYYSWZZZYKZX2020)

详细信息

作者简介:
司梦茹 (1995—)，女，硕士研究生，研究方向为水产动物遗传育种与分子生物学。E-mail: smryzykx@163.com

通讯作者:
周发林 (1975—)，男，研究员，博士，从事水产动物遗传育种研究。E-mail: zhoufalin0925@163.com

中图分类号: S 968.22+.7
计量
- 文章访问数: 162
- HTML全文浏览量: 26
- PDF下载量: 6
- 被引次数: 0
出版历程
- 收稿日期: 2021-07-05
- 修回日期: 2021-09-01
- 录用日期: 2021-10-09
- 网络出版日期: 2021-10-26

Characterization and expression analysis of PP2C from Penaeus monodon under acute low salt and ammonia nitrogen stress

SI Mengru^{1, 2
,},
LI Yundong^{2, 3},
YANG Qibin³,
JIANG Song²,
YANG Lishi²,
HUANG Jianhua²,
JIANG Shigui^{2, 3},
ZHOU Falin^{2, 3
, ,}

1.
College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China
2.
South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences/Key Laboratory of South China Sea Fishery Resources Exploitation & Utilization, Ministry of Agriculture and Rural Affairs, Guangzhou 510300, China
3.
Tropical Fishery Research and Development Center, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Sanya 572018, China

摘要

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。
- 斑节对虾 /
- PmPP2C /
- 分子克隆 /
- 低盐 /
- 氨氮
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.
- Penaeus monodon /
- PmPP2C /
- Molecular cloning /
- Low salt /
- Ammonia nitrogen

HTML全文

图 1 PmPP2C 核酸序列及氨基酸序列展示

Figure 1. Nucleotide and deduced amino acid sequence of PmPP2C

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

图 4 SOPMA软件对PmPP2C蛋白二级结构预测结果

Figure 4. Secondary structure of PmPP2C protein predicted by SOPMA

下载: 全尺寸图片幻灯片

图 5 斑节对虾PP2C三维结构空间示意图

Figure 5. Three-dimensional ribbon structure of PmPP2C

下载: 全尺寸图片幻灯片

图 6 PmPP2C在各组织的相对表达量

Figure 6. Expression of PmPP2C in different tissues

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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 Function
PP2C-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	3' RACE
PP2C-5'GSP1	TCCCTGCTGAGTTCGTCTTG	58.4	5' RACE
PP2C-5'GSP2	TTGGAAGGCATTTGTCGG	57.2	5' RACE
PP2C-qF	GCAAGCAGCCCGTAACCAAT	62.5	qPCR
PP2C-qR	ACCATCTCCAAGCACAACCC	59.4
EF-1α-qF	AAGCCAGGTATGGTTGTCAACTTT	57.3
EF-1α-qR	CGTGGTGCATCTCCACAGACT	59.2

下载: 导出CSV

参考文献(36)

[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, 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]. Aquac 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.