斑节对虾新型抗菌肽<i>ALF-like</i>基因克隆表达及其抗菌功能分析

梁子莹; 邱丽华; 王鹏飞; 张博; 闫路路; 乔秀亭; 赵超

doi:10.3969/j.issn.2095-1191.2023.09.016

斑节对虾新型抗菌肽ALF-like基因克隆表达及其抗菌功能分析

梁子莹^1,2,
邱丽华^2,3,
王鹏飞^2,3,
张博²,
闫路路²,
乔秀亭^1,,
赵超^2,3,

1 天津农学院水产学院, 天津 300384;
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:

国家自然科学基金青年基金项目(42006113)；海南省重点研发计划项目(ZDYF2021SHFZ268,ZDYF2022XDNY244)；海南省自然科学基金青年基金项目(322QN432)

详细信息

作者简介:
梁子莹(1996-),https://orcid.org/0000-0002-8633-5578,研究方向为海洋生物功能基因,E-mail:1416887836@qq.com

通讯作者:
乔秀亭(1965-),https://orcid.org/0000-0001-5111-1040,教授,主要从事水产动物营养与饲料研究工作,E-mail:qiao1230430@163.com

赵超(1987-),https://orcid.org/0000-0002-2654-6058,博士,副研究员,主要从事海洋生物功能基因研究工作,E-mail:zhaochao1018@outlook.com

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出版历程
- 收稿日期: 2023-04-12
- 网络出版日期: 2024-02-01

Cloning,expression and antibacterial function analysis of novel antibacterial peptide gene ALF-like in Penaeus monodon

1 College of Fishery Science, Tianjin Agricultural University, Tianjin 300384, China;
2 South China Sea Fisheries Re-search Institute, Chinese Academy of Fishery Sciences/Key Lab of South China Sea Fishery Resource Exploitation and Utilization, Ministry of Agriculture and Rural Affairs, Guangzhou, Guangdong 510300, China;
3 Tropical Fisheries Research and Development Center, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Sanya, Hainan 572018, China

Funds:

Youth Project of National Natural Science Foundation of China(42006113)；Hainan Key Research and Development Plan Project(ZDYF2021SHFZ268,ZDYF2022XDNY244)；Youth Project of Hainan Natural Science Foundation(322QN432)

摘要

摘要: 【目的】克隆并明确斑节对虾(Penaeus monodon)新型抗菌肽ALF-like基因(PmALF-like)的结构特征、组织表达分布规律及副溶血弧菌(Vibrio parahemolyticus)刺激后的表达变化,探究其在抗菌先天免疫中的作用与功能,为阐明斑节对虾抗菌免疫调控机制提供参考依据。【方法】依据PmALF-like基因表达序列标签(EST)序列,利用RACE扩增获得PmALF-like基因cDNA全长序列,通过ProtParam、SignalP 5.0、MegAlign和MEGA 7.0等在线软件进行生物信息学分析;利用实时荧光定量PCR检测PmALF-like基因在斑节对虾不同组织中的表达特征及其在副溶血弧菌刺激后的表达模式;利用原核重组方法获得PmALF-like成熟肽的重组蛋白(去信号肽),并通过液体培养基法和牛津杯法等探究Pm ALF-like成熟肽抑制细菌生长的作用。【结果】PmALF-like基因cDNA序列全长1242 bp,包括45 bp的5'端非编码区(5'-UTR)、798 bp的3'端非编码区(3'-UTR)和399 bp的开放阅读框(ORF),共编码132个氨基酸残基;分子量为15.26k D,理论等电点(pI)为7.67;利用SignalP5.0对PmALF-like蛋白进行信号肽预测,结果表明其第18个氨基酸处存在信号肽剪切位点。实时荧光定量PCR检测结果表明,PmALF-like基因在斑节对虾血淋巴、肝胰腺、鳃、肠道、肌肉等组织中均有不同程度的表达,以肝胰腺中的相对表达量最高,血淋巴次之,肌肉组织中的相对表达量较少。副溶血弧菌刺激后,斑节对虾肝胰腺组织PmALF-like基因产生免疫应答,其基因相对表达量在6~72 h显著(P<0.05)或极显著(P<0.01)上调。利用原核表达方法在体外成功获得不含信号肽的PmALF-like成熟肽,借助液体培养基法和牛津杯法发现PmALF-like成熟肽对维氏气单胞菌(Aeromonas veronii)、副溶血弧菌及嗜水气单胞菌(Aeromonas hydrophila)的生长均有明显抑制作用。【结论】PmALF-like基因在斑节对虾各组织中广泛表达,其成熟肽对维氏气单胞菌、副溶血弧菌及嗜水气单胞菌的生长具有明显抑制作用。
- 斑节对虾 /
- 抗菌肽 /
- PmALF-like基因 /
- 组织表达 /
- 功能分析
Abstract: 【Objective】To clone a novel antibacterial peptide gene from Penaeus monodon(PmALF-like),and to clarify its structural characteristics,tissue expression pattern,and its expression changes after stimulation of Vibrio parahemolyticus,so as to explore its role and function in antibacterial innate immunity,with an aim to provide reference for elucidating the disease-resistant immune mechanism of P. monodon. 【Method】Based on the expression sequence target(EST) sequence of PmALF-like gene, the full-length cDNA sequence of PmALF-like was obtained by RACE amplification. Bioinformatics analysis was performed using online softwares such as ProtParam,SignalP 5.0,MegAlign and MEGA 7.0. The expression characteristics of PmALF-like gene in different tissues and its expression changes after V. parahemolyticus stimulation were detected by real-time fluorescence quantitative PCR(qRT-PCR). The recombinant protein PmALF-like or mature peptide was obtained by prokaryotic recombination method(removing signaling peptide),and the inhibitory effect of PmALF-like mature peptide on bacterial growth was investigated by liquid medium method and Oxford cup method. 【Result】The total length of the cDNA sequence of PmALF-like gene was 1242 bp,including the 5' non-coding region(5'-UTR) of 45 bp,the 3' non-coding region(3'-UTR) of 798 bp and the open reading frame(ORF) of 399 bp,encoding 132 amino acids. The molecular weight was 15.26 kD and the theoretical isoelectric point(p I) was 7.67. A signal peptide of PmALF-like protein was predicted by the online software SignalP 5.0 with a splicing site at the 18^th amino acid.The results of qRT-PCR showed that PmALF-like was expressed in hemopancreas,hepatopancreas,gills,intestinal tract,muscle and other tissues to varying degrees,with the highest relative expression level in hepatopancreas,followed by hemopancreas,and less relative expression level in muscle tissue. After stimulation by V. parahemolyticus,the expression level of PmALF-like gene in hepatopancreas of P. monodon produced immune response,and its gene expression was significantly(P<0.05) or extremely significantly(P<0.01) up-regulated at 6-72 h. PmALF-like mature peptide without signal peptide was successfully obtained by prokaryotic expression method in vitro. It was found that the mature peptide of PmALF-like had obvious inhibitory effect on the growth of Aeromonas victori,V. parahemolyticus and Aeromonas hydrophila by liquid medium method and Oxford cup method. 【Conclusion】PmALF-like gene is widely expressed in various tissues of P. maculatus, its mature peptide has obvious inhibitory effects on the growth of A. vickeri,V. parahaemolyticus and A. hydrophila.
- Penaeus monodon /
- antibacterial peptide /
- PmALF-like gene /
- tissue expression /
- functional analysis

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