对虾急性肝胰腺坏死病病原毒素基因在不同环境下表达差异研究

doi:10.11924/j.issn.1000-6850.casb20200300228

摘要/Abstract

摘要：

急性肝胰腺坏死病(AHPND)是由副溶血弧菌引起的对虾病害,给世界养虾业造成了严重的经济损失。本文旨在研究主要致病因子Pir毒素基因在不同环境因素处理下的表达差异,初步探究抑制Pir毒素基因表达的可能措施。应用荧光定量RT-PCR技术,采用比较CT值法检测致病菌中Pir毒素基因在不同温度和pH条件下的表达量。结果表明,试验分离株为引发对虾AHPND毒素蛋白Pir的致病株,在18℃、24℃、30℃和36℃条件下,Pir毒素基因的表达量随温度的增加而升高,在pH 7、pH 8和pH 9条件下,Pir毒素基因的表达量随pH的上升而降低,且pH因素对其表达量差别较大。研究结果为探究副溶血弧菌致病力强弱和环境因素的关系提供了基础数据,同时也为养殖生产中AHPND防控策略的制定提供了科学依据。

关键词: 副溶血弧菌, 急性肝胰腺坏死病, Pir基因, 表达分析

Abstract:

Acute hepatopancreatic necrosis disease (AHPND) is caused by infection with Vibrio parahaemolyticus, and has cost huge economic losses to word shrimp culture industry. The aim is to study the differences in the expression of the main pathogenic factor Pir toxin gene under the treatment of different environmental factors, and to explore the possible measures to inhibit the expression of Pir toxin gene. In this study, the expression level of Pir toxin gene in pathogenic bacteria was detected by the application of fluorescence quantitative RT-PCR technology and the comparative CT value method under different environmental factors, such as temperature and pH. The results showed that the bacteria revealed positive amplification of the Pir toxin gene which was related to AHPND in a virulence plasmid harboring this strain. Under the condition of 18℃, 24℃, 30℃ and 36℃, the expression of Pir toxin gene increased with the rise of temperature, and under the condition of pH 7, pH 8, and pH 9, the expression level of Pir toxin gene decreased with the reduce of pH. Especially, the expression of Pir gene was remarkably influenced by the pH factor. The research results provide basic data for exploring the relationship between the pathogenicity of Vibrio parahaemolyticus and environmental factors, and also a scientific basis for the development of AHPND control in aquaculture.

Key words: Vibrio parahaemolyticus, AHPND, Pir gene, gene expression

中图分类号:

S917.1

安伟, 肖雨, 高晓华, 彭军辉, 汤茜. 对虾急性肝胰腺坏死病病原毒素基因在不同环境下表达差异研究[J]. 中国农学通报, 2020, 36(36): 132-136.

An Wei, Xiao Yu, Gao Xiaohua, Peng Junhui, Tang Qian. Different Expression Levels of Virulence Genes of Bacterial Pathogen Associated with Acute Hepatopancreatic Necrosis Disease Under Different Environments[J]. Chinese Agricultural Science Bulletin, 2020, 36(36): 132-136.

图/表 5

名称		序列(5’-3’)	片段大小/bp
外侧引物	AP4-F1	ATGAGTAACAATATAAAACATGAAAC	1269
外侧引物	AP4-R1	ACGATTTCGACGTTCCCCAA	1269
内侧引物	AP4-F2	TTGAGAATACGGGACGTGGG	230
内侧引物	AP4-R2	GTTAGTCATGTGAGCACCTTC	230

表1. 毒素Pir基因引物序列

图1. Pir毒素电泳结果 1：第一轮PCR;2：第一轮阴性对照;3：第二轮PCR;4：第二轮阴性对照;M：DL2000

图2. 不同试验处理组细菌提取总RNA电泳结果 1：18℃试验组;2：24℃试验组;3：30℃试验组;4：36℃试验组;5：pH 7试验组;6：pH 8试验组;7：pH 9试验组

图3. 不同温度处理组Pir基因相对表达差异

图4. 不同pH处理组Pir基因相对表达差异

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