Duox 2调控克氏原螯虾肠组织抗细菌先天免疫的分子机制

doi:10.13560/j.cnki.biotech.bull.1985.2024-0540

摘要/Abstract

摘要：

【目的】为揭示双氧化酶2（DUOX 2）在克氏原螯虾（Procambarus clarkii）肠道中对活性氧（reactive oxygen species，ROS）水平的调节功能，以及对体内抗菌肽基因表达的影响。【方法】首先通过实时荧光定量聚合酶链式反应（real-time quantitative PCR, RT-qPCR）检测金黄色葡萄球菌（Staphylococcus aureus）感染后的血细胞、肝胰腺、肠、鳃组织中Duox 2基因的表达。RNA干扰后金黄色葡萄球菌刺激克氏原螯虾，统计其存活率，以及对血淋巴中细菌清除能力涂板观察，并且利用荧光探针2', 7'-二氯二氢荧光素二乙酸酯（2',7'-dichlorodihydrofluorescein diacetate，DCFH-DA）测定克氏原螯虾肠道中的ROS水平。最后通过RT-qPCR检测肠道中抗菌肽相关基因（Pc-toll 1、Pc-toll 3、Pc-ALF 1、Pc-lysozyme）的表达。【结果】在金黄色葡萄球菌刺激下，克氏原螯虾的Duox 2基因表达在血细胞、肝胰腺、肠、鳃中显著升高。并且RNA干扰技术抑制Duox 2基因的表达后，克氏原螯虾对金黄色葡萄球菌的抵抗力下降，存活率降低，血淋巴中细菌数量增加。此外，Duox 2基因表达降低导致肠道ROS表达的上升水平受到抑制，并且抗菌肽相关基因的表达水平受到抑制，证实了Duox 2在调控克氏原螯虾免疫防御中的重要作用。【结论】Duox 2通过调节克氏原螯虾肠道内的ROS水平，对机体抗菌肽相关基因的表达产生影响，从而调控体内金黄色葡萄球菌的清除能力。Duox 2对克氏原螯虾抵御金黄色葡萄球菌感染的先天免疫反应有重要作用。

关键词: 克氏原螯虾, 双氧化酶2, 活性氧, RNA干扰, 金黄色葡萄球菌

Abstract:

【Objective】To reveal the regulatory function of Dual Oxidase 2（DUOX2）in the intestine of the Procambarus clarkii on the levels of reactive oxygen species（ROS）, as well as its impact on the expression of antimicrobial peptide genes.【Method】Initially, the expressions of the Duox 2 gene in hemocytes, hepatopancreas, intestine, and gill tissues following Staphylococcus aureus infection were detected using quantitative real-time PCR（RT-qPCR）. After RNA interference and subsequent stimulation with S. aureus, the survival rate of the P. clarkii was statistically analyzed, along with the bacterial clearance capability in the hemolymph observed on culture plates. Furthermore, the ROS levels in the P. clarkii intestine were measured using the fluorescent probe 2', 7'-dichlorodihydrofluorescein diacetate（DCFH-DA）. Finally, the expression changes of antimicrobial peptide-related genes（Pc-toll 1, Pc-toll 3, Pc-ALF 1, and Pc-lysozyme）in the intestine were detected through RT-qPCR.【Result】Under the stimulation of S. aureus, the expression of the Duox 2 gene in P. clarkii significantly increased in hemocytes, hepatopancreas, intestine, and gills. Furthermore, after the expression of the Duox 2 gene was suppressed using RNA interference technology, the resistance of P. clarkii to S. aureus decreased, the survival rate dropped, and the number of bacteria in the hemolymph increased. Moreover, the reduction in Duox 2 gene expression led to the suppression of ROS levels in the intestine, and the expressions of antimicrobial peptide-related genes were also suppressed, further confirming the important role of Duox 2 in regulating the immune defense of P. clarkii.【Conclusion】Duox 2 gene regulates the expression of antimicrobial peptide-related genes by modulating the levels of ROS in the P. clarkii intestine, thereby controlling the body's ability to clear S. aureus. Therefore, Duox 2 plays an important role in the innate immune response of P. clarkii against S. aureus infection.

Key words: Procambarus clarkii, dual oxidase 2, reactive oxygen species（ROS）, RNA interference, Staphylococcus aureus

文静, 李倩倩, 张明达, 谭茗月, 金博阳, 沈秀丽, 杜志强. Duox 2调控克氏原螯虾肠组织抗细菌先天免疫的分子机制[J]. 生物技术通报, 2025, 41(1): 324-332.

WEN Jing, LI Qian-qian, ZHANG Ming-da, TAN Ming-yue, JIN Bo-yang, SHEN XIU-li, DU Zhi-qiang. Molecular Mechanism of Duox 2 Regulating Innate Immunity against Bacteria in Procambarus clarkii Intestine[J]. Biotechnology Bulletin, 2025, 41(1): 324-332.

图/表 6

表1 引物列表

Table 1 Primer list

引物Primer	引物序列Primer sequence（5'-3'）
Pc-Duox 2-iF	GCGTAATACGACTCACTATAGGGTGATGAAAAATGCGTTT
Pc-Duox 2-iR	GCGTAATACGACTCACTATAGGAAAGATGTCTCCCTGAAG
Pc-GFP-iF	GCGTAATACGACTCACTATAGGCGAGCTGGACGGCGACGTAAAC
Pc-GFP-iR	GCGTAATACGACTCACTATAGGCTTGAAGTTCACCTTGATGCC
Pc-Duox 2-RT-F	GCTGCCCAAGTTTCACTA
Pc-Duox 2-RT-R	AAAGGAGGCTGCACCAGC
18 S rRNA-RT-F	TCTTCTTAGAGGGATTAGCGG
18 S rRNA-RT-R	AAGGGGATTGAACGGGTTA
Pc-toll 1-RT-F	GACTTGTCCAAAAACGATATACG
Pc-toll 1-RT-R	TGCGTTACAGTAGTGAGCGAA
Pc-toll 3-RT-F	TCATTTGGCATCTGGCTCAC
Pc-toll 3-RT-R	GCAGGTGGTGGCGTTGA
Pc-ALF 1-RT-F	CGAGAGGCTGTAGAGGATGC
Pc-ALF 1-RT-R	CCCAGTTTGTTGATGATGAG
Pc-lysozyme-RT-F	GTCAACCCACCCTCAATAAC
Pc-lysozyme-RT-R	CTTGTGAATCAGGGCGTA

图1 金黄色葡萄球菌刺激后克氏原螯虾Duox 2基因的表达模式不同字母代表差异显著（P < 0.05），误差线表示3个重复的标准差（SD），下同

Fig. 1 Expression patterns of Duox 2 gene in P. clarkii stimulated by S. aureus Different letters indicate significant differences（P < 0.05）. The error bar indicates three repeated standard deviations（SD）, the same below

图2 RNAi并且注射金黄色葡萄球菌之后克氏原螯虾存活率的统计结果

Fig. 2 Statistical results of P. clarkii survival after RNAi and S. aureus injection ** P < 0.01，**** P < 0.0001

图3 RNAi并且注射金黄色葡萄球菌之后克氏原螯虾血淋巴细菌清除实验的检测结果

Fig. 3 Results of the bacterial clearance assay in P. clarkii haemolymph after RNAi and injection of S. aureus

图4 RNAi并且注射金黄色葡萄球菌之后克氏原螯虾肠组织活性氧的检测结果

Fig. 4 Detection results of reactive oxygen species in P. clarkii intestinal tissue after RNAi and S. aureus injection

图5 RNAi并且注射金黄色葡萄球菌之后克氏原螯虾肠组织先天免疫相关抗菌肽基因的相对表达量

Fig. 5 Relative expressions of innate immune-related antimicrobial peptide genes in P. clarkii intestinal tissues after RNAi and S. aureus injection

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