小龙虾prx 6基因在对抗金黄色葡萄球菌感染中的分子作用机制研究

doi:10.13560/j.cnki.biotech.bull.1985.2023-1205

摘要/Abstract

摘要：

【目的】 旨在研究prx 6基因在小龙虾先天免疫中的调控作用机制，探索小龙虾prx 6基因在金黄色葡萄球菌（Staphylococcus aureus）感染后发挥免疫调控作用的可能分子机制。【方法】 选取prx 6基因作为研究对象，提取正常小龙虾各组织的总RNA，利用RT-qPCR分析目的基因在小龙虾各组织中的分布，并检测S. aureus免疫刺激后，prx 6基因在小龙虾相关组织中的表达模式。通过RNA干扰技术（RNAi）敲低prx 6基因表达量，在感染S. aureus后，测定小龙虾肝胰腺中Pc-crustin 3、Pc-crustin 4、Pc-ALF 9和Pc-lectin 1免疫效应基因的相对表达量。同时，进一步对小龙虾血淋巴中的细菌载量进行统计分析。【结果】 通过对prx 6基因在小龙虾各组织的表达水平分析，观察到该基因在肝胰腺的表达量显著高于其他组织。在S. aureus感染后，小龙虾肝胰腺、血细胞和肠组织中prx 6基因的表达量显著上调，在鳃组织仅有早期表达量有所增加。小龙虾存活率的检测结果表明RNAi下调prx 6基因表达量之后，其生存率显著低于dsGFP+S. aureus对照组。为了进一步探索死亡率升高的原因，研究了抗菌肽基因在抗细菌防御中的表达水平。RNAi实验结果显示，在prx 6基因表达量降低的情况下，小龙虾肝胰腺Pc-crustin 3、Pc-crustin 4、Pc-ALF 9以及Pc-lectin 1基因表达水平都出现了显著下降。此外，小龙虾血淋巴中的细菌载量检测结果显示，prx 6基因RNAi组的细菌载量显著高于dsGFP+S. aureus对照组。【结论】 小龙虾prx 6基因通过影响抗菌肽基因的表达，以及血淋巴细菌清除能力来参与抗细菌先天免疫应答。

关键词: 先天免疫, 过氧化物还原酶6, RNA干扰, 抗菌肽, 金黄色葡萄球菌

Abstract:

【Objective】 To unravel the molecular intricacies associated with the immune modulatory role of the crayfish prx 6 gene in response to Staphylococcus aureus infection and to expound its function in the orchestration of innate immunity in crayfish, this investigation was carried out. 【Method】 The study pivoted on the prx 6 gene and harvested total RNA from diverse unstimulated crayfish tissues. The RT-qPCR was utilized to pinpoint the tissue-specific distribution patterns of this gene. Following immune provocation by S. aureus, the prx 6 gene expression dynamics within crayfish’s immune-centric tissues were monitored. The research further endeavored to gauge the relative expression of immune executor genes, specifically, Pc-crustin 3, Pc-crustin 4, Pc-ALF 9, and Pc-lectin 1 in the hepatopancreas after RNAi-mediated silencing of prx 6, in the wake of S. aureus infection. Additionally, a quantitative appraisal of the bacterial burden in crayfish hemolymph was undertaken. 【Result】 The investigations underscored an amplified expression of the prx 6 gene within the hepatopancreas as compared to other tissues. Post S. aureus infection, the prx 6 gene expression showcased substantial augmentation in the hepatopancreas, hemocytes, and intestinal tissues, with a preliminary surge of expression detected in gill tissues. In terms of survival rates, a pronounced decrement in crayfish vitality was observed after prx 6 knockdown, relative to dsGFP + S. aureus controls. To elucidate the key factors linked to escalated mortality, the expression strata of antimicrobial peptide genes integral to bacterial defense were quantified. The outcomes revealed a significant downturn in the expression of Pc-crustin 3, Pc-crustin 4, Pc-ALF 9, and Pc-lectin 1 within the hepatopancreas, seemingly tied to prx 6 repression. Intriguingly, assessment of hemolymph bacterial loads unveiled a substantial elevation in bacterial load in the prx 6 knockdown group as set against controls. 【Conclusion】 The prx 6 gene in crayfish plays a pivotal role in the antibacterial innate immune response through modulating the expressions of antimicrobial peptide genes and enhancing the clearance capabilities of bacteria from the hemolymph.

Key words: innate immunity, PRX 6, RNA interference, antimicrobial peptides, Staphylococcus aureus

金博阳, 秦仕宇, 张明达, 李倩倩, 文静, 沈秀丽, 杜志强. 小龙虾prx 6基因在对抗金黄色葡萄球菌感染中的分子作用机制研究[J]. 生物技术通报, 2024, 40(7): 314-322.

JIN Bo-yang, QIN Shi-yu, ZHANG Ming-da, LI Qian-qian, WEN Jing, SHEN Xiu-li, DU Zhi-qiang. Research on the Molecular Mechanism of Crayfish prx 6 in the Process of Defending against Staphylococcus aureus Infection[J]. Biotechnology Bulletin, 2024, 40(7): 314-322.

图/表 8

表1 RT-qPCR引物序列

Table 1 RT-qPCR primer sequences

引物名称 Primer name	引物序列 Primer sequence（5'-3'）
prx 6-RT-F	CGGATCACTGGAGGGTCA AACA
prx 6-RT-R	GCA ATTTTCATCCTCGGCATCA
prx 6-iF	GCGTAATACGACTCACTATAGGATGGTTAACTTAGGCGAT
prx 6-iR	GCGTAATACGACTCACTATAGGGTCTATGGCTCTAAGAAT
GFP-iF	GCGTAATACGACTCACTATAGGCGAGCTGGACGGCGACGTAAAC
GFP-iR	GCGTAATACGACTCACTATAGGCTTGAAGTTCACCTTGATGCC
18S RNA-RT-F	TCTTCTTAGAGGGATTAGCGG
18S RNA-RT-R	AAGGGGATTGAACGGGTTA
Pc-ALF 9-RT-F	AGTGGCGTCATACAGGAAGGGG
Pc-ALF 9-RT-R	CCAAAGGATGGCGAGAAATAGT
Pc-crustin 3-RT-F	TACGTCTTGCCCTCGTCTTA
Pc-crustin 3-RT-R	CAGCGTCCTCCTCTTTGTAATC
Pc-crustin 4-RT-F	CTCTGACTGCCAGGTCTTT
Pc-crustin 4-RT-R	TGCGAGCTGTGATGGTTAG
Pc-lectin 1-RT-F	GGGGAGGGCTGCTACTACTTG
Pc-lectin 1-RT-R	CGTGCCACCCACCCATAAG

图1 prx 6基因的正常组织表达谱

Fig. 1 Expression patterns of prx 6 gene in normal tissues

图2 金黄色葡萄球菌刺激后prx 6的表达模式金黄色葡萄球菌刺激后prx 6在4种组织中的相对表达模式；不同小写字母表示差异显著（P<0.05）；相同字母表示差异不显著（P>0.05），下同

Fig. 2 Expression patterns of prx 6 after S. aureus stimulation Relative expression of prx 6 in tissues after S. aureus infection. Different lowercase letters showed significant differences（P<0.05）. The same letters indicate insignificant differences（P>0.05）. The same below

图3 RNAi并且注射金黄色葡萄球菌之后小龙虾存活率统计 ****表示组间差异极显著（P<0.000 1）

Fig. 3 Survival rates of P. clarkii statistics after S. aureus injection in RNAi assay **** indicates highly significant different with control group（P<0.000 1）

图4 RNAi试验中S. aureus刺激后相关抗菌肽基因在肝胰腺中的相对表达量分析结果 18S RNA作为内参对照

Fig. 4 Results for relative expression analysis of Amps genes in crayfish hepatopancreas after S. aureus challenge in RNAi assay 18S RNA is used as inner control

图5 RNAi试验中的小龙虾血淋巴细菌清除结果分别将1×PBS组、dsPrx 6+S. aureus组和dsGFP+S. aureus组小龙虾的血淋巴涂板在琼脂平板上。检测了12 h和24 h的细菌丰度

Fig. 5 Bacterial clearance experiments results in crayfish hemolymph in RNAi assay The hemolymph of crayfish from 1×PBS, dsPrx 6 + S. aureus and dsGFP + S. aureus groups was coated on the agar plate. Bacterial abundance at 12 and 24 h was detected

图6 细菌菌落数统计结果对LB平板中的单个菌落进行计数，使用公式进行计算

Fig. 6 Statistics results of bacterial colonies Individual colonies in the LB plates were counted and cakulated using the formula

图7 prx 6通过影响抗菌肽基因的表达来抵抗金黄色葡萄球菌侵染的理论推测小龙虾受到金黄色葡萄球菌刺激后，通过激活体内prx 6，进一步影响抗菌肽基因的转录与表达，分泌出抗菌肽抵抗外来病原菌

Fig. 7 Theoretical speculation for prx 6 affecting the expression of Amps genes to defend against S. aureus infection After S. aureus infection, crayfish can further modulate the transcription and expression of genes related to antimicrobial peptides by activating prx 6, thereby facilitating the secretion of antimicrobial peptides against foreign pathogens

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