克氏原螯虾prx6基因抗鲶爱德华氏菌侵染的分子机制研究  

Antimicrobial Function of Crayfish prx 6 on Edwardsiella ictaluri

作　　者：秦仕宇 金博阳 李倩倩 张明达 文静 沈秀丽[2] 杜志强 QIN Shiyu;JIN Boyang;LI Qianqian;ZHANG Mingda;WEN Jing;SHEN Xiuli;DU Zhiqiang(School of Life Science and Technology,Inner Mongolia University of Science and Technology,Baotou,Inner Mongolia Autonomous Region 014010,China;Library,Inner Mongolia University of Science and Technology,Baotou,Inner Mongolia Autonomous Region 014010,China)

机构地区：[1]内蒙古科技大学生命科学与技术学院,内蒙古包头014010 [2]内蒙古科技大学图书馆,内蒙古包头014010

出　　处：《福建农业学报》2024年第11期1228-1235,共8页Fujian Journal of Agricultural Sciences

基　　金：内蒙古自治区自然科学基金项目(2024MS03051);国家自然科学基金项目(32060834);内蒙古科技大学2022年基本科研业务费专项资金(028)。

摘　　要：【目的】以克氏原螯虾(Procambarus clarkii)为研究对象,探索过氧化物还原酶6(Peroxiredoxin 6,Prx6)基因(prx 6)在抗细菌先天免疫应答过程中的作用机制,为克氏原螯虾养殖过程病害问题的防治提供新思路。【方法】为了确定prx 6基因是否参与到克氏原螯虾抗细菌先天免疫过程,检测鲶爱德华氏菌(Edwardsiella ictaluri)免疫刺激后的克氏原螯虾血细胞、肝胰腺、鳃以及肠组织中prx 6的相对表达量。采用体外转录法制备Prx6双链RNA(double-strands RNA,dsRNA),使用无菌注射器注射至克氏原螯虾血窦处,通过qRT-PCR检测其干扰效果。随后,通过RNAi试验分析鲶爱德华氏菌免疫刺激后克氏原螯虾的存活率,并通过qRT-PCR检测克氏原螯虾内源抗菌肽(antimicrobial peptides,Amps)基因的变化情况。【结果】通过qRT-PCR试验,发现经鲶爱德华氏菌刺激后克氏原螯虾各组织中prx 6基因的表达量上调,表明其可能参与了克氏原螯虾抗鲶爱德华氏菌的先天免疫调节。为深入研究prx 6基因的免疫调节作用机制,体外制备了Prx 6双链RNA,并注射dsRNA后检测克氏原螯虾肝胰腺中prx 6的相对表达量,结果显示其表达水平被显著抑制。在RNAi试验中,注射病原菌之后,与dsGFP对照组相比,克氏原螯虾的存活率显著下降,证明prx 6在宿主抵抗病原菌感染过程中发挥了重要作用。进一步检测病原菌感染后克氏原螯虾抗菌肽基因的变化情况,结果显示,与dsGFP对照组相比较,克氏原螯虾内源的抗菌肽基因ALF4、ALF 1、crustin 1、crustin 2、lys和lectin 2表达量被明显抑制。推测prx 6基因可能通过调节抗菌肽基因的表达水平,影响了克氏原螯虾对病原菌的抵抗能力,进而导致存活率下降。【结论】prx 6在克氏原螯虾对鲶爱德华氏菌的先天免疫中起重要作用,可能通过调控抗菌肽的表达参与克氏原螯虾抵抗鲶爱德华氏菌的免疫应答过程。【Objective】Function of peroxiredoxin 6 gene(prx 6)in the antimicrobial innate immune response mechanism of Procambarus clarkii against Edwardsiella ictaluri was studied.【Methods】To confirm the involvement of prx 6 in the immunological response,gene expressions in the haemocytes,hepatopancreas,gills,and intestines of the P.clarkii were examined.Double-strand RNA(dsRNA)of the antimicrobial peptide(Amp)gene in P.clarkii,prx 6,was prepared by in vitro transcription in crayfish blood sinusoids using sterile syringes with the effect verified by qRT-PCR analysis.In a challenge test,survival rate of the E.ictaluri-inoculated crayfish was determined by a RNAi assay with the changes in prx 6 expression analyzed by qRT-PCR.【Results】The upregulated expressions of prx 6 in various crayfish tissues after the E.ictaluri stimulation indicated a positive association of the gene with the pathogenic invasion.Furthermore,an injection of the in vitro prepared dsRNA significantly suppressed the expression of prx 6 in the hepatopancreas confirming the response of the crayfish.The RNAi assay also showed a significant lower survival rate of the crayfish in the E.ictalurid-inoculation group than that in the dsGFP control indicating a significant effect of prx 6 in combating the bacterial infections.Additionally,in the challenge test some other endogenous Amp genes,such as ALF 4,ALF 1,crustin 1,crustin 2,lys,and lectin 2,were significantly downregulated in the E.ictaluri-inoculated crayfish in comparison with the dsGFP-inoculated counterparts.It was speculated that prx 6 contributed to the crayfish resistance to the infection by E.ictaluri through modulating Amp genes resulting in an improved survival.It was hypothesized that prx 6 participated in the innate immune response of crayfish against E.ictaluri through regulations on the endogenous Amp genes.

关 键 词：克氏原螯虾 RNA干扰 先天免疫 prx 6基因 抗菌肽基因 鲶爱德华氏菌 

分 类 号：S945.4[农业科学—水产养殖]