牙鲆rnd1基因克隆、表达模式及与抗病力的关系

Cloning and expression pattern of rnd1 and its involvement in disease resistance in Japanese flounder (Paralichthys olivaceus)

中文关键词: 牙鲆  rnd1  迟缓爱德华氏菌  抗病免疫  抑菌活性

英文关键词: Paralichthys olivaceus  rnd1  Edwardsiella tarda  Immunity  Antibacterial activity

中文摘要:

      迟缓爱德华氏菌病是海水鲆鲽鱼类的主要病害，发掘抗病分子标记辅助育种是十分有效的策略。本研究以牙鲆(Paralichthys olivaceus) rnd1基因(Pornd1)为对象，对该基因在牙鲆抗病免疫方面的作用进行系统分析。首先对Pornd1基因进行克隆鉴定和抗病相关单核苷酸多态性位点的定位，然后利用荧光定量PCR (qRT-PCR)对Pornd1基因的组织分布、细菌感染后表达情况以及在抗病和易感家系中的表达水平进行检测。结果显示，Pornd1 cDNA开放阅读框为699 bp，编码232个氨基酸。结合前期GWAS分析数据，本研究对Pornd1基因扩增和测序，确定Pornd1基因内含子2上存在一个抗迟缓爱德华氏菌病相关单核苷酸多态性位点，该位点在易感家系和抗病家系中分别是C/T，抗病家系(freqT=0.92)高于易感家系(freqT=0.20)，具有显著性差异(P<0.05)。Pornd1在心、肝和肾中的相对表达量较高；迟缓爱德华氏菌感染后肝、肾和脾中Pornd1的表达量在6 h降低后逐渐升高，在48 h达到最高。Pornd1在抗病家系肝脏中的表达量显著高于易感家系。在蛋白水平，利用大肠杆菌(Escherichia coli)表达系统表达PoRnd1重组蛋白，检测其抑菌活性，发现PoRnd1重组蛋白对金黄色葡萄球菌(Staphylococcus aureus)、大肠杆菌、迟缓爱德华氏菌(Edwardsiella tarda)和哈维氏弧菌(Vibrio harveyi)均具有一定抑菌活性。本研究揭示了Pornd1在牙鲆免疫抗病方面的作用，为开展牙鲆抗病分子育种提供了一个有效标记，并为解析其抗病性状的遗传机制提供理论基础。

英文摘要:

      Edwardsiella tarda is a major causative pathogen of bacterial ascites in Japanese flounder, leading to massive economic losses, and the discovery of molecular markers linked to disease resistance is an effective strategy in resistance breeding programs. The Rho GTPase family comprises small proteins with a molecular weight of 20~30 kDa. Rho GTPase family members are involved in diverse cellular processes, such as cytoskeleton, cell adhesion, vesicle transport, and proliferation. In addition, they play pivotal roles in infection by different pathogens. Rho-related GTP-binding protein Rho6 (Rnd1), a member of the Rho-GTPase family, participates in various biological functions, including neural junction formation, axonal extension, tumorigenesis, neuronal function, and apoptosis. Some members of the Rho family, such as Rac1 and Rac2, regulate immune response in grass carp, large yellow croaker, zebrafish, and half-smooth tongue sole. However, the function of Rnd1 in fish is poorly understood. Japanese flounder (Paralichthys olivaceus) is greatly affected by E. tarda infections during the breeding process. In previous studies, whole-genome sequencing and assembly of Japanese flounder were performed, and subsequently, various disease resistance genes were screened to support the improvement of Japanese flounder germplasm resources. To study the role of Pornd1 in resistance against E. tarda infection in Japanese flounder, Pornd1 was cloned and identified using PCR. The full-length Pornd1 cDNA was 699 bp, containing an open reading frame encoding a 232-amino acid protein. The predicted molecular weight of PoRnd1 was 26 kDa. Sequence and homology analyses showed that the Rnd1 protein harbors a Rho-GTP superfamily structural domain, which is highly conserved in various species. PoRnd1 shares the highest homology with Rnd1 from Hippoglossus hippoglossus (98.28%). On phylogenetic tree, PoRnd1 was clustered with Rnd1 from other fish species. The single-nucleotide polymorphism (SNP) locus associated with E. tarda resistance is located at 4 575 720 bp on chromosome 14 of Japanese flounder. The frequency of the T allele in disease-resistant families (freqT=0.92) was significantly higher than that in susceptible families (freqT=0.20). The SNP was located at the 2nd intron of Pornd1. Real-time quantitative PCR was employed to characterize the expression profiles of Pornd1 in the tissues of healthy and E. tarda-infected fish. Pornd1 expression was the highest in the heart, followed by the liver, kidney, head kidney, and spleen, but its expression was low in the skin, blood, gills, and muscle. In E. tarda-infected fish, the expression of Pornd1 mRNA decreased after 6 h, then gradually increased, and subsequently reached the highest level after 48 h in the liver, kidney, and spleen. Pornd1 expression in the kidney and spleen in the 48 h group was significantly higher than that in the 6 and 12 h groups. Furthermore, Pornd1 expression in the liver of resistant families was significantly higher than that in susceptible families. Based on its His tag, the PoRnd1 recombinant protein was purified using an Ni column and subjected to SDS-PAGE. The target band of PoRnd1 at 32 kDa was observed in the gel after Coomassie Blue staining. The PoRnd1 recombinant protein (0.5 mg/mL) was used to study antibacterial activity through the Oxford cup assay. PoRnd1 significantly inhibited the growth of Staphylococcus aureus, Escherichia coli, E. tarda, and Vibrio harveyi. In summary, Pornd1 may be closely linked to disease resistance in Japanese flounder and can thus serve as an effective gene marker for disease resistance breeding. Our findings provide a theoretical basis for further elucidating the molecular mechanisms of immunity in Japanese flounder.