| 刘依铭,许冬梅,刘思影,刘玉芬,刘鹏,赵文阁,陈辉.2023.黑龙江林蛙ferritin H基因克隆及其在细菌侵染下的表达变化分析.动物学杂志,58(6):881-890. |
| 黑龙江林蛙ferritin H基因克隆及其在细菌侵染下的表达变化分析 |
| Cloning of the ferritin H Gene from Rana amurensis and Analysis of Its Expression Changes Exposed to Bacterial Infection |
| 投稿时间：2022-11-29 |
| DOI：10.13859/j.cjz.202322299 |
| 中文关键词: 铁蛋白基因 黑龙江林蛙 嗜水气单胞菌 基因克隆 细菌感染 |
| 英文关键词:Ferritin gene Rana amurensis Aeromonas hydrophila Cloning Bacterial infection |
| 基金项目:黑龙江省自然科学基金联合引导项目(No. LH2021C053)； |
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| 中文摘要: |
| 铁蛋白广泛存在于生物体内，能够使细胞内的铁元素含量保持相对稳定且能参与机体免疫反应。近年来因细菌侵染，黑龙江林蛙(Rana amurensis)种群数量出现下降趋势，本研究探究铁蛋白基因在细菌感染的林蛙中的表达模式，期望可以为黑龙江林蛙抗细菌感染的机制研究提供参考。本研究首先采用PCR技术扩增黑龙江林蛙铁蛋白H亚基基因的编码区序列并对其进行生物信息学分析；再应用实时荧光定量PCR技术(RT-qPCR)检测黑龙江林蛙被嗜水气单胞菌(Aeromonas hydrophila)侵染后，铁蛋白H亚基基因在其肝、脾、肾、皮肤以及肌肉组织中的转录水平变化情况；最后采用免疫荧光检测技术，分析嗜水气单胞菌侵染后，黑龙江林蛙铁蛋白H亚基蛋白表达情况。结果表明，该基因编码区长度534 bp，编码177个氨基酸；对该基因氨基酸序列进行分析，其与欧洲林蛙(R. temporaria)相应基因同源性最高，达到98.37%；RT-qPCR结果显示，铁蛋白H亚基基因在黑龙江林蛙组织中广泛存在，在嗜水气单胞菌侵染后，铁蛋白H亚基mRNA在黑龙江林蛙肝、脾、肾、皮肤以及肌肉组织中的表达均显著上调(P < 0.01)。免疫荧光检测结果发现，在感染嗜水气单胞菌后，铁蛋白H亚基蛋白在黑龙江林蛙肝和肌肉组织的胞质中均有不同程度表达。综上结果表明，黑龙江林蛙铁蛋白H亚基基因会通过上调表达来响应细菌性感染，由此推测该基因参与了黑龙江林蛙的细菌性免疫应答。 |
| 英文摘要: |
| [Objectives] Ferritin exists widely in organisms, which can maintain a relatively stable iron content in cells and also participate in body’s immune response. In recent years, due to bacterial infection, the population of Rana amurensis has shown a downward trend. This study explores the expression pattern of ferritin H genes in bacteria-infected R. amurensis, hoping to provide a reference for the study of the mechanism of R. amurensis resistance to bacterial infection. [Methods] In this study, PCR was used to amplify the coding region of ferritin H(ferH) gene of R. amurensis and analyze its bioinformatics. Quantitative Real-time PCR (RT-qPCR) was used to detect the transcriptional changes of ferH gene in the liver, spleen, kidney, skin, and muscle tissue of R. amurensis after Aeromonas hydrophila (Ah) infection. The transcription level of ferHgene relative to the reference gene was calculated by 2﹣ΔΔCT using EXCEL 2019. Finally, immunofluorescence detection technology was used to analyze the expression of ferH protein of R. amurensis after Ah infection. Three positive regions were selected for each sample to take photos, and Image J software was used to analyze the relative average optical density. The above results were expressed as Mean ± SD. SPSS 26.0 was used for statistical analysis. One-way ANOVA was used to compare the difference between the treatment group and the control group, and the statistical significance was P < 0.05. GraphPad Prism 8 software was used for mapping. [Results] The results showed that the encoding region of ferHgene was 534 bp, encoding 177 amino acids (Fig. 1). The amino acid sequence analysis of this gene showed that it had the highest homology with R. temporaria, reaching 98.37% (Fig. 2). The results of RT-qPCR showed that ferHgene was widely transcribed in R. amurensis tissues (Fig. 3), and the transcription level of ferHgene was significantly up-regulated in liver, spleen, kidney, skin, and muscle tissues after Ah infection (P < 0.01). ferHgene in liver, skin and muscle tissues reached the peak of transcription 6 h after infection, which was 11.95, 24.31, and 24.72 times higher than that in the control group (P < 0.01). The peak transcription in spleen and kidney tissues was 18.22 and 18.19 times higher than that in the control group at 24 h after infection (P < 0.01) (Fig. 4). In addition, immunofluorescence assay results showed that the protein was expressed in the cytoplasm of the liver and muscle tissue of R. amurensis to varying degrees after infection with Ah. ferH protein expression was highest in liver and muscle tissue after Ah 6 h infection, 11.63 and 4.82 times higher than in control group, respectively (P < 0.01) (Fig. 5). [Conclusion] In conclusion, the ferHgene of R. amurensis is up-regulated in response to bacterial infection, suggesting that the gene is involved in bacterial immune response. |
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