鲤环指蛋白145基因克隆、生物信息学及功能分析

doi:10.16431/j.cnki.1671-7236.2023.10.002

摘要/Abstract

摘要： 【目的】试验旨在克隆鲤(Cyprinus carpio)环指蛋白145(ring finger protein145,RNF145)基因CDS区,并对其进行生物信息学分析及功能初探。【方法】对鲤RNF145基因进行了克隆并测序,通过在线软件对鲤RNF145蛋白进行生物信息学分析;采用实时荧光定量PCR分析鲤RNF145基因在不同发育阶段、不同组织中及在鲤春病毒血症病毒(Spring Viremia of Carp virus,SVCV)和Poly(I∶C)刺激下的mRNA表达变化。【结果】鲤RNF145基因CDS序列全长2 049 bp,编码682个氨基酸,其氨基酸序列与鲫相似性最高;鲤RNF145蛋白分子质量为76.56 ku,理论等电点为6.16,脂肪系数为115.32 %,不稳定指数为41.87,无信号肽,含有11个跨膜螺旋,属跨膜蛋白;亚细胞定位预测结果显示,其定位于细胞质膜(87％)和内质网(13％);该蛋白含有TRC8-N、RING_H2_RNF145和RAD18结构域,具有α-螺旋(56.45％)、延伸链(9.68％)、β-转角(2.35％)及无规则卷曲(31.52％),与MFN2、NUGGC.1、ASZ1和TRIM36相互作用的置信度较高。早期发育阶段鲤RNF145基因mRNA表达呈先显著下降后上升,再显著下降趋势(P＜0.05);组织表达谱分析表明,其在脾脏中表达量最高,在肝脏中表达量最低;SVCV1(5.6×10⁷ TCID₅₀/mL)在体感染24 h后,RNF145基因在肌肉、脾脏、头肾、心脏中表达量均显著升高,在肝脏中则显著下降(P＜0.05);该剂量感染鲤上皮瘤细胞(Epithelioma papulosum crprini,EPC)时,RNF145基因表达量在12 h时开始显著升高,于48 h时仍显著高于对照组(P＜0.05);SVCV2(5.6×10⁴ TCID₅₀/mL)感染EPC时,鲤RNF145基因 mRNA表达量在感染后24 h时显著高于对照组,48 h后显著低于对照组(P＜0.05)。与SVCV1刺激结果不同,在体注射Poly(I∶C)(1 000 μg/mL) 24 h后,该基因在脾脏和头肾中表达量均显著低于对照组(P＜0.05);Poly(I∶C)(10 μg/mL)感染EPC时,鲤RNF145基因mRNA表达量在感染后24 h时显著高于对照组,48 h后显著低于对照组(P＜0.05)。【结论】本试验成功克隆了鲤RNF145基因的CDS全长序列,揭示了其在不同组织、早期不同发育阶段及SVCV和Poly(I∶C)刺激下的表达变化,为进一步研究该基因功能提供了前期基础。

关键词: 鲤; RNF145基因; 克隆; 生物信息学; 鲤春病毒血症病毒; Poly(I:C)

Abstract: 【Objective】 The purpose of this study was to clone the CDS region of ring finger protein 145 (RNF145) gene in Cyprinus carpio,and conduct bioinformatics analysis and functional investigation on it.【Method】 RNF145 gene in Cyprinus carpio was cloned and sequenced,and bioinformatics analysis of RNF145 protein in Cyprinus carpio was performed using online softwares.The mRNA expression changes of RNF145 gene of Cyprinus carpio in different development stages,different tissues and stimulated by Spring Viremia of Carp virus (SVCV) and Poly(I∶C) were analyzed by Real-time quantitative PCR.【Result】 The full-length CDS sequence of RNF145 gene in Cyprinus carpio was 2 049 bp,encoding 682 amino acids.Its amino acid sequence had the highest similarity to that of Carassius auratus.The molecular weight of RNF145 protein in Cyprinus carpio was 76.56 ku,the theoretical isoelectric point was 6.16,the fat index was 115.32%,the instability index was 41.87,there was no signal peptide,and it contained 11 transmembrane helices,belonging to transmembrane protein.The results of subcellular localization prediction showed that it was located in the plasma membrane (87%) and endoplasmic reticulum (13%).This protein contained TRC8-N domain,RING_H2_RNF145 domain and RAD18 domain,and had typical RING-finger family characteristics,with alpha helix (56.45%),extended chain (9.68%),beta turn (2.35%) and random coil (31.52%).The confidence level for interactions with MFN2,NUGGC.1,ASZ1,and TRIM36 were high.In the early developmental stage,the mRNA expression of RNF145 gene in Cyprinus carpio showed a significant decrease followed by an increase,followed by a significant decrease (P＜0.05).The analysis of tissue expression profile showed that its expression was the highest in spleen and the lowest in liver.After 24 h of in vivo infection with SVCV1 (5.6×10⁷ TCID₅₀/mL),the expression of RNF145 gene was significantly increased in muscle,spleen,head kidney and heart tissues,but significantly decreased in liver (P＜0.05).When infecting carp epithelial tumor cells (EPC) with this dose,the expression of RNF145 gene began to significantly increase at 12 h and remained significantly higher than the control group at 48 h (P＜0.05).When EPC was infected with SVCV2 (5.6×10⁴ TCID₅₀/mL),the expression of RNF145 gene mRNA in Cyprinus carpio was significantly higher than that in control group at 24 h after infection,and significantly lower than that in control group after 48 h (P＜0.05).Different from the SVCV1 stimulation results,after 24 h of in vivo injection of Poly(I∶ C) (1 000 μg/mL),the expression of RNF145 gene in spleen and head kidney were significantly lower than that in control group (P＜0.05).When EPC was infected with Poly(I∶C),the mRNA expression of RNF145 gene in Cyprinus carpio was significantly higher than that in control group at 24 h after infection,and significantly lower than that in control group after 48 h (P＜0.05).【Conclusion】 The full-length CDS sequence of RNF145 gene in Cyprinus carpio was successfully cloned in this experiment,revealing its expression changes in different tissues,early stages of development,and under SVCV and Poly(I∶C) stimulation,providing a preliminary basis for further studying the function of RNF145 gene in Cyprinus carpio.

Key words: Cyprinus carpio; RNF145 gene; clone; bioinformatics; SVCV; Poly(I:C)

中图分类号:

S917.4

郭朝辉, 张子豪, 李海洁, 张美娜, 江鑫, 于光晴, 李明, 刘变枝. 鲤环指蛋白145基因克隆、生物信息学及功能分析[J]. 中国畜牧兽医, 2023, 50(10): 3882-3895.

GUO Chaohui, ZHANG Zihao, LI Haijie, ZHANG Meina, JIANG Xin, YU Guangqing, LI Ming, LIU Bianzhi. Cloning,Bioinformation and Function Analysis of RNF145 Gene in Cyprinus carpio[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(10): 3882-3895.

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