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

Abstract

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)

CLC Number:

S917.4

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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Cloning,Bioinformation and Function Analysis of RNF145 Gene in Cyprinus carpio

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