PolyI:C刺激猪PK15细胞后病毒感染应答基因未注释转录本鉴定及其特征分析

doi:10.16431/j.cnki.1671-7236.2024.04.030

摘要/Abstract

摘要： 【目的】鉴定猪PK15细胞在类病毒聚肌胞苷酸(PolyI:C)刺激后病毒感染应答基因的未注释转录本的数量、剪接类型、新蛋白编码与分子结构,为进一步研究这些未注释转录本的功能奠定基础。【方法】将猪PK15细胞分为对照组和试验组,每组3个重复;试验组加入终浓度为20 μg/mL的PolyI:C,对照组加入等体积(2 μL)的PBS,两组在37℃、5% CO₂条件下分别刺激6 h后进行Nanopore测序,鉴定两组的总转录本与差异表达基因。对差异表达基因进行GO功能分析,进一步筛选病毒感染的应答基因。对总转录本与Ensemble注释的转录本序列进行比较,发现未注释转录本。将病毒感染应答基因的未注释转录本与其对应的Ensemble注释转录本序列进行比对,分析未注释转录本的剪接类型和编码蛋白。【结果】PolyI:C刺激后,两组共鉴定蛋白编码的转录本61 505个,其中有Ensemble数据库注释的39 497个,未注释的转录本22 008个,未注释转录本数量占总数的35.78%。同时两组鉴定到71个差异蛋白编码基因,与对照组相比,试验组上调表达基因57个,下调表达基因14个。GO功能富集分析显示,这些差异表达基因富集到20个生物过程,其中前3个生物过程分别是防御病毒反应、Ⅰ型干扰素信号通路和病毒应答,均与病毒感染应答相关。24个病毒感染应答的基因有16个基因存在未注释转录本,其中CCL5、IFI6、BST2和MX1基因未注释转录本的数量多于其Ensemble注释的总转录本数量,且大部分未注释转录本产生新的蛋白序列。IFIT3、OAS2、RSAD2、CCL5、IFI44、CD40、IFI6、BST和MX1 9个基因的未注释转录本有差异表达。【结论】本研究系统地鉴定了猪PK15细胞受PolyI:C刺激后病毒感染应答基因的未注释转录本的分子特征,筛选的IFIT3、OAS2、RSAD2、CCL5、IFI44、CD40、IFI6、BST和MX1 9个基因的差异表达的未注释转录本可能具有重要生物学作用,为进一步解析宿主基因在抗病毒反应中的复杂转录调控机制提供了依据。

关键词: Nanopore测序; PolyI:C; 病毒感染; 转录本; 选择性剪接

Abstract: 【Objective】 The purpose of this study was to identify the number of unannotated transcripts,splicing types,new protein coding and molecular structure of genes response to viral infection in porcine PK15 cells after polyI:C stimulation,which would lay a foundation on the function of these unannotated transcripts for further study.【Method】 Porcine PK15 cells were divided into control group and experimental group,with 3 replicates in each group.The experimental group was added with PolyI:C at a final concentration of 20 μg/mL,and the control group was added with an equal volume (2 μL) of PBS.The two groups were stimulated at 37 ℃ and 5% CO₂ for 6 h,respectively.After then,Nanopore sequencing was performed to identify the total transcripts and differentially expressed genes of the two groups.GO function analysis was performed on the differentially expressed genes to further screen the genes response to viral infection.The total transcripts were compared with the Ensemble annotated transcript sequences,and unannotated transcripts were identified.The unannotated transcripts genes response to viral infection were compared with their corresponding Ensemble annotated transcripts,and the splicing types and encoded proteins of unannotated transcripts were analyzed.【Result】 After PolyI:C stimulation,a total of 61 505 protein-coding transcripts were identified in the two groups,of which 39 497 were annotated in the Ensemble database,and 22 008 were unannotated transcripts,accounting for 35.78% of the total.At the same time,71 differential protein-coding genes were identified in the two groups.Compared with control group,57 genes were up-regulated and 14 genes were down-regulated in experimental group.GO functional enrichment analysis showed that these differentially expressed genes were enriched into 20 biological processes,of which the first three most enriched biological processes were virus defense response,type Ⅰ interferon signaling pathway and response related to virus.Among the 24 genes response to viral infection,16 genes had unannotated transcripts.Among them,the number of unannotated transcripts of CCL5,IFI6,BST2 and MX1 genes was more than the total number of transcripts annotated by Ensemble,and most of the unannotated transcripts produced new protein sequences.Unannotated transcripts of 9 genes including IFIT3,OAS2,RSAD2,CCL5,IFI44,CD40,IFI6,BST and MX1 were differentially expressed.【Conclusion】 This study systematically identified the molecular characteristics of unannotated transcripts of genes response to viral infection in porcine PK15 cells stimulated by PolyI:C.The differentially expressed unannotated transcripts of 9 genes,including IFIT3,OAS2,RSAD2,CCL5,IFI44, CD40,IFI6,BST and MX1 might play an important biological role,which provided a basis for further analysis of the complex transcriptional regulation mechanism of host genes in antiviral response.

Key words: Nanopore sequencing; PolyI:C; viral infection; transcripts; alternative splicing

中图分类号:

S852.65

赵为民, 王红, 徐盼, 陈哲, 陶晓莉, 李碧侠, 付言峰, 程金花. PolyI:C刺激猪PK15细胞后病毒感染应答基因未注释转录本鉴定及其特征分析[J]. 中国畜牧兽医, 2024, 51(4): 1622-1631.

ZHAO Weimin, WANG Hong, XU Pan, CHEN Zhe, TAO Xiaoli, LI Bixia, FU Yanfeng, CHENG Jinhua. Identification and Characterization Analysis of Unannotated Transcripts of Genes Response to Viral Infection in Porcine PK15 Cells Stimulated by PolyI:C[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(4): 1622-1631.

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