干扰素调节因子2结合蛋白2在斑马鱼胚胎早期发育过程中的表达谱分析

doi:10.3969/j.issn.1674-8115.2021.03.005

摘要/Abstract

摘要：

目的·探究干扰素调节因子2结合蛋白2（interferon regulatory factor 2 binding protein 2，irf2bp2）基因的2个横向同源物（irf2bp2a和irf2bp2b）在斑马鱼胚胎早期发育过程中的表达异同。方法·选取发育不同时间点（胚胎期和幼虫期）的斑马鱼胚胎，通过mRNA全胚胎原位杂交技术明确irf2bp2a和irf2bp2b的时空表达谱，并通过实时定量PCR检测其表达水平。结果·irf2bp2a和irf2bp2b在神经系统、眼及胸鳍等部位都有着类似的高表达，而各自在肝脏或斯坦尼小体出现特异性表达，提示其在斑马鱼胚胎早期发育过程中具有独特的表达模式。实时定量PCR检测结果显示irf2bp2a和irf2bp2b在胚胎发育早期都有表达，但表达水平有所不同。结论·尽管斑马鱼irf2bp2a和irf2bp2b这2个横向同源物具有高度保守的功能结构域，但二者在胚胎早期发育过程中的表达谱不尽相同，提示其功能并不完全冗余。

关键词: 干扰素调节因子2结合蛋白2, 斑马鱼胚胎, 表达谱, mRNA全胚胎原位杂交, 实时定量PCR

Abstract:

Objective·To explore the expression similarities and differences of the two paralogs of interferon regulatory factor 2 binding protein 2 (irf2bp2) gene (irf2bp2a and irf2bp2b) during the early embryonic development of zebrafish.

Methods·Zebrafish embyos at different developmental stages (embryonic and larval stages) were chosen as research object. The mRNA whole mount in situ hybridization was used to detect the temporal and spatial expression patterns of irf2bp2a and irf2bp2b, and real-time quantitative PCR (qPCR) was used to detect their expression levels.

Results·Although irf2bp2a and irf2bp2b shared similar expression patterns in the nervous system, eye and pectoral fin, irf2bp2a and irf2bp2b were specifically expressed in liver and stannius corpuscles, respectively, suggesting their expressions were quite unique in certain organs. The results of qPCR showed that both irf2bp2a and irf2bp2b were expressed in the early embryonic development stage, but their expression levels were different.

Conclusion·Although the functional domains of irf2bp2a and irf2bp2b are highly conserved, the two paralogs of zebrafish irf2bp2 display some distinctions in the expression patterns during the early embryonic development of zebrafish, which suggests that their functions might not be redundant.

Key words: interferon regulatory factor 2 binding protein 2 (irf2bp2), zebrafish embyo, expression pattern, mRNA whole mount in situ hybridization, real-time quantitative PCR (qPCR)

中图分类号:

R321

王海红, 高硕, 朱军, 周隽. 干扰素调节因子2结合蛋白2在斑马鱼胚胎早期发育过程中的表达谱分析[J]. 上海交通大学学报(医学版), 2021, 41(3): 314-319.

Hai-hong WANG, Shuo GAO, Jun ZHU, Jun ZHOU. Expression patterns of interferon regulatory factor 2 binding protein 2 during the early embryonic development of zebrafish[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(3): 314-319.

图/表 5

表1 WISH探针引物序列

Tab 1 Primer sequences for WISH probe

Gene	Primer sequence (5' →3')
irf2bp2a	F：ACTGCTCCACATTGAGCTCCAGCCTG
	R：GGCTCCCAATCTGGGCGGACGCTC
irf2bp2b	F：CGCGGGCTGGAGCGCACACTGCC
	R：CGGGATCCCATTCGCCTGGCGTTG

表2 qPCR引物序列

Tab 2 Primer sequences for qPCR

Gene	Primer sequence (5'→3')
irf2bp2a	F：GTACGAGACTGGTCCTGCAT
	R：TGCTGTCCGTCTCCGTTAAT
irf2bp2b	F：GGTAAGCACTCTCGGGGAAT
	R：ACGGCATCTTGAGCACTTCT
β-actin	F：TGCTGTTTTCCCCTCCATTG
	R：TTCTGTCCCATGCCAACCA

图1 WISH检测斑马鱼irf2bp2a基因在胚胎中的表达Note: A. 1-cell stage. B. 6 hpf. C. 12 hpf. D. 24 hpf. E. 30 hpf. F. 36 hpf. G. 48 hpf. H. 72 hpf. I. 5 dpf. D1, E1, F1, G1 and H1 showed side view of the whole embryo; D2, E2, F2, G2 and H2 showed partial enlarged view of the head. C—cerebellum; E—eyes; Ep—epidermis; HB—hindbrain; J—jaw; L—lens; LL—lateral line; Lv—liver; M—muscle; MB—midbrain; MHB—midbrain-hindbrain boundary; MTc—midbrain tectum; MTg—midbrain tegmentum; OfV—olfactory vesicle; OV—otic vesicle; PBI—posterior blood island; PF—pectoral fin; PP—pharyngeal pouch; R—retina; r—rhombomere; r1—rhombomere 1; SB—swimming bladde; T—telencephalon; PA—pharyngeal arch.

Fig 1 Expression of irf2bp2a analyzed by WISH during zebrafish embryogenesis

图2 WISH检测斑马鱼irf2bp2b基因在胚胎中的表达Note: A. 1-cell stage. B. 6 hpf. C. 12 hpf. D. 24 hpf. E. 36 hpf. F. 48 hpf. G. 72 hpf. H. 5 dpf. D1, E1 and F1 showed side view of the whole embryo; D2, E2 and F2 showed partial enlarged view of the head; D3 showed top view of the back of the head; E3 showed view of transverse section of the tail indicated in E1; F3 showed ventral view. Ed—endoderm; H—heart; Pr—proctodeum; SC—stannius corpuscle.

Fig 2 Expression of irf2bp2b analyzed by WISH during zebrafish embryogenesis

图3 irf2bp2a和irf2bp2b基因表达水平定量分析Note: A/B. Transcription level of zebrafish irf2bp2a (A) and irf2bp2b (B) detected by qPCR. C. Statistical anaylsis of their relative expression levels. ①P=0.005, ②P=0.002, ③P=0.003, ④P=0.004, compared with irf2bp2b group.

Fig 3 Quantitative analysis of irf2bp2a and irf2bp2b gene expression

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