长牡蛎(Crassostrea gigas)环GMP-AMP合酶(cyclic GMP-AMP synthase, cGAS)的基因克隆与功能研究

doi:10.11978/2024067

热带海洋学报 ›› 2025, Vol. 44 ›› Issue (1): 24-34.doi: 10.11978/2024067CSTR: 32234.14.2024067

长牡蛎(Crassostrea gigas)环GMP-AMP合酶(cyclic GMP-AMP synthase, cGAS)的基因克隆与功能研究

白晶¹^,²(), 毛帆², 刘客林², 宋菁晨², 喻子牛², 张扬²()

1.暨南大学, 广东广州 510632
2.中国科学院热带海洋生物资源与生态重点实验室, 中国科学院南海海洋研究所, 广东广州 510301

收稿日期:2024-03-22 修回日期:2024-04-10 出版日期:2025-01-10 发布日期:2025-02-10
通讯作者: 张扬
作者简介:
白晶(1998—), 女, 黑龙江省牡丹江市人, 硕士, 从事贝类免疫研究。email: baijing4729@163.com
基金资助:
国家重点研发项目(2022YFD2400301); 国家自然科学基金(32073002); 国家自然科学基金(U22A20533); 第九届青托工程项目(2023QNRC001); 广州市科技计划项目(2023A04J0096); 广州市科技计划项目(2024A04J6278)

Molecular cloning and functional study of cyclic GMP-AMP synthase from Crassostrea gigas

BAI Jing¹^,²(), MAO Fan², LIU Kelin², SONG Jingchen², YU Ziniu², ZHANG Yang²()

1. Jinan University, Guangzhou 510632, China
2. Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Science, Guangzhou 510301, China

Received:2024-03-22 Revised:2024-04-10 Online:2025-01-10 Published:2025-02-10
Contact: ZHANG Yang
Supported by:
National Key Research and Development Program of China(2022YFD2400301); National Natural Science Foundation of China(32073002); National Natural Science Foundation of China(U22A20533); 9th Young Elite Scientists Sponsorship Program(2023QNRC001); Science and Technology Planning Project of Guangdong Province, China(2023A04J0096); Science and Technology Planning Project of Guangdong Province, China(2024A04J6278)

摘要/Abstract

摘要：

环GMP-AMP合酶(cyclic GMP-AMP synthase, cGAS)是一种关键的细胞内传感器, 能够识别细胞质内异常存在的DNA并触发免疫反应。为了揭示cGAS在软体动物先天性免疫调控中的重要作用, 本研究成功克隆了长牡蛎中的cGAS基因(CgcGAS), 其开放阅读框(open reading frame, ORF)全长1623bp, 编码540个氨基酸, 理论相对分子质量为62.3kDa, 并具有保守的Mab21结构域。系统进化分析表明了CgcGAS为软体动物cGAS家族中的一员。定量逆转录聚合酶链式反应(qRT-PCR)结果显示CgcGAS广泛表达于各组织, 并在消化腺的相对表达量最高。亚细胞定位实验观察到CgcGAS蛋白在细胞核和细胞质中都有分布, 主要定位于细胞核, 提示其可能在细胞核内的DNA感应以及细胞质内的DNA结合和信号传递中发挥作用。另外, 双荧光素酶报告基因系统和RNA干扰实验结果显示, CgcGAS能够显著激活核因子κB(nuclear factor kappa-B, NF-κB)和干扰素刺激性反应元件(interferon-sensitive response element, ISRE)信号通路, 及其下游的炎症相关因子干扰素诱导病毒抑制蛋白(virus inhibitory protein endoplasmic reticulum-associated interferon-inducible, viperin)、肿瘤坏死因子(tumor necrosis factor, TNF)、白细胞介素-17 (interleukin-17, IL-17)及转录因子干扰素调节因子2/8 (interferon regulatory factor 2/8, IRF2/8)的表达。综上所述, CgcGAS在长牡蛎的先天性免疫反应中的信号传递过程中发挥了关键作用。

关键词: 长牡蛎, 先天性免疫, 环GMP-AMP合酶, 基因克隆, 功能

Abstract:

Cyclic GMP-AMP synthase (cGAS) is a critical intracellular sensor that can recognize abnormally located DNA in the cytoplasm and trigger immune responses. To elucidate the critical role of cGAS in the regulation of innate immunity in mollusks, we successfully cloned and analyzed Crassostrea gigas cGAS (CgcGAS). The open reading frame (ORF) of CgcGAS was 1623bp and encoded 540 amino acids with a theoretical molecular weight of 62.3 kDa and a conserved Mab21 domain. Phylogenetic analysis confirmed that CgcGAS was a member of the molluscan cGAS family. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) results revealed widespread expression of CgcGAS in various tissues, with the highest relative expression in the digestive glands. Subsequently, subcellular localization experiments showed that CgcGAS was observed in both the nucleus and cytoplasm, with a predominant nuclear localization, suggesting that CgcGAS may have played a role in DNA sensing in the nucleus and DNA binding and signaling in the cytoplasm. Furthermore, dual-luciferase reporter gene assays and RNA interference experiments revealed that CgcGAS could activate the NF-κB and ISRE signaling pathways, as well as the expression of downstream inflammation-related factors, such as virus inhibitory protein endoplasmic reticulum-associated interferon-inducible (viperin), tumor necrosis factor (TNF), interleukin-17 (IL-17), and the transcription factor interferon regulatory factor 2/8 (IRF2/8). In conclusion, CgcGAS played a critical role in the signal transduction process of innate immune responses in Crassostrea gigas.

Key words: Crassostrea gigas, innate immunity, cyclic GMP-AMP synthase, gene cloning, function

中图分类号:

P735

白晶, 毛帆, 刘客林, 宋菁晨, 喻子牛, 张扬. 长牡蛎(Crassostrea gigas)环GMP-AMP合酶(cyclic GMP-AMP synthase, cGAS)的基因克隆与功能研究[J]. 热带海洋学报, 2025, 44(1): 24-34.

BAI Jing, MAO Fan, LIU Kelin, SONG Jingchen, YU Ziniu, ZHANG Yang. Molecular cloning and functional study of cyclic GMP-AMP synthase from Crassostrea gigas[J]. Journal of Tropical Oceanography, 2025, 44(1): 24-34.

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引物名称	序列(5′-3′)	用途
cGAS-F1	ATGGTAATCAAATGTCCTAATTGTG	ORF克隆
cGAS-R1	TTATTGTAAGAGACCCTCTAGTTCC	ORF克隆
pEGFP-N1-cGAS-F	TCAGATCTCGAGCTCAAGCTTGCCACCATGGTAATCAAATGTCC	pEGFP-N1-cGAS重组质粒
pEGFP-N1-cGAS-R	ATGGTGGCGACCGGTGGATCCGATTGTAAGAGACCCTCTAGTTCCCT	pEGFP-N1-cGAS重组质粒
PCDNA3.1/V5-His-cGAS-F	GCACAGTGGCGGCCGCTCGAGATGGTAATCAAATGTCCTAATTGTGAC	pcDNA3.1/V5-His-cGAS重组质粒
PCDNA3.1/V5-His-cGAS-R	AGGCTTACCTTCGAAGGGCCCTTGTAAGAGACCCTCTAGTTCCCTG	pcDNA3.1/V5-His-cGAS重组质粒
dscGAS-F	CGACAAAACAAAGATCGACTACAAC	cGAS-RNAi
dscGAS-R	CAGTCTGGATTTCTCTTGCATCCTT	cGAS-RNAi
dscGAS-F-T7	TAATACGACTCACTATAGGCGACAAAACAAAGATCGACTACAAC	cGAS-RNAi
dscGAS-R-T7	TAATACGACTCACTATAGGCAGTCTGGATTTCTCTTGCATCCTT	cGAS-RNAi
dsGFP-F	GCAAGGGCGAGGAGCTGTTCACCGG	GFP-RNAi
dsGFP-R	TTGCCGTCCTCCTTGAAGTCGATGC	GFP-RNAi
dsGFP-F-T7	TAATACGACTCACTATAGGGCAAGGGCGAGGAGCTGTTCACCGG	GFP-RNAi
dsGFP-R-T7	TAATACGACTCACTATAGGTTGCCGTCCTCCTTGAAGTCGATGC	GFP-RNAi
cGAS-F2	GGAAAGACGACAGGGACGG	qRT-PCR
cGAS-R2	TGTCTGGAGAACCCCTTTGG	qRT-PCR
viperin-F	CTGAAACCCATCAGTGTCAACTACC	qRT-PCR
viperin-R	GACAATGAAGGGCTCGCCAC	qRT-PCR
IRF2-F	ACTTCCGCTGTGCCCTGAAT	qRT-PCR
IRF2-R	TATGACCTTTGGCACTGTCGTTC	qRT-PCR
IL-17-F	AAACATGCTGGAATACCTCGAGT	qRT-PCR
IL-17-R	GTGGGACGCTACGAGGAAATA	qRT-PCR
TNF-F	GTATTACTGAGGAATGGCGTGAAAC	qRT-PCR
TNF-R	CAAAATCCCTGTCACTACAACCG	qRT-PCR
IRF8-F	GGACAGCGGTCAGACACGAC	qRT-PCR
IRF8-R	CCTTGAATATTGTGGAGTCTGCCT	qRT-PCR
β-actin-F	GGATTGGCGTGGTGGTAGAG	qRT-PCR
β-actin-R	GTATGATGCCCCTTTGTTGAGTC	qRT-PCR

长牡蛎(Crassostrea gigas)环GMP-AMP合酶(cyclic GMP-AMP synthase, cGAS)的基因克隆与功能研究

Molecular cloning and functional study of cyclic GMP-AMP synthase from Crassostrea gigas

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组别	pNF-κB-luc/ISRE-luc/ng	目的质粒/ng	对照空质粒/ng	内参质粒RT-TK/ng
对照组	200	0	400	20
实验组1	200	100	300	20
实验组2	200	200	200	20
实验组3	200	400	0	20

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