长牡蛎新型模式识别受体CgLRRC69的克隆和功能鉴定

doi:10.11978/2020024

热带海洋学报 ›› 2021, Vol. 40 ›› Issue (1): 65-74.doi: 10.11978/2020024CSTR: 32234.14.2020024

长牡蛎新型模式识别受体CgLRRC69的克隆和功能鉴定

张翔宇^1,³(), 宋菁晨², 刘坤娜^1,³, 毛帆¹, 肖述¹, 向志明¹, 张扬¹(), 喻子牛¹()

1.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
2.华南农业大学海洋学院, 广东广州 510642
3.中国科学院大学, 北京 100049

收稿日期:2020-02-28 修回日期:2020-04-28 出版日期:2021-01-10 发布日期:2020-05-19
通讯作者: 张扬,喻子牛
作者简介:张翔宇(1995—), 女, 山东省烟台市人, 硕士, 从事贝类免疫研究。email: loginlv@163.com
基金资助:
国家自然科学基金项目(31572640);国家自然科学基金项目(31572661)

Cloning and functional analysis of a new pattern recognition receptor CgLRRC69 in Pacific oyster, Crassostrea gigas

ZHANG Xiangyu^1,³(), SONG Jingchen², LIU Kunna^1,³, MAO Fan¹, XIAO Shu¹, XIANG Zhiming¹, ZHANG Yang¹(), YU Ziniu¹()

1. Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Science, Guangzhou 510301, China
2. College of Oceanology, South China Agricultural University, Guangzhou 510642, China
3. University of Chinese Academy of Sciences, Beijing 100049, China

Received:2020-02-28 Revised:2020-04-28 Online:2021-01-10 Published:2020-05-19
Contact: ZHANG Yang,YU Ziniu
Supported by:
National Natural Science Foundation of China(31572640);National Natural Science Foundation of China(31572661)

摘要/Abstract

摘要：

在双壳类软体动物中, 血淋巴细胞介导的吞噬作用是清除入侵微生物的主要方式。本文在长牡蛎中鉴定了一个包含富含亮氨酸重复序列(Leucine-rich repeat, LRR)结构域的新型基因, 命名为CgLRRC69。对该基因的组织分布分析表明, CgLRRC69 mRNA在血淋巴细胞、鳃、肌肉、外套膜、心脏、消化腺和性腺中广泛表达。副溶血弧菌感染可以显著地刺激CgLRRC69在血淋巴细胞中表达, 并且在感染后6h达到峰值。同时, 酶联免疫吸附实验发现CgLRRC69可以特异性结合脂多糖(lipopolysaccharide, LPS), 表明它可能在免疫防御中有功能。吞噬实验结果显示, CgLRRC69重组蛋白可以显著地提高血淋巴细胞的吞噬能力; RNAi干扰CgLRRC69在牡蛎体内的表达, 显著降低了血淋巴细胞对细菌的清除能力。因此, 这些结果揭示了CgLRRC69作为一种新型模式识别受体, 可以特异性识别革兰氏阴性菌的主要成分LPS, 通过调理作用有效地清除细菌。

关键词: 长牡蛎, LRR富含亮氨酸重复序列, 模式识别, 吞噬作用, 免疫防御

Abstract:

In bivalve mollusks, circulating hemocytes phagocytosis is one basic way to eliminate invading pathogens. In this study, we identi?ed a novel Leucine-rich repeat (LRR) domain containing gene (i.e., CgLRRC69) in the Pacific oyster, Crassostrea gigas, which plays an important role in recognizing bacteria and promoting hemocyte phagocytosis. Tissue distribution analysis showed its constitutive expression in various tissues, including hemocytes, gills, muscles, mantle, heart, digestive glands, and gonads. Moreover, CgLRRC69 mRNA was signi?cantly increased and peaked at six hours of post-infection with V. parahaemolyticus in hemocytes. Furthermore, functional assay revealed that recombinant CgLRRC69 protein can specifically bind lipopolysaccharide (LPS), indicating its function in immune defense. Meanwhile, CgLRRC69 protein can also significantly promote phagocytosis of hemocytes. Finally, CgLRRC69 depletion via RNAi in vivo resulted in obvious reduction of bacterial clearance in hemocytes. Therefore, our study demonstrates that CgLRRC69 may function as a pattern recognition receptor for Vibrio spp. and promotes hemocyte phagocytosis. This study contributes to our better understanding of the mechanism of immune defense and disease management in the Paci?c oyster.

Key words: Crassostrea gigas, LRR-rich leucine repeats, pattern recognition, phagocytosis, immune defense

中图分类号:

P735.2

张翔宇, 宋菁晨, 刘坤娜, 毛帆, 肖述, 向志明, 张扬, 喻子牛. 长牡蛎新型模式识别受体CgLRRC69的克隆和功能鉴定[J]. 热带海洋学报, 2021, 40(1): 65-74.

ZHANG Xiangyu, SONG Jingchen, LIU Kunna, MAO Fan, XIAO Shu, XIANG Zhiming, ZHANG Yang, YU Ziniu. Cloning and functional analysis of a new pattern recognition receptor CgLRRC69 in Pacific oyster, Crassostrea gigas[J]. Journal of Tropical Oceanography, 2021, 40(1): 65-74.

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引物	序列
DsGFP-F	TAATACGACTCACTATAGGNGTCAGTGGAGAGGGTGAAGG	RNAi
DsGFP-R	TAATACGACTCACTATAGGNAGGCATGGCACTCTTGAAAA	RNAi
DsLRR-F	TAATACGACTCACTATAGGNTCCGCTTGAGTTTGGATTCC	RNAi
DsLRR-R	TAATACGACTCACTATAGGNTTCCTTCAGTGAGAGGACCT	RNAi
LRRi-F	AGAACTACCCGAAGTCCTTGA	qRT-PCR
LRRi-R	CCAAACTCAAGCGGAAGACT	qRT-PCR
GAPDH-F	CTGCCAACGTGTCAGTGGTG	qRT-PCR
GAPDH-R	TCAGTGTAGCCCAGGATGCC	qRT-PCR
LRR-F	AGCATTGATAGTTGAGATCA	ORF
LRR-R	TCGAAGAGCAAATCACATGA	ORF

长牡蛎新型模式识别受体CgLRRC69的克隆和功能鉴定

Cloning and functional analysis of a new pattern recognition receptor CgLRRC69 in Pacific oyster, Crassostrea gigas

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