香港牡蛎(Crassostrea hongkongensis)新型凝集素ChPerlucin的基因克隆与功能研究

doi:10.11978/2021029

热带海洋学报 ›› 2022, Vol. 41 ›› Issue (1): 42-51.doi: 10.11978/2021029CSTR: 32234.14.2021029

香港牡蛎(Crassostrea hongkongensis)新型凝集素ChPerlucin的基因克隆与功能研究

赵泽慧^1,²(), 张爱娇^1,², 杨雨澄^1,², 毛帆¹, 肖述¹, 李军¹, 张扬¹, 向志明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. 中国科学院大学, 北京100049

收稿日期:2021-03-04 修回日期:2021-04-19 出版日期:2022-01-10 发布日期:2021-04-25
通讯作者: 向志明,喻子牛,姚衍桃
作者简介:赵泽慧(1993—), 女, 河北人, 硕士研究生, 主要从事贝类免疫与抗逆的研究。email: zhaozehui18@mails.ucas.ac.cn
基金资助:
广东省自然科学基金项目(2021A1515010541)

Molecular cloning and functional studies of ChPerlucin in Crassostrea hongkongensis

ZHAO Zehui^1,²(), ZHANG Aijiao^1,², YANG Yucheng^1,², MAO Fan¹, XIAO Shu¹, LI Jun¹, ZHANG Yang¹, XIANG Zhiming¹(), YU Ziniu¹()

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

Received:2021-03-04 Revised:2021-04-19 Online:2022-01-10 Published:2021-04-25
Contact: XIANG Zhiming,YU Ziniu,YAO Yantao
Supported by:
Natural Science Foundation of Guangdong Province, China(2021A1515010541)

摘要/Abstract

摘要：

C-型凝集素(C-type lectin)是一类能识别并结合多糖的免疫蛋白质超家族, 本文首次克隆了一种新型香港牡蛎C-型凝集素基因——甘露聚糖结合凝集素, 并获得了该基因的全长序列, 命名为ChPerlucin。结果显示, ChPerlucin基因cDNA全长577bp, 包括21bp的5′末端非翻译区(untranslated region, UTR)、73bp的 3′UTR, 以及483bp的开放阅读框(open reading frame, ORF), 编码160个氨基酸, ChPerlucin蛋白理论分子量为18kD, 等电点为5.95。ChPerlucin蛋白含有保守的C-型凝集素样结构域(C-type lectin or carbohydrate-recognition domain, CLECT); 邻接法(Neighbor-Joining, NJ)进化树分析表明, ChPerlucin与其他贝类的Perlucin聚为一支, 说明该基因是软体动物Perlucin家族的新成员。qRT-PCR结果显示, ChPerlucin在香港牡蛎所检测组织和各个胚胎发育时期中均有表达; 病原菌刺激后, ChPerlucin表达量显著升高; 利用毕赤酵母真核成功表达了ChPerlucin重组蛋白, 发现ChPerlucin重组蛋白具有抑菌效果。以上结果表明ChPerlucin在香港牡蛎的先天性免疫过程中发挥了重要的作用。

关键词: 香港牡蛎, 先天性免疫, C-型凝集素, ChPerlucin

Abstract:

The C-type lectin superfamily is a class of immune proteins that can recognize and bind to polysaccharides. In this study, a new type C-type lectin gene of Crassostrea hongkongensis was cloned for the first time, and the full-length sequence of the gene was obtained. The results show that the full length of ChPerlucin gene cDNA is 577 bp, including 5′-untranslated region (UTR) in 21 bp, 3′-UTR in 73 bp, and an open reading frame (ORF) in 483 bp open reading that code 160 amino-acid polypeptide. The predicted molecular mass is 18kD, and the isoelectric point is 5.95. Amino acid sequence alignment showed that the Perlucin gene of Crassostrea hongkongensis contains a conserved carbohydrate recognition domain (CLECT). Neighbor-Joining (NJ) evolutionary tree analysis showed that ChPerlucin clustered with Perlucin from other shellfish, indicating that this gene is a new member of the Perlucin family of mollusks. The results of qRT-PCR showed that ChPerlucin was broadly expressed in various tissues and during different stages of the oyster’s embryonic and larval development; the expression of ChPerlucin increased significantly after bacteria stimulation; the recombinant protein ChPerlucin was successfully expressed using Pichia pastoris eukaryotic; and we find that ChPerlucin recombinant protein has antibacterial effect. The above results indicate that ChPerlucin plays an important role in the innate immunity of Crassostrea hongkongensis.

Key words: Crassostrea hongkongensis, innate immunity, C-type lectin, ChPerlucin

中图分类号:

S944.4

赵泽慧, 张爱娇, 杨雨澄, 毛帆, 肖述, 李军, 张扬, 向志明, 喻子牛. 香港牡蛎(Crassostrea hongkongensis)新型凝集素ChPerlucin的基因克隆与功能研究[J]. 热带海洋学报, 2022, 41(1): 42-51.

ZHAO Zehui, ZHANG Aijiao, YANG Yucheng, MAO Fan, XIAO Shu, LI Jun, ZHANG Yang, XIANG Zhiming, YU Ziniu. Molecular cloning and functional studies of ChPerlucin in Crassostrea hongkongensis[J]. Journal of Tropical Oceanography, 2022, 41(1): 42-51.

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引物名称	序列(5'-3')	用途
ChPerlucin F₁	GCTACTGCCAGGCTTACCATTCCAA	ChPerlucin 3′RACE引物
ChPerlucin F₂	ATAGGAATAACGGACCTTACAGCAG	ChPerlucin 3′RACE引物
ChPerlucin R₁	GTTGGATGAATACACCCACTCGTTT	ChPerlucin 5′RACE引物
ChPerlucin R₂	TAAGGTCCGTTATTCCTATCCAAAA	ChPerlucin 5′RACE引物
ChPerlucin F₃	GGAAAGAATGTAAGGTAAACAATGG	ChPerlucin ORF引物
ChPerlucin R₃	TGACGCTTGGTTAATCAATTCTTTT	ChPerlucin ORF引物
ChPerlucin F₄	CTGCCAGGCTTACCATTCCAA	ChPerlucin实时荧光定量PCR
ChPerlucin R₄	GGCTTGCCCAGTTTGTCGTCT	ChPerlucin实时荧光定量PCR
ChPerlucin F₅	AAGGATCCACCATGGGTTCCGGCTGCCGCTCGGGATGGACTCAGTACAAA	pPIC3.5K-ChPerlucin载体构建
ChPerlucin R₅	TTGAATTCTCAATGATGATGATGATGATGTCCAATGATCACCGGGCCACT	pPIC3.5K-ChPerlucin载体构建
GAPDH-F	GGATTGGCGTGGTGGTAGAG	GAPDH实时荧光定量PCR
GAPDH-R	GTATGATGCCCCTTTGTTGAGTC	GAPDH实时荧光定量PCR

香港牡蛎(Crassostrea hongkongensis)新型凝集素ChPerlucin的基因克隆与功能研究

Molecular cloning and functional studies of ChPerlucin in Crassostrea hongkongensis

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