七彩神仙鱼C型凝集素家族成员CD302和MBL的克隆及在病原刺激下的免疫应答

马腾飞; 温彬; 刘鑫; 缪琳; 高建忠; 陈再忠

doi:10.12131/20220018

七彩神仙鱼C型凝集素家族成员CD302和MBL的克隆及在病原刺激下的免疫应答

doi: 10.12131/20220018

马腾飞^1,,
温彬^{1, 2},
刘鑫¹,
缪琳¹,
高建忠^{1, 2},
陈再忠^{1, 2, ,}

1.
上海海洋大学/水产种质资源发掘与利用教育部重点实验室/农业农村部淡水水产种质资源重点实验室，上海 201306
2.
上海海洋大学/水产科学国家级实验教学示范中心，上海 201306

基金项目: 上海市自然科学基金项目 (20ZR1423600)；上海扬帆人才计划项目 (19YF1419400)；上海海洋大学科技发展专项基金 (A2-2006-22-200202)

详细信息

作者简介:
马腾飞 (1998—)，男，硕士研究生，研究方向为观赏鱼养殖。E-mail: tfmalh@163.com

通讯作者:
陈再忠 (1972—)，男，教授，博士，研究方向为观赏鱼养殖。E-mail: chenzz@shou.edu.cn

中图分类号: S 917.4
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- 被引次数: 0
出版历程
- 收稿日期: 2022-01-17
- 修回日期: 2022-03-31
- 录用日期: 2022-05-07
- 网络出版日期: 2022-05-13
- 刊出日期: 2022-12-05

Molecular cloning and characterization in immune response of two C-type lectin genes (SaCD302 andSaMBL) from Symphysodon aequifasciatus

MA Tengfei^1
,,
WEN Bin^{1, 2},
LIU Xin¹,
MIAO Lin¹,
GAO Jianzhong^{1, 2},
CHEN Zaizhong^{1, 2
, ,}

1.
Shanghai Ocean University/Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education/Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture and Rural Affairs, Shanghai, 201306, China
2.
Shanghai Ocean University/Shanghai Collaborative Innovation for Aquatic Animal Genetics and Breeding, Shanghai 201306, China

摘要

摘要: 七彩神仙鱼 (Symphysodon aequifasciatus) 具独特的亲代抚育行为，前期研究发现2种C型凝集素在育幼亲本皮肤中高表达，但表达模式不同，预示着潜在的功能分化。为探究两者潜在免疫功能的差异性，克隆获得了七彩神仙鱼C型凝集素基因 SaCD302 和 SaMBL，并分析了病原刺激下的免疫应答模式。结果显示，SaCD302 和 SaMBL 的开放阅读框 (ORF) 长度分别为741和795 bp，分别编码246和264个氨基酸。SaCD302的氨基酸序列包含一个信号肽 (aa 1—29)，一个CTL结构域 (aa 32—169)和一个跨膜结构域 (aa 184—206)。SaMBL的氨基酸序列包含两个低复杂度区域 (aa 32—92和 aa 104—121) 和一个典型的CTL结构域 (aa 137—263)。SaCD302 和 SaMBL 的氨基酸序列与尼加拉瓜湖始丽鱼 (Archocentrus centrarchus) 对应氨基酸序列的同源性最高。SaCD302 在各组织中均有表达，头肾中表达量最高，其次为肝脏和鳃；SaMBL 在皮肤中高表达，在肠道、肝脏、脾脏和性腺中低表达。嗜水气单胞菌 (Aeromonas hydrophila) 感染后，SaCD302 在脾脏、头肾、肠道和皮肤中的表达量均显著上调，SaMBL 仅在头肾、肠道和皮肤中表达上调，且时序性不同，表明二者可能在七彩神仙鱼免疫防御中发挥不同作用。
- 七彩神仙鱼 /
- C型凝集素基因 /
- CD302 /
- MBL /
- qRT-PCR
Abstract: Symphysodon aequifasciatus has a unique parental rearing behavior. In the previous study, two C-type lectins (CTLs) were found to be highly expressed in the skin of parent fish, but with different expression patterns. In order to investigate the difference in the potential immune functions of the specific CTLs, we cloned two CTL genes (SaCD302 and SaMBL), then simulated and analyzed their immune esponse patterns under pathological infection. The results show that the ORF region ofSaCD302 and SaMBL were 741 and 795 bp in length, encoding 246 and 264 amino acids (aa), respectively. The SaCD302 sequence contained a signal peptide (aa 1–29), a C-type lectin-like domain (CTLD) (aa 32–169) and a transmembrane domain (aa 184–206). Two low complexity regions (aa 32–92 and aa 104–121) and a CTLD (aa 137–263) were expressed in SaMBL sequence. Moreover, SaCD302 and SaMBL shared the highest similarity with that of Archocentrus centrarchus. SaCD302 was highly expressed in the head kidney, followed by the liver and gill. Compared with the intestine, liver, spleen and gonads, the skin had the higher expression of SaMBL. After Aeromonas hydrophila infection, SaCD302 expression was significantly upregulated in the spleen, head kidney, intestine and skin, whileSaMBL was only upregulated in the head kidney, intestine and skin, and these two genes were expressed in different temporal manners, indicating that they might play different roles in the immune defense of S. aequifasciatus.
- Symphysodon aequifasciatus /
- C-type lectins gene /
- CD302 /
- MBL /
- qRT-PCR

HTML全文

图 1 SaCD302 (a) 和SaMBL (b)基因的核酸序列及氨基酸序列

Figure 1. Nucleotide and deduced amino acid sequence of SaCD302 (a) andSaMBL (b)

下载: 全尺寸图片幻灯片

图 2 SaCD302 (a) 和SaMBL (b) 结构域示意图

Figure 2. Domain of SaCD302 (a) andSaMBL (b)

下载: 全尺寸图片幻灯片

图 3 SaCD302 (a) 和SaMBL (b) 氨基酸的多重序列比对

Figure 3. Multiple sequence alignment of amino acid sequences of SaCD302 (a) andSaMBL (b)

下载: 全尺寸图片幻灯片

图 4 基于NJ法构建的SaCD302 (a) 和SaMBL (b) 氨基酸序列系统进化树

Figure 4. Neighbor-joining phylogenetic analysis of SaCD302 (a) and SaMBL (b)

下载: 全尺寸图片幻灯片

图 5 SaCD302 (a) 和SaMBL (b) 在七彩神仙鱼不同组织中的表达特征

注：图中不同字母表示在不同组织中存在差异性显著 (P<0.05)；后图同此。

Figure 5. Relative expression of SaCD302 (a) and SaMBL (b) in different tissues of S. aequifasciatus

Note: Different letters indicate significant difference in expression of different tissues (P<0.05). The same case in following figures.

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图 6 嗜水气单胞菌感染后SaCD302在七彩神仙鱼不同组织中的表达分析

Figure 6. Expression analysis of indifferent tissues of SaCD302 fromS. aequifasciatus infected byA. hydrophila

下载: 全尺寸图片幻灯片

图 7 嗜水气单胞菌感染后SaMBL在七彩神仙鱼不同组织中的表达分析

Figure 7. Expression analysis of SaMBL in different tissues from S. aequifasciatus infected by A. hydrophila

下载: 全尺寸图片幻灯片

表 1 七彩神仙鱼 SaCD302 和 SaMBL 基因克隆及荧光定量PCR引物

Table 1. Cloning and fluorescence quantitative primers of SaCD302 and SaMBL gene in S. aequifasciatus

引物 Primer	引物序列 (5'—3') Primer sequence (5'–3')	用途 Function
SaCD302-F	ATGGAGTCGCGGAAGAAAAGT	基因克隆
SaCD302-R	CTATGGCACGCTGTCAGTTTC	基因克隆
SaCD302-qPCR-F	TGTGAAGCTGCTCAGGATGA	qRT-PCR
SaCD302-qPCR-R SaMBL-F SaMBL-R SaM BL-qPCR-F SaMBL-qPCR-R	GCTTCTGGTGCAGAAACCAA ATGTGCATTGTTCTCTCGGA CTATAACTGACAAATGATGT GGTCACGGTACTTTCTGGGA AGCCTCTCTCCTTGTTGGAC	qRT-PCR 基因克隆基因克隆 qRT-PCR qRT-PCR
β-actin-qPCR-F	GTGCGTGACATCAAGGAGAAG	qRT-PCR
β-actin-qPCR-R	GGAAGGAAGGCTGGAAGAGG	qRT-PCR

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