壳寡糖对中华绒螯蟹非特异性免疫的影响

doi:10.14012/j.cnki.fjsc.2023.06.002

摘要/Abstract

摘要：

为了研究免疫增强剂壳寡糖对中华绒螯蟹(Eriocheir sinensis)非特异性免疫的影响及机制,实验选用45只(20±2)g 附肢完整、个体均匀、健康的中华绒螯蟹,随机分为3组,每组15只,分别注射浓度为 0(对照组)、50、200 μg·mL^-1的壳寡糖0.1 mL,24 h后收集血清和血淋巴细胞,分析免疫酶活力和免疫基因的变化。结果显示,与对照组相比,壳寡糖显著提高了中华绒螯蟹血清中超氧化物歧化酶、过氧化氢酶、谷胱甘肽过氧化物酶和溶菌酶的活性,并且显著增加了血清中一氧化氮的含量,降低了血清中丙二醛的含量。实时荧光定量PCR结果显示,与对照组相比,壳寡糖显著增强了Toll信号通路中EsToll1、EsToll2、EsMyD88、EsTube、EsPelle和EsDorsal的表达,上调抗脂多糖因子EsALF和EsALF3的表达。研究表明,壳寡糖能够提高中华绒螯蟹非特异性免疫和抗氧化能力,开启Toll信号通路,促进抗脂多糖因子的表达。本研究为壳寡糖在中华绒螯蟹中的应用提供理论支撑。

关键词: 壳寡糖, 中华绒螯蟹, 免疫, 分子机制

Abstract:

This study aimed to investigate the effect and mechanism of immune enhancer chitosan oligosaccharides (COS) on non-specific immunity of E.sinensis. A total of 45 crabs (20±2) g with intact appendages and uniform individuals were selected for the experiments, and they were randomly divided into three groups, with fifteen crabs in each group. Each crab was injected with 0.1 mL of COS at concentrations of 0 (control group), 50 and 200 μg·mL^-1, respectively. Serum and blood lymphocytes were collected 24 hours later to analyze the changes in immune enzyme activity and immune genes, etc. The results showed that compared with the control group, COS treatment significantly increased the activities of superoxide dismutase, catalase, glutathione peroxidase and lysozyme in the serum of crab, significantly increased the content of nitric oxide, and decreased the content of malondialdehyde in serum. Quantitative real-time PCR results showed that compared with the control group, COS significantly increased the expression of EsToll1, EsToll2, EsMyD88, EsTube, EsPelle and EsDorsal in the Toll signaling pathway, and up-regulated the expression of anti-lipopolysaccharide factors EsALF and EsALF3. These results suggested that COS could improve the non-specific immunity and antioxidant capacity of E.sinensis, activate the Toll signaling pathway, and promote the expression of anti-lipopolysaccharide factors. This study provided theoretical support for the application of COS in E.sinensis.

Key words: chitosan oligosaccharides, Eriocheir sinensis, immunity, molecule mechanism

中图分类号:

高榆嘉, 徐美, 许云舒, 徐田田, 张敏娜, 许青松. 壳寡糖对中华绒螯蟹非特异性免疫的影响[J]. 渔业研究, 2023, 45(6): 530-536.

GAO Yujia, XU Mei, XU Yunshu, XU Tiantian, ZHANG Minna, XU Qingsong. Effect of chitosan oligosaccharides on non-specific immunity of Eriocheir sinensis[J]. Journal of Fisheries Research, 2023, 45(6): 530-536.

图/表 5

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引物名称 Primer name		序列(5’-3’) Sequence
EsToll1-F	CCACTGTCTTGCTCGTCGTCTT
EsToll1-R	CAATGCTCTGGTCAATCTGGTTCTG
EsToll2-F	GCATACCAGGACGACGAACAAG
EsToll2-R	TCAAGGAGGTCACAGTCACAGT
EsMyD88-F	GAACAGGATGCCATTGGTGAAA
EsMyD88-R	GGTGATCTTGAGGTGGATGTAGAGT
EsTube-F	ATTGTGCTGCTGGAGTTGCTGAC
EsTube-R	CATCGTCGGTCGCTTCTTCTTGG
EsPelle-F	TAAGCCAGCAAACAACGGAGCA
EsPelle-R	GAGTCACAGGCAAAGAAGGGGA
EsDorsal-F	CGTCAGCAGCACAGCAGAGAAT
EsDorsal-R	CCCGTATTTCCTCCCTCAACTTCAG
EsSOD-F	CCCAGTTGATGACGTTGAAG
EsSOD-R	CTCGGCTACTGCTCCAAGAA
EsCAT-F	ATCAAGTGTCATTCCTCTTCTCTG
EsCAT-R	CCTTCCCTTCTTTGTTCACCA
EsALF-F	GACGCAGGAGGATGCTAAC
EsALF-R	TGATGGCAGATGAAGGACAC
EsALF3-F	GACGAGGAAGTAGGCTTAGTGGT
EsALF3-R	GGGCTGCTGTTCTCTCTGGA
Esβ-actin-F	GCATCCACGAGACCACTTACA
Esβ-actin-R	CTCCTGCTTGCTGATCCACATC

引物名称 Primer name		序列(5’-3’) Sequence
EsToll1-F	CCACTGTCTTGCTCGTCGTCTT
EsToll1-R	CAATGCTCTGGTCAATCTGGTTCTG
EsToll2-F	GCATACCAGGACGACGAACAAG
EsToll2-R	TCAAGGAGGTCACAGTCACAGT
EsMyD88-F	GAACAGGATGCCATTGGTGAAA
EsMyD88-R	GGTGATCTTGAGGTGGATGTAGAGT
EsTube-F	ATTGTGCTGCTGGAGTTGCTGAC
EsTube-R	CATCGTCGGTCGCTTCTTCTTGG
EsPelle-F	TAAGCCAGCAAACAACGGAGCA
EsPelle-R	GAGTCACAGGCAAAGAAGGGGA
EsDorsal-F	CGTCAGCAGCACAGCAGAGAAT
EsDorsal-R	CCCGTATTTCCTCCCTCAACTTCAG
EsSOD-F	CCCAGTTGATGACGTTGAAG
EsSOD-R	CTCGGCTACTGCTCCAAGAA
EsCAT-F	ATCAAGTGTCATTCCTCTTCTCTG
EsCAT-R	CCTTCCCTTCTTTGTTCACCA
EsALF-F	GACGCAGGAGGATGCTAAC
EsALF-R	TGATGGCAGATGAAGGACAC
EsALF3-F	GACGAGGAAGTAGGCTTAGTGGT
EsALF3-R	GGGCTGCTGTTCTCTCTGGA
Esβ-actin-F	GCATCCACGAGACCACTTACA
Esβ-actin-R	CTCCTGCTTGCTGATCCACATC

项目Items	壳寡糖/(μg·mL^-1) Chitosan oligosaccharides
项目Items	0		50		200
超氧化物歧化酶/(U·mL^-1) SOD		14.90±0.75		20.48±1.01^**		21.31±0.61^**
过氧化氢酶/(U·mL^-1) CAT		4.69±1.71		13.62±3.60^**		22.03±4.16^**
谷胱甘肽过氧化物酶/(U·mL^-1) GSH-PX		134.76±18.36		215.40±49.79		241.04±23.78^**
丙二醛/(nmol·mL^-1) MDA		7.33±1.01		4.67±0.83^*		4.53±0.61^*
溶菌酶/(U·mL^-1) LZM		207.46±21.70		246.14±39.40		293.30±17.06^*
碱性磷酸酶/(U·mL^-1) AKP		1.34±0.09		1.43±0.18		1.59±0.51
酸性磷酸酶/(U·mL^-1) ACP		2.08±0.20		2.33±0.52		2.25±0.47
一氧化氮/(μmol·L^-1) NO		14.00±3.92		23.36±2.25^*		34.57±3.53^**

项目Items	壳寡糖/(μg·mL^-1) Chitosan oligosaccharides
项目Items	0		50		200
超氧化物歧化酶/(U·mL^-1) SOD		14.90±0.75		20.48±1.01^**		21.31±0.61^**
过氧化氢酶/(U·mL^-1) CAT		4.69±1.71		13.62±3.60^**		22.03±4.16^**
谷胱甘肽过氧化物酶/(U·mL^-1) GSH-PX		134.76±18.36		215.40±49.79		241.04±23.78^**
丙二醛/(nmol·mL^-1) MDA		7.33±1.01		4.67±0.83^*		4.53±0.61^*
溶菌酶/(U·mL^-1) LZM		207.46±21.70		246.14±39.40		293.30±17.06^*
碱性磷酸酶/(U·mL^-1) AKP		1.34±0.09		1.43±0.18		1.59±0.51
酸性磷酸酶/(U·mL^-1) ACP		2.08±0.20		2.33±0.52		2.25±0.47
一氧化氮/(μmol·L^-1) NO		14.00±3.92		23.36±2.25^*		34.57±3.53^**