RNA干扰PhCatC1/2基因对波纹龙虾相关免疫基因表达的影响

doi:10.3969/j.issn.1004-1524.2023.05.07

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (5): 1037-1047.DOI: 10.3969/j.issn.1004-1524.2023.05.07

RNA干扰PhCatC1/2基因对波纹龙虾相关免疫基因表达的影响

梁妃爽(), 梁华芳(), 黄佳宇, 王潘妹, 温崇庆

广东海洋大学水产学院,广东湛江524088

收稿日期:2021-12-27 出版日期:2023-05-25 发布日期:2023-06-01
作者简介:梁妃爽(1994—),男,广东湛江人,硕士,从事甲壳动物遗传育种与增养殖研究。E-mail:1083286453@qq.com
通讯作者: *梁华芳,E-mail: hfliang@126.com
基金资助:
广东省科技厅农业攻关项目(2013B020308009);广东海洋大学科研项目(C16394)

Effect of RNA interference with PhCatC1/2 gene on expression of related immune genes in Panulirus homarus

LIANG Feishuang(), LIANG Huafang(), Huang Jiayu, WANG Panmei, WEN Chongqing

Fisheries College, Guangdong Ocean University, Zhanjiang 524088, Guangdong, China

Received:2021-12-27 Online:2023-05-25 Published:2023-06-01

摘要/Abstract

摘要：

初步研究RNA干扰PhCatC1/2基因对波纹龙虾(Panulirus homarus)相关免疫基因表达的影响,采用qPCR技术检测PhCatC1/2、PhTlr13、PhLys和Phβ-G基因的表达,并观察肝胰腺组织的变化情况。结果表明:注射PhCatC1-dsRNA后,PhCatC2表达量比对照组显著下降(P<0.05),与对照组相比PhTlr13基因表达量在前期呈现显著升高,后下降的趋势,PhLys和Phβ-G基因的表达量均随dsRNA干扰时效的增加而增加;沉默PhCatC2基因后PhTlr13和Phβ-G基因的表达量随dsRNA干扰时效的增加而增加,PhCatC1基因表达量呈现下上调交替的趋势,PhLys基因表达量表现出先下调后上调的趋势。通过PhCatC1/2基因沉默后的分析初步推测,波纹龙虾可能通过促进Toll样受体和溶酶体通路来参与免疫应答,得出PhCatC1/2基因具有免疫功能。

关键词: 波纹龙虾, RNA干扰, PhCatC1/2, PhTlr13, PhLys, Phβ-G, 表达分析

Abstract:

Preliminary study of the effect of RNA interference with the PhCatC1/2 gene on the expression of immune genes associated with Panulirus homarus,the expression of PhCatC1/2, PhTlr13, PhLys and Phβ-G genes were detected by qPCR and changes in hepatopancreatic tissue were observed. The results showed that the expression of PhCatC2 was significantly different from the control group after PhCatC1-dsRNA injection (P<0.05), the PhTlr13 gene expression showed a significant increase in the first period and then a decrease compared with the control group,expression of both PhLys and Phβ-G genes increased with increasing duration of dsRNA interference. Expression of PhTlr13 and Phβ-G genes increased with increasing duration of dsRNA interference after silencing of the PhCatC2 gene, PhCatC1 gene expression showed an alternating trend of down- and up-regulation, PhLys gene expression showed a trend of down-regulation followed by up-regulation. Preliminary speculation from analysis of the PhCatC1/2 gene after silencing that the lobster Panulirus homarus may be involved in the immune response by promoting Toll-like receptor and lysosomal pathways and concluding that the PhCatC1/2 gene has an immune function.

Key words: Panulirus homarus, RNA interference, PhCatC1/2, PhTlr13, PhLys, Phβ-G, expression analysis

中图分类号:

S917

梁妃爽, 梁华芳, 黄佳宇, 王潘妹, 温崇庆. RNA干扰PhCatC1/2基因对波纹龙虾相关免疫基因表达的影响[J]. 浙江农业学报, 2023, 35(5): 1037-1047.

LIANG Feishuang, LIANG Huafang, Huang Jiayu, WANG Panmei, WEN Chongqing. Effect of RNA interference with PhCatC1/2 gene on expression of related immune genes in Panulirus homarus[J]. Acta Agriculturae Zhejiangensis, 2023, 35(5): 1037-1047.

图/表 6

表1 引物序列

Table 1 Primer sequences

引物名称 Primer name	引物序列 Primer sequence(5'→3')	用途 Application
EGFP-F	ATGGTGAGCAAGGGCGAGGAG
EGFP-R	TTACTTGTACAGCTCGTCCATGCC
dsRNA-EGFP-F	TAATACGACTCACTATAGGGATGGTGAGCAAGGGCGAGGAG	dsRNA合成dsRNA synthesis
dsRNA-EGFP-R	TAATACGACTCACTATAGGGTTACTTGTACAGCTCGTCCATGCC	dsRNA合成dsRNA synthesis
PhCatC1-F	TCCTGGAGACGGAACGCTT	qPCR
PhCatC1-R	CCCTGGGATAACACTGAACACG
dsRNA-PhCatC1-F	TAATACGACTCACTATAGGGTCCTGGAGACGGAACGCTT	dsRNA合成dsRNA synthesis
dsRNA-PhCatC1-R	TAATACGACTCACTATAGGGCCCTGGGATAACACTGAACACG	dsRNA合成dsRNA synthesis
PhCatC2-F	CGACCTCCTATTGTGACCGC	qPCR
PhCatC2-R	TGACCAACGCCTCCAGCAT
dsRNA-PhCatC2-F	TAATACGACTCACTATAGGGCGACCTCCTATTGTGACCGC	dsRNA合成dsRNA synthesis
dsRNA-PhCatC2-R	TAATACGACTCACTATAGGGTGACCAACGCCTCCAGCAT	dsRNA合成dsRNA synthesis
β-actin-F	CCATCCAGGCTGTGCTCTCT	内参基因Reference gene
β-actin-R	GCGGTGGTTGTTGAAGGTG
PhTlr13-F	AGTCCTCTTGCCCACCTTACA	qPCR
PhTlr13-R	TCGGATGACCACTCGCTAACT
PhLys-F	TGTCTCCTCCTGGCTGTTCT	qPCR
PhLys-R	CCCACCCATCATTTCTGC
Phβ-G -F	TGTGGGTCTCAGTGGTTTTGG	qPCR
Phβ-G -R	GGTCTGGTTGGATCTGCTTCTT

图1 PhCatC1和PhCatC2基因的RNA干扰实验结果柱状图上方“*”代表与对照组的数据相比差异显著(P<0.05)。

Fig.1 RNA interference test results of PhCatC1 and PhCatC2 gene “*”above the bars represented significant difference(P<0.05)compared with the control group.

图2 波纹龙虾PhCatC1基因沉默后不同免疫相关基因的表达变化图中不同小写字母表示差异显著(P<0.05)。下同。

Fig.2 Expression of different immune-related genes in PhCatC1 silenced Panulirus homarus Different lowercase letters in the figure represented significant difference(P<0.05).The same as below.

图3 波纹龙虾PhCatC2基因沉默后不同免疫相关基因的表达变化

Fig.3 Expression of different immune-related genes in PhCatC2 silenced Panulirus homarus

图4 波纹龙虾实验前肝胰腺组织结构

Fig.4 Histological structure of hepatopancreas before experiment in Panulirus homarus

图5 波纹龙虾PhCatC1/2-dsRNA干扰后的肝胰腺石蜡切片 A-D分别表示PhCatC1-dsRNA干扰后波纹龙虾肝胰腺24、48、72和96 h的组织结构;E-H分别表示PhCatC2-dsRNA干扰后波纹龙虾肝胰腺24、48、72和96 h的组织结构。

Fig.5 Paraffin sections of the hepatopancreas after interference of PhCatC2-dsRNA in Panulirus homarus A-D showed the structure of hepatopancreas from 24 to 96 h after PhCatC1-dsRNA interference;E-H showed the structure of hepatopancreas from 24 to 96 h after PhCatC2-dsRNA interference.

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RNA干扰PhCatC1/2基因对波纹龙虾相关免疫基因表达的影响

Effect of RNA interference with PhCatC1/2 gene on expression of related immune genes in Panulirus homarus

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