日本沼虾桩蛋白基因的克隆与镉胁迫对其表达的影响

doi:10.3969/j.issn.1004-1524.20230104

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (2): 247-253.DOI: 10.3969/j.issn.1004-1524.20230104

日本沼虾桩蛋白基因的克隆与镉胁迫对其表达的影响

彭佳诚¹(), 吴越¹, 徐洁皓², 夏美文¹, 齐天鹏¹, 徐海圣¹^,³^,^*()

1.浙江大学动物科学学院,浙江杭州 310058
2.浙江万里学院生物与环境科学学院,浙江宁波 315100
3.浙江大学湖州市南太湖现代农业科技推广中心,浙江湖州 313000

收稿日期:2023-02-06 出版日期:2024-02-25 发布日期:2024-03-05
作者简介:彭佳诚(1998—),男,回族,湖南长沙人,硕士,研究方向为水产动物免疫学。E-mail:enoemos47@163.com
通讯作者: *徐海圣,E-mail:hsxu@zju.edu.cn
基金资助:
国家科技重大专项(2017ZX07401-002);宁波市海洋生物资源高价值利用公共服务平台(NBHY-2017-P2);湖州市农业科学与技术研究院技术创新团队(2019HN02)

Cloning of paxillin gene from Macrobrachium nipponense and effect of cadmium stress on its expression

PENG Jiacheng¹(), WU Yue¹, XU Jiehao², XIA Meiwen¹, QI Tianpeng¹, XU Haisheng¹^,³^,^*()

1. College of Animal Sciences, Zhejiang University, Hangzhou 310058, China
2. College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, Zhejiang, China
3. Zhejiang University Huzhou South Taihu Lake Modern Agricultural Science and Technology Promotion Center, Huzhou 313000, Zhejiang, China

Received:2023-02-06 Online:2024-02-25 Published:2024-03-05

摘要/Abstract

摘要：

镉(Cd)是蓄积性和毒性较强的生物非必需重金属元素之一,会对水生生物造成较大的危害。为更好地了解镉对日本沼虾的分子毒性作用,为水环境镉污染的生态风险预警和评估提供生物标志物,克隆了日本沼虾(Macrobrachium nipponenes)桩蛋白(paxillin)基因MnPXN,并检测了镉胁迫对其表达的影响。MnPXN基因全长为3 655 bp,包含1 491 bp的编码区,编码496个氨基酸;MnPXN蛋白具有6个低复杂区域和3个保守的LIM结构域,与凡纳滨对虾(Litopenaeus vannamei)聚成一支,与家蚕(Bombyx mori)、赤拟谷盗(Tribolium castaneum)等昆虫的亲缘关系较近,与线虫(Nematoda)亲缘关系最远。实时荧光定量PCR(qRT-PCR)结果显示,MnPXN在日本沼虾的肝胰腺、胃、鳃中表达水平较高,在心脏和肌肉中表达水平较低。在0.242 7 mg·L^-1 CdCl₂中暴露24 h,肝胰腺中MnPXN基因表达水平较对照组均显著升高,0.020 0 mg·L^-1 CdCl₂胁迫6 h后MnPXN基因表达水平显著升高,24 h表达量达到最大值,96 h恢复至起始水平。RNA干扰能显著(P<0.05)降低MnPXN在肝胰腺中的表达水平,显著(P<0.001)增加日本沼虾的死亡率,表明MnPXN基因能够降低镉胁迫对日本沼虾的毒性作用,其作用机理尚需进一步研究。

关键词: 镉胁迫, 日本沼虾, 桩蛋白, 基因表达, RNA干扰

Abstract:

Cadmium (Cd), with strong accumulation and toxicity, is one of the non-essential heavy metal elements, which could cause great harm to aquatic organisms. To better understand the molecular toxicity of cadmium to Macrobrachium nipponense, and provide biomarkers for ecological risk warning and assessment of cadmium pollution in water environment. In this paper, paxillin gene (MnPXN) of M. nipponense was cloned, and the effect of cadmium stress on its expression was examined. The total length of MnPXN gene is 3 655 bp, including 1 491 bp coding region, encoding 496 amino acids; 6 low complexity regions and 3 conservative LIM domains are included in MnPXN, forming a branch with Litopenaeus vannamei. It is closely related to insects such as silkworm and Tribolium castaneum, and is farthest related to nematodes. The results of quantitative reverse transcriptase-mediated PCR (qRT-PCR) showed that the expression level of MnPXN was high in hepatopancreas, stomach and gill, but low in heart and muscle. The expression level of MnPXN gene in hepatopancreas that after 24 h of treatment with 0.242 7 mg·L^-1 CdCl₂ was significantly higher than that in control group; After 6 h of 0.020 0 mg·L^-1 CdCl₂ stress, the expression level of MnPXN gene increased significantly, reaching the maximum at 24 h, and gradually recovered to the initial level at 96 h. Besides, RNA interference could significantly reduce the expression level of MnPXN in hepatopancreas and significantly increase the mortality of M. nipponense, indicating that MnPXN could reduce the toxic effect of cadmium stress on M. nipponense, and its mechanism needs further study.

Key words: cadmium stress, Macrobrachium nipponense, paxillin, gene expression, RNA interference

中图分类号:

S917.4

彭佳诚, 吴越, 徐洁皓, 夏美文, 齐天鹏, 徐海圣. 日本沼虾桩蛋白基因的克隆与镉胁迫对其表达的影响[J]. 浙江农业学报, 2024, 36(2): 247-253.

PENG Jiacheng, WU Yue, XU Jiehao, XIA Meiwen, QI Tianpeng, XU Haisheng. Cloning of paxillin gene from Macrobrachium nipponense and effect of cadmium stress on its expression[J]. Acta Agriculturae Zhejiangensis, 2024, 36(2): 247-253.

图/表 6

表1 基因克隆所用引物

Table 1 Primers for gene clone

引物 Primer	引物序列 Primer sequence(5'→3')	用途 Purpose
MnPXN-3'-F₁	CAATGCCAGCAGCCTCCAGACAC	RACE
MnPXN-3'-R₁	AAGCCGTCCTCGCCGAAGGTGTT	RACE
MnPXN-3'-F₂	TGGACGAGTTGAACAGCGTTGACA	RACE
MnPXN-3'-R₂	TCCCTTCTGGGTCGTCGTGATGC	RACE
MnPXN-3'-F₃	GAATGACCTGAGTGGCACCTGAC	RACE
MnPXN-3'-R₃	TGCCGTGAAGAATGACATAGAAT	RACE
MnPXN-3'-F₄	GAGGAAGTGCGGACGGAGCAGAGGT	RACE
MnPXN-3'-R₄	GATCTCGATGTGCCGTGAAGAAT	RACE
MnPXN-3'-F₅	GTTATGTCATTGGCTCAAGAGGA	RACE
MnPXN-3'-R₅	GTGTATTTATACTGTATGCCTAACC	RACE
MnPXN-3'-F₆	GCTACAAGAGGCTCCCAACTACAT	RACE
MnPXN-3'-R₆	CAGGAGGTAAAGACTACTGATGT	RACE
MnPXN-3'-F₇	AAAAGGTTTGGTAAATGTCCAGGTT	RACE
MnPXN-3'-R_UPM	CTAATACGACTCACTATAGGGC	RACE
MnPXN-5'-F₁	TTGTTCATCCAGGAGGTAAAGAC	RACE
MnPXN-5'-R₁	GCCAATAAGGCATCAATGGTATCCTG	RACE
MnPXN-5'-F₂	GAAGTTTGGCAGTATCTTATGGA	RACE
MnPXN-5'-R₂	TTGTGAATGAGGGTGGCGTTTGG	RACE
MnPXN-5'-F₃	CAATAAGGCATCAATGGTATCCTGCA	RACE
MnPXN-5'-R₃	AATTTCAAAAATCTAATT	RACE
MnPXN-5'-F_UPM	CTAATACGACTCACTATAGGGCAA GCAGTGGTATCAACGCAGAGT	RACE
MnPXN-5'-R₄	GGGCATCATTCTTTGTCTTTCTT	RACE
MnPXN-RT-F	CCCCATCTTGGATAAATGCGTGAC	qRT-PCR
MnPXN-RT-R	AAGCCGTCCTCGCCGAAGGTGTTGC	qRT-PCR
β-actin-F	GTGCCCATCTACGAGGGTTA	qRT-PCR
β-actin-R	CGTCAGGGAGCTCGTAAGAC	qRT-PCR

图1 不同物种PXN的氨基酸序列比对图中红色方框为6个物种共有的3个LIM结构域,黑色方框为智人、小鼠、斑马鱼、凡纳滨对虾拥有的第4个LIM结构域。

Fig.1 Multiple amino acid sequence alignments of PXN from different species The red box in the figure is the 3 LIM domains shared by 6 species, and the black box is the 4th LIM domain possessed by Homo sapiens, mouse, zebrafish and Litopenaeus vannamei.

图2 MnPXN与其他物种PXN蛋白的系统进化树

Fig.2 Phylogenetic analysis of MnPXN with PXN protein from other species

图3 MnPXN基因在日本沼虾不同组织中的表达情况 “*”表示表达水平与心脏组相比差异显著(P<0.05);“**”表示差异极显著(P<0.01);“***”表示差异极显著(P<0.001).

Fig.3 Expression level of MnPXN in different tissues of Macrobrachium nipponense “*” indicates that the expression level of this group is significantly different from that of the heart group (P<0.05); “**” indicates significant difference at 0.01 level; “***” indicates significant difference at 0.001 level.

图4 不同浓度镉胁迫24 h和镉胁迫不同时间对MnPXN mRNA表达水平的影响 “*”表示差异显著(P<0.05);“**”表示差异极显著(P<0.01);“***”表示差异极显著(P<0.001)。下同。

Fig.4 The relative expression level of MnPXN mRNA in hepatopancreas of Macrobrachium nipponense under different concentration of CdCl2 after 24 h and exposed to CdCl2 for different time “*” indicates significant difference at 0.05 level; “**” indicates significant difference at 0.01 level; “***” indicates significant difference at 0.001 level. The same as below.

图5 RNA干扰对MnPXN基因表达和日本沼虾存活率的影响

Fig.5 Effect of RNA interference on MnPXN gene expression and survival rate of Macrobrachium nipponense

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日本沼虾桩蛋白基因的克隆与镉胁迫对其表达的影响

Cloning of paxillin gene from Macrobrachium nipponense and effect of cadmium stress on its expression

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