三疣梭子蟹（<i>Portunus trituberculatus</i>）<i>foxl2</i>基因功能初探及相关miRNA分析

张梦倩; 张景琰; 葛红星; 刘萍; 李健; 孟宪亮

doi:10.12284/hyxb2022060

三疣梭子蟹（Portunus trituberculatus）foxl2基因功能初探及相关miRNA分析

doi: 10.12284/hyxb2022060

张梦倩^1,,
张景琰²,
葛红星¹,
刘萍^{2, 3},
李健^{2, 3},
孟宪亮^{3, 4, ,}

1.
江苏海洋大学江苏省海洋生物技术重点实验室，江苏连云港 222005
2.
中国水产科学研究院黄海水产研究所农业农村部海洋渔业可持续发展重点实验室，山东青岛 266071
3.
青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室，山东青岛 266237
4.
中国水产科学研究院黄海水产研究所农业农村部水生动物基因组学重点实验室，山东青岛 266071

基金项目: 国家自然科学基金（41976106，41306178）；青岛市市南区科技计划（2020-2-001-QT）；国家现代农业产业技术体系（CARS-48）；中国水产科学研究院基本科研业务费（2020TD46，2018HY-ZD0201）。

详细信息

作者简介:
张梦倩（1997-），女，安徽省马鞍山市人，研究方向为甲壳动物繁殖生物学。E-mail：2080855236@qq.com

通讯作者:
孟宪亮，男，副研究员，主要从事甲壳动物生理学研究。E-mail：xlmeng@ysfri.ac.cn

中图分类号: S917.4
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出版历程
- 收稿日期: 2021-10-18
- 修回日期: 2021-11-16
- 刊出日期: 2022-04-14

Preliminary functional study of foxl2 in Portunus trituberculatus and analysis of its related miRNA

Zhang Mengqian^1
,,
Zhang Jingyan²,
Ge Hongxing¹,
Liu Ping^{2, 3},
Li Jian^{2, 3},
Meng Xianliang^{3, 4
, ,}

1.
Jiangsu Provincial Key Laboratory of Marine Biotechnology, Jiangsu Ocean University, Lianyungang 222005, China
2.
Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture and Rural Affairs, Yellow Sea Fisheries Research Institute, Chinese Academy of Fisheries Science, Qingdao 266071, China
3.
Laboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266237, China
4.
Key Laboratory of Aquatic Genomics, Ministry of Agriculture and Rural Affairs, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China

摘要

摘要: foxl2在脊椎动物卵巢分化、发育和功能维持等方面具有重要作用，然而其在三疣梭子蟹（Portunus trituberculatus）卵巢发育中的功能尚不明确。本研究首先克隆了三疣梭子蟹foxl2（Ptfoxl2）基因cDNA全长序列，该基因5′和3′非编码区域（UTR）长度分别为701 bp和211 bp，开放阅读框的长度为1 590 bp。基因表达分析结果显示，foxl2在三疣梭子蟹不同组织中均有表达，但在卵巢中表达量最高；其在卵巢发育不同时期的表达存在显著差异，在V期表达量最高；切除眼柄后，该基因的表达出现显著下降；干扰该基因表达后，卵巢vtg基因的表达显著上调。上述结果表明，foxl2可能在三疣梭子蟹卵巢发育调控中发挥重要功能，能够抑制卵巢组织中卵黄蛋白的合成。为进一步分析该基因的表达调控方式，利用生物信息学方法，预测了靶向foxl2的miRNA，并通过双荧光素酶报告基因检测实验，从细胞水平验证了这些miRNA对Ptfoxl2的调控作用；分析了其在卵巢发育不同时期以及切除眼柄后的表达模式。结果显示，共转染miR-9类似物和包含foxl2 3′UTR的pmirGLO质粒组，萤火虫酶与海肾荧光素酶活性比值出现显著下降，且在卵巢发育过程及切除眼柄后与foxl2表达模式相反。该结果初步证实miR-9可以从转录后水平调控三疣梭子蟹foxl2基因的表达。
- 三疣梭子蟹 /
- foxl2 /
- 切除眼柄 /
- 卵巢发育 /
- miRNA
Abstract: The foxl2 plays essential roles in regulating ovarian differentiation, development and functional maintenance of vertebrate. However, its function in ovarian development of the swimming crab Portunus trituberculatus remains unknown. In the present study, we firstly cloned the full-length cDNA of P. trituberculatus foxl2 (Ptfoxl2) which contained a 5′ untranslated region (UTR) of 701 bp, a 3′ UTR of 211 bp, and an open reading frame (ORF) 1590 bp. Gene expression analysis in different tissues showed that Ptfoxl2 exhibited the highest expression in ovary. There were significant differences in its expression in different stages of ovarian development, and its expression was the highest in stage V. After eyestalk ablation, Ptfoxl2 expression decreased significantly. In addition, RNAi of Ptfoxl2 resulted in a significant reduction in vitellogenin expression of ovary. Taken together, these results indicated that Ptfoxl2 played an important role in the regulation of ovarian development of P. trituberculatus by inhibiting endogenous vitellogenesis in maturation stage. In order to further analyze the post-transcriptional regulation of Ptfoxl2, the miRNAs targeting Ptfoxl2 was predicted by bioinformatics analysis and the interaction between the miRNAs and Ptfoxl2 was verified using the dual luciferase reporter assay. The results showed that the relative luciferase activity was significantly reduced after co-transfection of miR-9 mimics and pmirGLO-Ptfoxl2-3′ UTR. Expression analysis showed that miR-9 exhibited opposite pattern at ovarian maturation stage and after eyestalk ablation. Collectively, the results demonstrated that miR-9 can regulate Ptfoxl2 expression in the swimming crab.
- Portunus trituberculatus /
- foxl2 /
- eyestalk ablation /
- ovarian development /
- miRNA

HTML全文

图 1 Ptfoxl2基因cDNA全长序列以及推导的氨基酸序列

FH结构域用阴影表示；起始密码子和终止密码子用方框标出；下划线标注区域依次分别为miR-9、novel-68和novel-52的预测结合位点；左侧数字为核苷酸和氨基酸位置

Fig. 1 Nucleotide sequence and deduced amino acid sequence of Ptfoxl2 gene

FH domain was marked with shadow; initiation codon and stop codon were marked with black box; the underlined regions were the predicted binding sites for miR-9, novel-68, and novel-52 in turn; the numbers on the left indicate the positions of nucleotide and amino acid

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图 2 4种甲壳动物foxl2氨基酸序列多重比对

右侧数字为氨基酸位置；各物种foxl2基因序列GenBank登录号：三疣梭子蟹（OK413951）、拟穴青蟹（MN412580.1）、中华绒螯蟹（KF806733.1）、斑节对虾（XM_037939236.1）；黑色代表氨基酸残基同源性100%；红色代表氨基酸残基同源性75%；蓝色代表氨基酸残基同源性50%

Fig. 2 Multiple alignments of the amino acid sequences of foxl2 in four crustacean species

The numbers on the right indicate the positions of the amino acids; the GenBank accession numbers of foxl2 gene were as follows: Portunus trituberculatus (OK413951), Scylla paramamosain (MN412580.1), Eriocheir sinensis (KF806733.1), Penaeus monodon (XM_037939236.1); black represents 100% homology of amino acid residues; red represents 75% homology of amino acid residues; blue represents 50% homology of amino acid residues

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图 3 Ptfoxl2基因在三疣梭子蟹不同组织（A）、不同卵巢发育时期（B）以及切除眼柄后（C）的表达

不同字母代表数据差异显著（p<0.05）；*表示对照组和切除眼柄组表达存在显著差异（p<0.05）

Fig. 3 The expression of Ptfoxl2 in different tissues (A), in different ovarian developmental stages (B) and after eyestalk ablation (C)

Different letters indicate significant difference (p<0.05); * indicates a significant difference between the control group and eyestalk ablation group (p<0.05)

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图 4 注射Ptfoxl2 dsRNA 和GFP dsRNA后卵巢Ptfoxl2（A）和vtg（B）基因的表达

*表示存在显著差异（p<0.05）

Fig. 4 Relative expression of Ptfoxl2 (A) and vtg (B) in ovary after injecting Ptfoxl2 dsRNA and GFP dsRNA

* indicates a significant difference (p<0.05)

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图 5 共转染野生型和突变型质粒和miRNA类似物后荧光素酶的相对活性

+表示实验体系中存在该miRNA类似物或质粒；−表示实验体系中不存在该miRNA类似物或质粒；不同字母代表数据差异显著（p<0.05）

Fig. 5 Luciferase activity of the reporter plasmid containing wild-type or mutant target site after co-transfection of plasmid and miRNA mimics

+ indicates that the miRNA mimics or plasmid exists in the reaction system; − indicates that the miRNA mimics or plasmid doesn’t exist in the reaction system; different letters indicate significant difference (p<0.05)

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图 6 miR-9在三疣梭子蟹不同组织（A）、不同卵巢发育时期（B）以及切除眼柄后（C）的表达

不同字母代表数据差异显著（p<0.05）；*表示对照组和切除眼柄组表达存在显著差异（p<0.05）

Fig. 6 The expression of miR-9 in different tissues (A), in different ovarian developmental stages (B) and after eyestalk ablation (C)

Different letters indicate significant difference (p<0.05) ; * indicates a significant difference between the control group and eyestalk ablation group (p<0.05)

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表 1 实验所用引物的序列

Tab. 1 The sequences of the primers used in this study

引物名称	序列（5′-3′）	用途
foxl2-3′	CGGGAACTTCGCAAGCTACACACAG	RACE
foxl2-5′	GGTAGTCGTCTTTCATCGTGCCGTAGG	RACE
UPM	CTAATACGACTCACTATAGGGC	RACE
foxl2-F	CGTTGTCCTGATCTCACTGC	qRT-PCR
foxl2-R	CGTCTTTCATCGTGCCGTAG	qRT-PCR
β-actin-F	CGAAACCTTCAACACTCCCG	qRT-PCR
β-actin-R	GGGACAGTGTGTGAAACGCC	qRT-PCR
dsRNA-foxl2-T7-F	GGATCCTAATACGACTCACTATAGGGCAGATGCAAAGCGGGAACTT	RNAi
dsRNA-foxl2-T7-R	GGATCCTAATACGACTCACTATAGGGGGAAAGCGTCTCCAGTCATC	RNAi
dsRNA-GFP-T7-F	GGATCCTAATACGACTCACTATAGGCGACGTAAACGGCCACAAGTT	RNAi
dsRNA-GFP-T7-R	GGATCCTAATACGACTCACTATAGGATGGGGGTGTTCTGCTGGTAG	RNAi
miR-9	GCCTCTTTGGTTATCTAGCTGTAT	qRT-PCR

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