无效基因<i>fruitless</i>参与卤虫<i>Artemia franciscana</i>的生殖调控研究

李科; 任翊卓; 韩学凯; 刘雪; 隋丽英

doi:10.12284/hyxb2023139

无效基因fruitless参与卤虫Artemia franciscana的生殖调控研究

doi: 10.12284/hyxb2023139

李科^{1, 3,},
任翊卓²,
韩学凯^{1, 3, ,},
刘雪^{1, 3},
隋丽英^{1, 3, ,}

1.
天津科技大学亚洲区域卤虫参考中心，天津 300457
2.
河北环境工程学院入海河流及近岸海域生态修复河北省工程研究中心，河北秦皇岛 066102
3.
天津科技大学海洋资源化学与食品技术教育部重点实验室，天津 300457

基金项目: 天津市自然科学基金项目（18JCQNJC78500）；天津市科技支撑计划项目（17ZXZYNC00060）；教育部长江学者和创新团队发展计划项目（IRT-17R81）。

详细信息

作者简介:
李科（1998—），男，山东省菏泽市人，研究方向为水产动物遗传育种。E-mail：li_ke@mail.tust.edu.cn

通讯作者:
韩学凯，男，博士，助理研究员，研究方向为水产动物遗传育种。E-mail：hanxk@tust.edu.cn

隋丽英，女，博士，教授，研究方向为卤水生物资源开发与利用。E-mail：suily@tust.edu.cn

中图分类号: Q74；P735
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出版历程
- 收稿日期: 2023-04-09
- 修回日期: 2023-06-15
- 网络出版日期: 2023-12-27
- 刊出日期: 2023-10-30

Reproductive regulation of fruitless gene in brine shrimp Artemia franciscana

Li Ke^{1, 3
,},
Ren Yizhuo²,
Han Xuekai^{1, 3
, ,},
Liu Xue^{1, 3},
Sui Liying^{1, 3
, ,}

1.
Asia Regional Artemia Reference Center, Tianjin University of Science and Technology, Tianjin 300457, China
2.
Hebei Engineering Research Center for Ecological Restoration of Rivers and Coastal Areas, Hebei University of Environmental Engineering, Qinhuangdao 066102, China
3.
Key Laboratory of Marine Resource Chemistry and Food Technology, Ministry of Education, Tianjin University of Science and Technology, Tianjin 300457, China

摘要

摘要: fruitless （fru）是在昆虫与甲壳动物求偶、交配行为以及生殖发育过程中发挥关键作用的基因。卤虫不仅是水产育苗的重要开口饲料，而且是生物学研究的重要实验生物。本实验通过对卤虫Artemia franciscana转录组的筛选，获得了fru基因的开放阅读框（ORF）并进行生物信息学分析，利用qPCR技术研究了它在卤虫生殖腺发育的不同阶段的表达情况，最后利用RNAi显微注射技术，深入探索了它的功能。经过生物信息学分析，fru基因的ORF长度为1 215 bp，其中包含了404个氨基酸，其分子量和等电点分别为45.19 kDa和5.28，属于酸性亲水性蛋白，且不含信号肽和跨膜结构；结构域预测显示fru存在BTB_POZ和HTH两个结构域，二级结构主要由α-螺旋和无规则卷组成，三级结构与其对应。qPCR结果显示，fru基因的表达量在卵囊的晚期胚胎中表现出明显的增加趋势，其表达量显著高于其他发育阶段（P < 0.01）；而在精巢的未成熟期，fru基因的表达量也有所增加，其表达量显著高于成熟早期、中期和晚期（P < 0.01）。经过RNA干扰，我们发现fru基因的表达量有了显著的下降（P < 0.01），并且经过受到干扰的雌虫后代都为休眠卵。这表明fru基因对于A. franciscana的生殖和发育有着重要的影响，甚至可能是影响卤虫繁殖方式的关键因素。通过本次研究，我们获得了关于fru基因在卤虫生殖发育中的作用及其相关分子机制的重要信息，可以帮助我们更好地理解这一重要的生物学过程。
- 卤虫 /
- fru基因 /
- 性腺 /
- 基因表达特征 /
- RNAi /
- 生殖发育
Abstract: fruitless (fru) gene plays an important role in courtship, mating behavior and reproductive development of insectsand crustaceans. Artemia is not only the crucial live food in fish and crustacean larviculture, but also an ideal experimental organism for biological study. In this experiment, the open reading frame (ORF) of fru gene was obtained from the transcriptome of Artemia franciscana and analyzed bioinformatically. qPCR was used to study the expression characteristics of this gene at different stages of gonad development in the Artemia, and its function was explored by RNAi microinjection. Bioinformatics analysis showed that the ORF length of fru gene was 1 215 bp, which contains 404 amino acids, while its molecular weight and isoelectric point were 45.19 kDa and 5.28, and it was an acidic hydrophilic protein with no signal peptide or transmembrane structure; structural domain prediction showed that there are two structural domains of fru, BTB_POZ and HTH. The secondary structure is dominated by α-helix and irregular coil, and the tertiary structure corresponds to it. The qPCR results showed that the expression of fru gene showed a significant increase in late embryos stage, and its expression was significantly higher than that of early oocyte, later oocyte and early embryo stages in the ovary (P < 0.01); while the expression of fru gene also increased in immature stage of the testis, and its expression was significantly higher than that of early, middle and late maturation stages (P < 0.01). After RNA interference, we found that there was a significant decrease in the expression of the fru gene (P < 0.01), and all the offspring produced were cysts. This suggests that the fru gene has an important effect on reproduction and development of A. franciscana, and may be a key factor influencing the reproductive mode of Artemia. Through this study, we obtained important information about the role of fru gene in the reproductive development of Artemia and their related molecular mechanisms, which can help us better understand this important biological process.
- Artemia /
- fru gene /
- gonad /
- gene expression character /
- RNAi /
- reproductive development

HTML全文

图 1 卤虫fru基因编码蛋白的氨基酸序列系统进化树

Fig. 1 Phylogenetic tree of amino acid sequence of the protein encoded by the fru gene of Artemia franciscana

下载: 全尺寸图片幻灯片

图 2 卤虫fru基因编码蛋白生物信息学分析

a. 亲水性分析；b. 信号肽分析；c. fru蛋白跨膜结构预测；d. fru蛋白保守结构域预测

Fig. 2 Bioinformatics analysis of the protein encoded by the fru gene of Artemia franciscana

a. Hydrophilic analysis; b. signal peptide analysis; c. prediction of fru protein transmembrane structure; d. prediction of fru protein conserved domain

下载: 全尺寸图片幻灯片

图 3 卤虫fru基因编码蛋白的高级结构

a. fru蛋白二级结构; b. fru蛋白三级结构

Fig. 3 Advanced structure of the protein encoded by fru gene in Artemia franciscana

a. Secondary structure of fru protein; b. tertiary structure of fru protein

下载: 全尺寸图片幻灯片

图 4 卤虫fru基因生殖腺不同发育时期的表达结果

相同字母表示差异不显著（P > 0.05）；不同字母表示差异极显著（P < 0.01）

Fig. 4 Expression results of fru gene in gonads of Artemia franciscana at different developmental stages

The same letter indicate the difference is not significant (P > 0.05); different letters indicate significant differences (P < 0.01)

下载: 全尺寸图片幻灯片

图 5 RNA干扰后fru基因在卤虫不同时期卵巢中的表达

**表示与对照有极显著性差异（P < 0.01）

Fig. 5 Expression of fru gene in ovary of Artemia franciscana at different developmental stages after RNA interference

** a significant difference from the control (P < 0.01)

下载: 全尺寸图片幻灯片

表 1 fru基因的引物信息

Tab. 1 The primer information of fru gene

引物名称	序列（5'—3'）	退火温度/°C	片段大小/bp	用途
fru-F1	ACTTTGGTTCAACTTCTTA	55	1 117	基因扩增
fru-R1	TTCTTGTCATTCCATCAT
fru-F	TGTTTCCAAGTGAGCCATGC	58	105	qPCR
fru-R	TTGCTGAGTACTGCTGACCT
GAPDH-F	GGTCGTGACTTGACGGACTATCT	57	120	内参基因
GAPDH-R	AGCGGTTGCCATTTCTTGTT
dsfru-F	T7- TAGTGACCAGACCCAAGA	58	431	dsRNA合成
dsfru-R	T7- TGTTTCTGTCTCCCGTTA
dsEGFP-F	T7-CAGTGCTTCAGCCGCTACCC	58	350	dsRNA合成
dsEGFP-R	T7-AGTTCACCTTGATGCCGTTCTT

下载: 导出CSV

表 2 生物信息学在线分析软件

Tab. 2 Online analysis software in bioinformatics

在线软件	用途	网址
ORF Finder-NCBI	开放阅读框分析	https://www.ncbi.nlm.nih.gov/orffinder/
ExPASy-ProtParam	理化性质分析	https://web.expasy.org/protparam/
ExPASy-ProtScale	亲水性/疏水性分析	https://web.expasy.org/protscale/
TMHMM Server V 2.0	跨膜结构预测	https://www.cbs.dtu.dk/services/TMHMM/
SignallP-5.0 Server	信号肽分析	https://www.cbs.dtu.dk/services/SignalP/
CDD	保守结构域预测	https://www.ncbi.nlm.nih.gov/Structure/cdd/docs/cdd_search.html
PSORT II Prediction	亚细胞定位	https://psort.hgc.jp/form2.html
SOPMA	二级结构预测	https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl? page=npsa_sopma.html
SWISS-MODEL	三级结构预测	https://swissmodel.expasy.org/

下载: 导出CSV

表 3 卤虫fru基因编码蛋白的氨基酸序列与其他物种的相似性比较

Tab. 3 Comparison of amino acid sequence similarities of proteins encoded by Artemia franciscanna and other species fru gene

序列号	物种名称	序列相似性/%
RXG68647.1	鼠妇（Armadillidium vulgare）	45.95
XP_020279362.1	牛角刺槐蚁（Pseudomyrmex gracilis）	42.11
KOB68495.1	冬尺蠖蛾（Operophtera brumata）	41.44
XP_015115225.1	胡峰（Diachasma alloeum）	41.23
XP_012350829.1	小蜜蜂（Apis florea）	40.35
XP_014274254.1	茶翅蝽（Halyomorp hahalys）	40.35
XP_032451896.1	寄生蜂（Nasonia vitripennis）	40.35
OXU22454.1	灿金小蜂（Trichomalopsis sarcophagae）	40.35
XP_016919575.1	中华蜜蜂（Apis cerana）	40.35
CAA9998384.1	烟盲蝽（Nesidiocoris tenuis）	39.67
ALC42845.1	果蝇（Drosophila busckii）	39.64
KYN44668.1	北方皱切叶蚁（Trachymyrmex septentrionalis）	38.62
XP_050694347.1	中华绒螯蟹（Eriocheir sinensis）	44.35
XP_053653254.1	红螯螯虾（Cherax quadricarinatus）	35.57

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