光裸星虫<i>SERCA</i>基因克隆及在卵母细胞中的表达分析

苏泳霖; 钟如卓; 郭志诚; 王庆恒

doi:10.12131/20220226

光裸星虫SERCA基因克隆及在卵母细胞中的表达分析

doi: 10.12131/20220226

苏泳霖^1,,
钟如卓¹,
郭志诚¹,
王庆恒^{1, 2, 3, ,}

1.
广东海洋大学水产学院，广东湛江 524088
2.
广东省海水养殖生物育种工程实验室，广东湛江 524088
3.
广东省水产动物病害防控与健康养殖重点实验室，广东湛江 524088

基金项目: 湛江市科技计划项目 (2021A05190)；广东省科技计划项目 (GDKTP2020046400, 163-2019-XMZC-0009-02-0059)

详细信息

作者简介:
苏泳霖 (1998—)，女，学士，研究方向为水产养殖。E-mail: syl12342@163.com

通讯作者:
王庆恒 (1977—)，男，教授，硕士，研究方向为无脊椎动物生物学与养殖技术。E-mail: wangqingheng@163.com

中图分类号: S 917.4
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- 被引次数: 0
出版历程
- 收稿日期: 2022-08-24
- 修回日期: 2022-10-13
- 录用日期: 2022-11-18
- 网络出版日期: 2022-12-03
- 刊出日期: 2023-04-05

Cloning and expression analysis of SERCA gene of Sipunculus nudus in oocytes

1.
Fisheries College, Guangdong Ocean University, Zhanjiang 524088, China
2.
Guangdong Provincial Engineering Laboratory for Mariculture Organism Breeding, Zhanjiang 524088, China
3.
Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture, Zhanjiang 524088, China

摘要

摘要: 细胞内钙离子 (Ca²⁺) 浓度的变化是介导卵母细胞成熟的重要因素。肌质网/内质网Ca²⁺-ATP酶 (Sarco/endoplasmic reticulum calcium adenosine triphosphatase, SERCA) 属P型ATP酶家族成员，是细胞内Ca²⁺转运的重要调控蛋白。前期转录组分析显示，光裸星虫 (Sipunculus nudus) SERCA (Sn-SERCA) 的表达量在卵母细胞发育过程中差异显著，为进一步研究SERCA在卵母细胞不同发育阶段中的作用，利用RACE技术得到Sn-SERCA cDNA全长，采用实时荧光定量PCR检测Sn-SERCA在各卵母细胞发育时期的相对表达量。结果表明，Sn-SERCA全长为3 840 bp，5'非编码区 (UTR) 为196 bp，3'UTR为581 bp，开放阅读框3 060 bp，编码1 020 个氨基酸，Sn-SERCA具有P型ATP酶家族进行催化反应所需的两个保守基序“TGES”和“DKTGT”。多序列比对、Motif分析及三级结构预测结果显示SERCA同源蛋白具有较高保守性。系统进化树分析表明Sn-SERCA与粉正蚓 (Lumbricus rubellus)、鸭嘴海豆芽 (Lingula anatine) 等无脊椎动物同源蛋白序列聚为一支。荧光定量结果显示：卵母细胞在体腔液中发育时，Sn-SERCA在卵黄旺盛合成后期高表达；当卵母细胞进入肾管后，Sn-SERCA的表达量大幅上升，显著高于其他时期 (P<0.05)。结合前期卵母细胞发育的超微结构观察，研究结果表明Sn-SERCA在光裸星虫卵母细胞的卵黄积累和生发泡破裂发生过程中起重要作用。
- 光裸星虫 /
- SERCA基因 /
- 卵母细胞 /
- 表达分析
Abstract: The change of intracellular calcium (Ca²⁺) concentration is an important factor in oocyte maturation. As a member of the P-type ATPase family and a crucial regulator of intracellular calcium transport, sarco/endoplasmic reticulum calcium adenosine triphosphatase (SERCA) is a key enzyme. To investigate the function of SERCA in the development of the oocytes of Sipunculus nudus, we obtained the full length of S. nudus SERCA (Sn-SERCA) cDNA by using RACE technique, and determined the Sn-SERCA relative expression level in different oocyte developmental periods by using real-time fluorescence quantitative PCR (qRT-PCR). The results indicate that the overall length of the Sn-SERCA was 3 840 bp, the 5'UTR was 196 bp, the 3'UTR was 581 bp, and the open reading frame was 3 060 bp, encoding 1 020 amino acids. Sn-SERCA had TGES and DKTGT which were the P-type ATPase family's two conserved motifs and were necessary for catalytic reactions. According to multiple sequence alignment, motif analysis, and predictions of tertiary structures, the SERCA homologous proteins exhibited great conservatism. The phylogenetic tree analysis shows that the Sn-SERCA formed a broad branch with homologous protein sequences from invertebrates such as Lumbricus rubellus and Lingula anatine. The result of qRT-PCR demonstrates that the Sn-SERCA was significantly expressed in the late yolk vigorous synthesis period and had the maximum value during coelomic fluid development. When the oocytes moved to the nephridioduct, the Sn-SERCA expression was considerably higher than that at other time (P<0.05). The variable expression of Sn-SERCA in different periods of oocyte development suggests that Sn-SERCA is crucial in the development and germinal vesicle breakdown in S. nudus oocytes.
- Sipunculus nudus /
- SERCA gene /
- Oocytes /
- Expression analysis

HTML全文

图 1 光裸星虫卵母细胞各时期总RNA和cDNA琼脂糖凝胶电泳图

Figure 1. Agarose gel electrophoresis diagram of total RNA and cDNA from oocytes of S. nudus

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图 2 Sn-SERCA cDNA序列全长及推导的氨基酸序列

Figure 2. Full length and deduced amino acid sequence of Sn-SERCA cDNA sequence

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图 3 Sn-SERCA蛋白跨膜螺旋

Figure 3. Transmembrane helix of Sn-SERCA

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图 4 Sn-SERCA蛋白结构域

Figure 4. Protein domains of Sn-SERCA

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图 5 光裸星虫 (a)、粉正蚓 (b)、鸭嘴海豆芽 (c) 和加州海兔 (d) SERCA蛋白三级结构

Figure 5. Tertiary structure of protein of SERCA of S. nudus (a), L. rubellus (b), L. anatina (c) and A. californica (d)

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图 6 SERCA同源氨基酸序列多序列比对

Figure 6. Multiple sequence alignment of SERCA homologous protein

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图 7 SERCA同源氨基酸序列 Motif 分析

Figure 7. Motif analysis of SERCA homologous protein

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图 8 SERCA同源氨基酸序列系统发育树

Figure 8. Phylogenetic trees of SERCA homologous protein

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图 9 光裸星虫各发育时期卵母细胞中Sn-SERCA的相对表达量

Figure 9. Relative expression of Sn-SERCA in different developmental periods of oocytes

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表 1 Sn-SERCA基因克隆及实时荧光定量引物序列

Table 1. Primer sequence used in gene cloning and qRT-PCR of Sn-SERCA gene

引物Primer	序列 (5'—3')Sequence (5'–3')	用途Function
5'-outer	GTCTGCTGGAACCTTGTC	5'RACE
5'-inner	CTGCCATACTCCAACAACT	5'RACE
3'-outer	CAACTCAACTACTACCAACTG	3'RACE
3'-inner	GTCACTTCTTGTCTCCTCAT	3'RACE
M13-F	CGCCAGGGTTTTCCCAGTCACGAC	菌落PCR
M13-R	AGCGGATAACAATTTCACACAGGA	菌落PCR
qRT-PCR-S	TGCCTTGTAGCCTCCTTCC	qRT-PCR
qRT-PCR-A	ACTGTCCTCATCTTCCATTGTG	qRT-PCR
60SL7-S	CAGGCTAACAACTTCTTATGG	qRT-PCR (内参)
60SL7-A	TGGCTTGACAGATAACACTT	qRT-PCR (内参)

下载: 导出CSV

表 2 Sn-SERCA序列分析所用软件及在线工具

Table 2. Software and online tool for sequence analysis of Sn-SERCA

名称Name	网址Website	用途Function
DNAMAN 8		拼接测序结果及多序列比对
ORF Finder	https://www.ncbi.nlm.nih.gov/orffinder	预测ORF及氨基酸序列
ProtParam	https://web.expasy.org/protparam	预测蛋白的理化性质
NCBI Blast	https://blast.ncbi.nlm.nih.gov/Blast.cgi	搜索同源蛋白
SMART	http://smart.embl-heidelberg.de	预测保守结构域
SOPMA	https://npsa-prabi.ibcp.fr/NPSA/npsa_sopma.html	预测蛋白二级结构
phyre2	http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index	预测蛋白三级结构
MEME Suit	http://meme-suite.org/index.html	Motif 搜索

下载: 导出CSV

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