长棘海星(Acanthaster planci)幼体特异性PCR检测技术与应用*

doi:10.11978/2022011

热带海洋学报 ›› 2022, Vol. 41 ›› Issue (6): 125-131.doi: 10.11978/2022011CSTR: 32234.14.2022011

长棘海星(Acanthaster planci)幼体特异性PCR检测技术与应用^*

张颖¹(), 杨栎潼², 刘冰¹^,³, 郑凡昱¹, 罗鹏¹, 陈偿¹^,⁴()

1.中国科学院南海海洋研究所, 中国科学院热带海洋生物资源与重点实验室, 广东省应用海洋生物学重点实验室, 广东广州 510301
2.厦门大学马来西亚分校, 中国东盟海洋技术学院, 马来西亚雪兰莪州雪邦 43900
3.中国科学院大学, 北京 100049
4.中国科学院南海海洋研究所, 海南西沙海洋环境国家野外科学观测研究站, 广东广州 510301

收稿日期:2022-01-19 修回日期:2022-05-09 出版日期:2022-11-10 发布日期:2022-05-19
通讯作者: 陈偿
作者简介:张颖(1992—), 女, 湖南省郴州市人, 助理研究员, 从事海洋生物分子生态学研究。email: zhangy@scsio.ac.cn
基金资助:
财政部和农业农村部国家现代农业产业技术体系

Specific PCR detection for Acanthaster planci larvae and its application^*

ZHANG Ying¹(), YANG Litong², LIU Bing¹^,³, ZHENG Fanyu¹, LUO Peng¹, CHEN Chang¹^,⁴()

1. CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China
2. China-ASEAN College of Marine Sciences, Xiamen University Malaysia, Sepang 43900, Malaysia
3. University of Chinese Academy of Sciences, Beijing 100049, China
4. Xisha Marine Environment National Observation and Research Station, Hainan, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China

Received:2022-01-19 Revised:2022-05-09 Online:2022-11-10 Published:2022-05-19
Contact: CHEN Chang
Supported by:
China Agriculture Research System of MOF and MARA

摘要/Abstract

摘要：

长棘海星(Acanthaster planci)的暴发是导致我国南海乃至印度—太平洋海域珊瑚礁退化的主要原因之一。浮游幼体的密度是决定成体种群是否暴发的重要指标, 但是由于幼体肉眼不可见且不易分辨, 常规调查和显微镜观察均无法有效检测到自然海域的长棘海星幼体, 因此迫切需要开发一种高灵敏性且特异性的长棘海星幼体检测技术。本研究针对长棘海星幼体线粒体细胞色素氧化酶亚基I(mtCOI, mitochondrial cytochrome oxidase subunit I)基因序列, 建立了基于聚合酶链式反应(PCR, polymerase chain reaction)的长棘海星幼体特异性检测技术, 并对西沙七连屿珊瑚礁海域的长棘海星幼体进行了检测。结果表明, 设计筛选的4对特异性引物均可以扩增长棘海星ApmtCOI基因片段, 且与蓝指海星(Linckia laevigata)、面包海星(Culcita novaeguineae)、粒皮海星(Choriaster granulatus)和吕宋棘海星(Echinaster luzonicus)没有交叉反应。在退火温度为58.5℃时, 引物2aooniF/2aooniR的特异性最佳, 具有较高的灵敏度, 可检测到皮克级的长棘海星基因组DNA。利用该技术检测了西沙七连屿海域长棘海星幼体分布情况, 发现10月底西沙七连屿珊瑚礁海域能检测到长棘海星幼体, 且幼体分布并不均匀。因此, 该检测技术可作为今后长棘海星幼体种群监测的有效方法。

关键词: 长棘海星, 细胞色素氧化酶亚基I, 聚合酶链式反应, 幼体

Abstract:

The outbreak of crown-of-thorns seastar (CoTS, Acanthaster planci) is one of the main causes to coral reef degradation in the South China Sea and the Indo-Pacific region. The density of CoTS larvae is an important indicator to determine whether the outbreak of CoTS adult population occurs or not. However, as the larvae are not visible and thus difficult to be distinguished, conventional investigation and microscope observation cannot effectively detect the CoTS larvae in natural seawaters. Therefore, it is needed to develop a sensitive and specific method for detecting CoTS larvae. This study established specific polymerase chain reaction (PCR) method to detect the CoTS larvae based on CoTS mitochondrial cytochrome oxidase subunit I gene (ApmtCOI), and this method was applied to detect the CoTS larvae in the coral reef around Qilianyu Island, Xisha. The results showed that the designed and screened four pairs of specific primers could succeed in amplifying ApmtCOI gene, and had no cross-reaction with Linckia laevigata, Culcita novaeguineae, Choriaster granulatus and Echinaster luzonicus. Moreover, the primer 2aooniF/2anooiR had the best specificity and higher sensitivity when annealing temperature was 58.5°C, which could detect the CoTS genomic DNA in pictogram grade. Furthermore, this method had succeeded in detecting the CoTS larvae in the Qilianyu Island of Xisha at the end of October, and we found that the distribution of CoTS larvae was not uniform. Therefore, this detection technology can be used as an effective method for monitoring CoTS larvae population in the future.

Key words: Acanthaster planci, cytochrome oxidase subunit I, polymerase chain reaction, larvae

中图分类号:

Q953

张颖, 杨栎潼, 刘冰, 郑凡昱, 罗鹏, 陈偿. 长棘海星(Acanthaster planci)幼体特异性PCR检测技术与应用^*[J]. 热带海洋学报, 2022, 41(6): 125-131.

ZHANG Ying, YANG Litong, LIU Bing, ZHENG Fanyu, LUO Peng, CHEN Chang. Specific PCR detection for Acanthaster planci larvae and its application^*[J]. Journal of Tropical Oceanography, 2022, 41(6): 125-131.

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站位	坐标	地理位置	样品名
1	16°57.708′ N, 112°13.080′ E	西沙洲	XSZ
2	16°58.117′ N, 112°14.848′ E	赵述岛	ZS-1
3	16°58.315′ N, 112°16.300′ E	赵述岛	ZS-2
4	16°57.112′ N, 112°19.432′ E	中岛	ZD
5	16°56.794′ N, 112°19.801′ E	南岛	ND
6	16°56.086′ N, 112°20.221′ E	北沙洲	BSZ

序号	引物名称	引物序列(5'—3')	熔解温度(Tm)/℃		目的片段大小/bp	来源
1	COTS-F-1321	TCCGACTACCCGGACGCC TATAC	62.9	5985—6007	126	Doyle et al, 2017
1	COTS-R-1446	AGTGGT TCGCTGGGAAGTGAAGG	62.2	6008—6110	126	Doyle et al, 2017
2	2aooniF	ATTTGGGGCCTGAGCAGGAATG	60.6	4727—4748	301	Suzuki et al, 2016
2	2aooniR	GCCTCTTTCTACTCCGGCTGATG	60.2	5005—5027	301	Suzuki et al, 2016
3	AcanP-TF	GCACGATTTGTCTCTGCCAAA	56.1	3074—3094	785	本研究
3	AcanP-TR	AGTCCTTCTCTCGCCAGGT	58.4	3840—3858	785	本研究
4	COTS-F-69	GGCCTGAGCAGGAATGGTTGGAA	62.6	4733—4755	919	Uthicke et al, 2015
4	COTS-R-987	GCCTTGTAGCGTTGCCATTCACC	61.9	5629—5651	919	Uthicke et al, 2015

序号	引物名称	PCR 反应条件
1	COTS-F-1321 COTS-R-1446	95℃ 3min; 95℃ 15s, 60℃ 15s, 72℃ 30s, 34个循环; 72℃ 10min;
2	2aooniF 2aooniR	95℃ 3min; 95℃ 15s, 60℃ 15s, 72℃ 30s, 34个循环; 72℃ 10min;
4	AcanP-TF AcanP-TR	95℃ 3min; 95℃ 15s, 57℃ 15s, 72℃ 1min, 34个循环; 72℃ 10min;
5	COTS-F-69 COTS-R-987	95℃ 3min; 95℃ 15s, 57℃ 15s, 72℃ 1min, 34个循环; 72℃ 10min;

长棘海星(Acanthaster planci)幼体特异性PCR检测技术与应用^*

Specific PCR detection for Acanthaster planci larvae and its application^*

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