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浙江大学学报（农业与生命科学版）
浙江大学学报（农业与生命科学版）  2023, Vol. 49 Issue (2): 149-158    DOI: 10.3785/j.issn.1008-9209.2022.04.261
昆虫生理生化与害虫生物防治专题     
东方黏虫［Mythimna separata (Walker)］田间自然种群的性信息素受体MsepOR6功能鉴定
张永乐1(),程建军1,郑静君2,杜永均1()
1.浙江大学农业与生物技术学院农药与环境毒理研究所，浙江 杭州 310058
2.广东省农业有害生物预警防控中心，广东 广州 510500
Functional identification of the sex pheromone receptor MsepOR6 in the natural population of Mythimna separata (Walker)
Yongle ZHANG1(),Jianjun CHENG1,Jingjun ZHENG2,Yongjun DU1()
1.Institute of Pesticide and Environmental Toxicology, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, Zhejiang, China
2.Agricultural Pest Precaution and Management Center of Guangdong Province, Guangzhou 510500, Guangdong, China
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摘要：

为进一步理解东方黏虫[Mythimna separata (Walker)]的求偶行为，探明雄蛾对种内和种间性信息素的嗅觉分子识别机制，本研究以顺11-十六碳烯乙酸酯（Z11-16：Ac）和顺11-十六碳烯醇（Z11-16：OH）为性信息素进行田间诱捕到的东方黏虫作为研究对象，根据文献报道的MsepOR6基因片段序列，采用cDNA末端快速扩增（rapid amplification of cDNA ends, RACE）技术扩增出其全长，采用双电极电压钳技术鉴定MsepOR6对东方黏虫及其同作物生境害虫的性信息素的电流反应，并通过田间诱捕试验验证雄性东方黏虫对其中的活性化合物的行为反应。电生理测定结果表明：MsepOR6是东方黏虫雄蛾的性信息素受体基因，其cRNA对种内性信息素顺11-十六碳烯醛（Z11-16：Ald）、十六醛（16：Ald），以及种间性信息素顺9-十四碳烯乙酸酯（Z9-14：Ac）、顺7-十二碳烯乙酸酯（Z7-12：Ac）、顺9-十六碳烯乙酸酯（Z9-16：Ac）、顺9-十四碳烯醇（Z9-14：OH）有显著的电生理反应。田间生物测定结果表明，Z9-14：Ac可显著抑制东方黏虫性信息素的引诱活性，Z7-12：Ac则显著提高东方黏虫性信息素对雄蛾的引诱效果。本研究有助于理解东方黏虫的性信息素行为调控机制，为开发应用性信息素技术提供了理论支撑。
关键词： 东方黏虫;  嗅觉受体;  双电极电压钳;  性信息素;  种间化学通信    
Abstract:

In order to further understand the calling behavior of Mythimna separata (Walker) (Lepidoptera, Noctuidae) and explore the olfactory molecular recognition mechanism of male moths to intra- and inter-specific sex pheromones, the male moths collected by sex pheromone trapping with the mixture of Z11-16：Ac and Z11-16：OH in the field were used in this study. According to the reported fragment, we amplified the full-length sequence of MsepOR6 by rapid amplification of cDNA ends (RACE) technology. The responses of MsepOR6 cRNA to pheromone compounds were identified by two-electrode voltage clamp. The behavioral responses of male moths to active compounds were further verified by sex pheromone trapping experiment in the field. Electrophysiological assay results showed that MsepOR6 is one of the sex pheromone receptor genes, which was sensitive to two intra-specific sex pheromones of Z11-16：Ald and 16：Ald, and four inter-specific sex pheromones of Z9-14：Ac, Z7-12：Ac, Z9-16：Ac, and Z9-14：OH. Field bioassay results showed that Z9-14：Ac was antagonist and Z7-12：Ac was synergist in sex pheromone trapping of male moths. This study identified the function of MsepOR6 in the natural population of male moths, which is helpful to understand the regulatory mechanism of calling behavior and provides theoretical support for further development of sex pheromone application technology.
Key words: Mythimna separata (Walker)    olfactory receptor    two-electrode voltage clamp    sex pheromone    inter-specific chemical communication
收稿日期: 2022-03-15 出版日期: 2023-04-27
CLC:  S433.4  
基金资助: 浙江省重点研发计划项目(2018C02027)
通讯作者: 杜永均     E-mail: 2908085596@qq.com;yongjundu@zju.edu.cn
作者简介: 张永乐（https://orcid.org/0000-0003-4551-3873），E-mail：2908085596@qq.com
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引用本文:

张永乐,程建军,郑静君,杜永均. 东方黏虫［Mythimna separata (Walker)］田间自然种群的性信息素受体MsepOR6功能鉴定[J]. 浙江大学学报（农业与生命科学版）, 2023, 49(2): 149-158.

Yongle ZHANG,Jianjun CHENG,Jingjun ZHENG,Yongjun DU. Functional identification of the sex pheromone receptor MsepOR6 in the natural population of Mythimna separata (Walker). Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(2): 149-158.

链接本文:

https://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2022.04.261    或    https://www.zjujournals.com/agr/CN/Y2023/V49/I2/149

序号

No.

基因

Gene

引物序列（5′→3′）

Primer sequence (5′→3′)

用途

Usage

1MsepOR6F: TACTCTGATTACGCAATGGAqPCR
R: AGTTGACCACATACGCATT
2RPS3F: AATGAGTTCTTGACCAGGGAGqPCR
R: GTGTCCTCGTCGCCATAAT
3ActinF: ATCAAGGAGAAGCTCTGCTACGqPCR
R: ATCGTTCGTTTCCGATGGTG
4MsepOR6F: ACTTTGGCAGATCTGATAgccaccATGCAAAGATTGAAAAGAAAGTT

同源重组

Homologous recombination

R: CCGGGCCCACTAGTGATCTACTTGCTTCGAAGGAAAGT
5MsepORcoF: GGCAGATCTGATATCgccaccATGATGACCAAAGTGAAGGCCCA

同源重组

Homologous recombination

R: ACCGGGCCCACTAGTTTACTTGAGTTGCACCAACACCA
表1  试验中所用引物

化合物

Compound

性信息素

组分类型

Type of sex

pheromone

component

CAS登录号

CAS

accession

number

纯度

Purity/%

顺11-十六碳烯醛

Z11-16：Ald

种内53939-28-9＞97

顺11-十六碳烯醇

Z11-16：OH

种内56683-54-6＞97

顺11-十六碳烯乙酸酯

Z11-16：Ac

种内34010-21-4＞98

十六醛

16：Ald

种内629-80-1＞98

十六碳烯乙酸酯

16：Ac

种内629-70-9＞98

顺9-十六碳烯醛

Z9-16：Ald

种间56219-04-6＞97

顺9-十六碳烯乙酸酯

Z9-16：Ac

种间56219-10-4＞97

顺7-十二碳烯乙酸酯

Z7-12：Ac

种间14959-86-5＞97

顺9-十四碳烯乙酸酯

Z9-14：Ac

种间16725-53-4＞98

顺13-十八碳烯醛

Z13-18：Ald

种间58594-45-9＞93

顺12-十四碳烯乙酸酯

Z12-14：Ac

种间35153-20-9＞93

顺9-十四碳烯醇

Z9-14：OH

种间35153-15-2＞97
表2  电生理实验中所用化合物
图1  MsepOR6 多序列比对结果
图2  东方黏虫MsepOR6与MsepORco的系统进化树Hvir：烟芽夜蛾；Harm：棉铃虫；Hass：烟青虫；Aseg：黄地老虎；Msep：东方黏虫；Slitu：斜纹夜蛾；Psau：疆夜蛾；Sfru：草地贪夜蛾；Bmor：家蚕。
图3  MsepOR6 在东方黏虫组织中的特异性表达An：触角；He：头；Th：胸；Ab：腹；Le：足；Wi：翅。数值表示为平均值±标准误。短栅上的不同小写字母表示在P＜0.05水平差异有统计学意义；n=6。
图4  非洲爪蟾卵母细胞中共表达 MsepOR6/MsepORco 对化合物的反应A～B. MsepOR6/MsepORco对1.0×10－4 mol/L种内及种间性信息素的电流反应（n=6）；C. MsepOR6/MsepORco对种内性信息素Z11-16：Ald的剂量反应（n=4）；D. MsepOR6/MsepORco对种间性信息素Z9-14：Ac的剂量反应（n=4）。
图5  不同比例性信息素混合物对东方黏虫雄蛾的引诱力比较A.添加不同比例Z9-14：Ac后的相对诱蛾量；B.添加不同比例Z7-12：Ac后的相对诱蛾量。数值表示为平均值±标准误。短栅上的不同小写字母表示在P＜0.05水平差异有统计学意义。
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