| 粘虫生物钟timeless基因的克隆及时空和昼夜表达分析 |
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| 引用本文：冀佳悦,张蕾,程云霞,罗礼智,仵均祥,江幸福.粘虫生物钟timeless基因的克隆及时空和昼夜表达分析.植物保护学报,2018,45(4):670-678 |
| DOI：10.13802/j.cnki.zwbhxb.2018.2017044 |
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| 作者 | 单位 | E-mail | | 冀佳悦 | 西北农林科技大学, 旱区作物逆境生物学国家重点实验室, 陕西 杨凌 712100
中国农业科学院植物保护研究所, 植物病虫害生物学国家重点实验室, 北京 100193 | | | 张蕾 | 中国农业科学院植物保护研究所, 植物病虫害生物学国家重点实验室, 北京 100193 | | | 程云霞 | 中国农业科学院植物保护研究所, 植物病虫害生物学国家重点实验室, 北京 100193 | | | 罗礼智 | 中国农业科学院植物保护研究所, 植物病虫害生物学国家重点实验室, 北京 100193 | | | 仵均祥 | 西北农林科技大学, 旱区作物逆境生物学国家重点实验室, 陕西 杨凌 712100 | junxw@nwsuaf.edu.com | | 江幸福 | 中国农业科学院植物保护研究所, 植物病虫害生物学国家重点实验室, 北京 100193 | xfjiang@ippcaas.cn |
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| 中文摘要:为明确生物钟timeless基因对粘虫Mythimna separata迁飞、交配等周期性活动的调节作用，利用PCR技术克隆了粘虫timeless基因，命名为Mstim，采用生物信息方法分析其序列特征，使用实时荧光定量PCR技术检测该基因在不同发育阶段、组织及昼夜的表达特征。结果表明，Mstim的cDNA全长为3 132 bp，编码1 043个氨基酸，其预测的蛋白质MsTIM分子量为117 kD，等电点5.14，具有timeless保守的PIS、NLS等结构域，与甜菜夜蛾Spodoptera exigua及棉铃虫Helicoverpa armigera的氨基酸序列相似性高达97%。荧光定量PCR结果显示成虫期的Mstim表达量最高，卵期和蛹期次之，6龄幼虫的表达量最低；在雌、雄蛾中均以触角及头部中的表达量较高，飞行肌、足及生殖腺中的表达量较低。粘虫雌、雄蛾触角中Mstim的昼夜表达模式相似，均为光期表达量低于暗期，光期5 h的表达量最低，光期末期逐渐升高，至暗期后3 h达到最高值，暗期末期表达量开始下降。说明Mstim基因在粘虫中的表达不仅具有发育时期和组织的时空特异性，而且表现出明显的昼夜特性，推测其可能参与粘虫生长发育和迁飞与生殖等行为、生理节律的调节。 |
| 中文关键词:粘虫 永恒蛋白基因 克隆 时空及昼夜表达 |
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| Molecular cloning and spatiotemporal and diurnal expression of the clock gene timeless in the oriental armyworm, Mythimna separata (Walker) |
| Author Name | Affiliation | E-mail | | Ji Jiayue | State Key Laboratory of Crop Stress Biology in Arid Areas, College of Plant Protection, Northwest A & F University, Yangling, 712100, Shaanxi Province, China
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China | | | Zhang Lei | State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China | | | Cheng Yunxia | State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China | | | Luo Lizhi | State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China | | | Wu Junxiang | State Key Laboratory of Crop Stress Biology in Arid Areas, College of Plant Protection, Northwest A & F University, Yangling, 712100, Shaanxi Province, China | junxw@nwsuaf.edu.com | | Jiang Xingfu | State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China | xfjiang@ippcaas.cn |
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| Abstract:To investigate the regulatory effect of timeless gene on migration, mating and other rhythmic activities of Mythimna separata, PCR was used to clone M. separata timeless gene (Mstim). Bioinformatic methods were used to analyze the characteristics of Mstim. The expression patterns of Mstim at different developmental stages, tissues, and zeitgeber were quantified by real-time quantitative PCR (qPCR). The results showed that the full-length cDNA of Mstim was 3 132 bp, encoding 1 043 amino acid residues with a predicted molecular mass and theoretical isoelectric point of 117 kD and 5.14, respectively. MsTIM had the conserved PIS and NLS domains of timeless family, and the sequence similarity was 97% in comparison with Spodoptera exigua and Helicoverpa armigera. The qPCR analysis showed that the relative expression level of Mstim mRNA was the highest at adult stage, followed by egg and pupal stages, and 6th instar larva was the lowest. The higher relative expression of Mstim was recorded from antenna and head whereas lower expression was recorded from the flight muscle, leg and gonad in both female and male. The expression patterns of Mstim mRNA in antennae of female and male were similar at different zeitgeber time points. The expression of Mstim was lower in photophase than in scotophase, and the expression was lowest at the 5th hour of photophase but increased at the late photophase. The highest expression occurred at 3 h after light-out and then declined at the late scotophase. The expression of Mstim had not only spatiotemporal but also diurnal specificity, suggesting that this gene may be involved in regulating growth, migratory and reproductive rhythm in M. separata. |
| keywords:Mythimna separata timeless gene cloning spatiotemporal and diurnal expression |
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