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2025年8月12日 星期二
农业生物技术学报  2022, Vol. 30 Issue (7): 1357-1367    DOI: 10.3969/j.issn.1674-7968.2022.07.013
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
棉铃虫正性调节区锌指类似蛋白 10 基因的克隆及表达分析
张亚林, 胡得琴, 李源, 皇甫慧琳, 加恩力斯尼•那扎尔, 余睿, 刘小宁*
新疆大学 生命科学与技术学院/新疆生物资源基因工程重点实验室,乌鲁木齐 830046
Cloning and Expression Analysis of the Positive Regulatory Domain Zinc Finger Protein 10 Like Gene of Helicoverpa armigera
ZHANG Ya-Lin, HU De-Qin, LI Yuan, HUANGFU Hui-Lin, JIAENLISINI•Na-Zha-Er, YU Rui, LIU Xiao-Ning*
Xinjiang Key Laboratory of Biological Resources and Genetic Engineering/College of Life Science and Technology, Xinjiang University, Urumqi 830046, China
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摘要 棉铃虫(Helicoverpa armigera)是一种杂食性害虫 ,可对多种农作物产量造成严重损失。目前 Bt 作物的大面积种植和化学农药的大量使用使得棉铃虫进化出应对杀虫剂和植物次生物质而保护自身的 防 御 机 制 。 为 了 开 发 绿 色 杀 虫 剂 ,对 棉 铃 虫 进 行 无 害 化 防 控 ,本 研 究 以 棉 铃 虫 细 胞 色 素 P450 (cytochrome P450 proteins, CYP) CYP6B6 启动子上响应 2-十三烷酮(2-Tridecanone, 2-TD)的核心序列 HE1 做 探 针 ,利 用 DNA pull down 技 术 筛 选 出 1 个 棉 铃 虫 正 性 调 节 区 锌 指 类 似 蛋 白 10 (positive regulatory domain zinc finger protein 10 like, PRDM10l)基因。对该基因进行克隆、生信分析和融合蛋白的原核表达, 同时利用 qPCR 检测其时空表达谱及 2-TD 胁迫下该基因在 6 龄棉铃虫中肠中的表达规律。结果显示 , HaPRDM10l (GenBank No. XM_021334552.1)的 ORF 为 2 067 bp,编码 688 个氨基酸 ,蛋白分子质量和理 论等电点分别为 77.96 kD 和 8.84。HaPRDM10l 在棉铃虫的不同发育阶段均有表达,在 5 龄幼虫中表达量 最高;在 6 龄幼虫不同组织中,中肠表达量最高。不同浓度的 2-TD 处理后,HaPRDM10lCYP6B6 均能响 应 2-TD 的 诱 导 。 HaPRDM10l 在 10 mg/g 的 2-TD 处 理 12 h 时 表 达 量 达 到 最 大 值 ,为 对 照 组 的 7.6 倍 ; CYP6B6 在 5 mg/g 的 2-TD 处理 6 h 后表达量达到最大值,为对照组的 4.6 倍。相关性分析显示在 6 h 处理 组中 HaPRDM10lCYP6B6 的表达呈正相关(r=0.749, P<0.01)。上述结果表明 HaPRDM10l 可能调控 CYP6B6 的表达以应对 2-TD 的胁迫。本研究为明晰棉铃虫应对植物次生物质的胁迫机制提供参考。 关键词 棉铃虫;正性调节区锌指类似蛋白 10 (PRDM10l);克隆;时空表达;2-十三烷酮(2-TD)
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AbstractHelicoverpa armigera is an omnivorous pest that causes severe yield loss of many crops. The current planting of Bt crops and the extensive use of chemical pesticides have enabled the cotton bollworm to evolve a defense mechanism that protects itself against pesticides and plants secondary substances. In order to develop an environment friendly insecticide for harmless control of H. armigera, the core sequence HE1, which can respond to 2-tridecanone (2-TD), of the cytochrome P450 CYP6B6 promoter was used as a probe to screen out a positive regulatory domain zinc finger protein 10 like (PRDM10l) gene by using DNA pull down technology. The sequence of HaPRDM10l were cloned from 6th instar larvae of H. armigera, analysed by bioinformatics and its fusion protein expressed by using a prokaryotic expression system. qPCR was used to detect the spatial-temporal expression profile of HaPRDM10l in H. armigera, and its expression pattern in the midgut of the 6th instar larvae of H. armigera was detected after 2-TD treatment. The results showed that the ORF of HaPRDM10l (GenBank No XM_021334552.1) was 2 067 bp, encoded 688 amino acids. The molecular weight and theoretical isoelectric point of the protein were 77.96 kD and 8.84, respectively. HaPRDM10l was expressed at all developmental stages of H. armigera, with the highest expression in 5th instar larvae, and among different tissues the highest expression in the midgut of 6th instar larvae. After the cotton bollworm was treated with different concentrations of 2-TD, both HaPRDM10l and CYP6B6 could respond to the induction of 2-TD. The expression of HaPRDM10l reached the maximum when 10 mg/g of 2-TD treatment for 12 h, which was 7.6 folds of that in the control group, and the expression level of CYP6B6 reached the maximum at 6 h after 5 mg/g 2-TD treatment, which was 4.6 folds that of the control group. Further analysis revealed that there was a positive correlation between the expression of HaPRDM10l and CYP6B6 in the 6 h treatment group (r=0.749, P<0.01). The above results suggested that HaPRDM10l may regulate the expression of CYP6B6 in response to the stress of 2-TD. This study provides a reference for clarifying the mechanism of the H. armigera in response to the stress of plant secondary substances.
收稿日期: 2021-09-17     
ZTFLH:  S435.6  
基金资助:国家自然科学基金(31972279); 国家级大学生创新训练计划项目(202110755087); 新疆维吾尔自治区天山雪松计 划(2017xs02)
通讯作者: *liuxn0103@sina.com   
引用本文:   
张亚林, 胡得琴, 李源, 皇甫慧琳, 加恩力斯尼·那扎尔, 余睿, 刘小宁. 棉铃虫正性调节区锌指类似蛋白 10 基因的克隆及表达分析[J]. 农业生物技术学报, 2022, 30(7): 1357-1367.
ZHANG Ya-Lin, HU De-Qin, LI Yuan, HUANGFU Hui-Lin, JIAENLISINI�Na-Zha-Er, YU Rui, LIU Xiao-Ning. Cloning and Expression Analysis of the Positive Regulatory Domain Zinc Finger Protein 10 Like Gene of Helicoverpa armigera. 农业生物技术学报, 2022, 30(7): 1357-1367.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2022.07.013     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2022/V30/I7/1357
 
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