CN116716296B - 转录因子c-Jun在昆虫防治中的应用 - Google Patents
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Abstract
本发明公开了转录因子c‑Jun在昆虫防治中的应用；本发明通过对广州、深圳和海南的草地贪夜蛾进行筛选和鉴定，发现转录因子c‑Jun为毒死蜱等杀虫剂的抗性基因，而且根据上述基因设计dsRNA并注入草地贪夜蛾，发现可以增加杀虫剂的敏感性，提高杀虫剂的杀虫作用、增加害虫死亡率，因此可以用于防治草地贪夜蛾或制备杀虫剂的增效剂，在草地贪夜蛾的防治过程中具有重要作用。
Description
技术领域
本发明属于生物医药技术领域，具体涉及转录因子c-Jun在昆虫防治中的应用。
背景技术
目前，草地贪夜蛾，也称秋粘虫，属于鳞翅目夜蛾科，起源于美洲热带和亚热带地区，具有迁飞能力强、寄主范围广、为害重、防控难度大的特点，是一种杂食性害虫，是联合国粮农组织(FAO)全球预警的重要的农业害虫。自入侵我国以来，已经在全国大面积发生，对玉米及其他农作物的安全生产造成了极大的威胁。
目前对草地贪夜蛾的防治方法主要是应用化学农药，但由于草地贪夜蛾主要在玉米心叶处藏身危害，具有较好的隐藏能力，常规喷雾药液的流失量较大，昆虫不易接触到药液，难以达到较好的防治效果；而且目前田间为害调查需要人工踏查，并且容易调查不准备，影响化学农药的合理使用，而化学农药的长期大量不合理使用会引发农药残留、食品安全、土壤环境污染、抗药性以及天敌的破坏问题，因此，研发新型绿色分子靶标对于防治草地贪夜蛾是迫在眉睫的。
RNA干扰(RNAi)是一种由双链RNA(dsRNA)分子引起的特异性转录后基因沉默的现象，是研究基因功能的有力工具，同时在害虫防治方面也具有极大潜力。基于RNAi技术的害虫防治手段具有专一性，且对非靶标生物安全。因此，筛选出防治草地贪夜蛾的关键靶标，对于加速RNAi技术在防治草地贪夜蛾中的应用具有重要意义。
c-Jun是激活蛋白-1(activator protein-1，AP-1)的重要构成因子，参与增殖、凋亡、存活、组织形态发生和肿瘤发生等多种细胞生理和病理活动。在氧化应激过程中，c-Jun氨基末端激酶(c-Jun N-terminal kinase，JNK)和细胞外调节蛋白激酶(extracellularregulated protein kinase，ERK)蛋白激酶通过磷酸化Jun和Fos，激活AP-1启动细胞抗氧化活动。在果蝇中，MBF1蛋白可以结合和保护Jun的半胱氨酸残基免受氧化破坏，以维持AP-1的激活以及虫体和细胞的抗氧化能力。但是在昆虫中，尽管有着相关功能的报道，但大多集中于昆虫氧化应激功能等方面，在昆虫调控抗性相关的功能上c-jun所起到的作用目前还未有报道。
发明内容
本发明的目的在于提供转录因子c-Jun在防治昆虫中的应用。
本发明所采取的技术方案是：
本发明的第一方面，提供转录因子c-Jun在(a1)～(a8)任一项中的应用；
(a1)提高杀虫剂的杀虫效率；
(a2)提高昆虫对杀虫剂的敏感性；
(a3)制备杀虫增效剂；
(a4)制备杀虫剂；
(a5)降低昆虫存活率；
(a6)制备降低昆虫成活率的产品；
(a7)防治有害昆虫；
(a8)制备防治有害昆虫的产品。
优选地，所述昆虫为鳞翅目昆虫。
优选地，所述鳞翅目昆虫为田间草地贪夜蛾。
本发明的第二方面，提供转录因子c-Jun抑制剂在(a1)～(a8)任一项中的应用；
(a1)提高杀虫剂的杀虫效率；
(a2)提高昆虫对杀虫剂的敏感性；
(a3)制备杀虫增效剂；
(a4)制备杀虫剂；
(a5)降低昆虫存活率；
(a6)制备降低昆虫成活率的产品；
(a7)防治有害昆虫；
(a8)制备防治有害昆虫的产品。
优选地，所述转录因子c-Jun抑制剂包括下调昆虫体内基因的表达或活性的物质。
优选地，所述物质包括抑制或沉默转录因子c-Jun；或下调转录因子c-Jun转录、多肽表达或多肽活性的物质。
优选地，所述物质包括抑制或沉默转录因子c-Jun的dsRNA、反义核酸、小干扰RNA、微小RNA的构建物，或者能表达或形成dsRNA、反义核酸、小干扰RNA、微小RNA的构建物。
优选地，所述dsRNA由SEQ ID NO.1所示的核苷酸和反向互补的核苷酸编码而成。
本发明的第三方面，提供一种产品，所述产品包括本发明第二方面所述的转录因子c-Jun抑制剂。
优选地，所述产品还包括杀虫剂。
优选地，所述杀虫剂包括毒死蜱、溴氰菊酯。
优选地，所述产品为喷洒式制剂、饵剂、注射剂。
本发明的第四方面，提供一种防治昆虫的方法，其特征在于，所述方法包括向昆虫施用本发明第三方面所述的产品。
本发明的有益效果是：
本发明通过对广州、深圳和海南的草地贪夜蛾进行筛选和鉴定，发现转录因子c-Jun为毒死蜱等杀虫剂的抗性基因，而且根据上述基因设计dsRNA并注入草地贪夜蛾，发现可以增加杀虫剂的敏感性，提高杀虫剂的杀虫作用、增加害虫死亡率，因此可以用于防治草地贪夜蛾或制备杀虫剂的增效剂，在草地贪夜蛾的防治过程中具有重要作用。
附图说明
图1为广州、深圳、海南地区的田间草地贪夜蛾转录因子c-jun mRNA表达水平。
图2为田间草地贪夜蛾的转录因子c-jun的蛋白表达水平。
图3为室内毒死蜱、溴氰菊酯汰选品系中转录因子c-jun mRNA表达水平。
图4在毒死蜱抗性品系中RNAi干扰c-jun后，幼虫的致死率和体重统计。
图5为毒死蜱抗性品系RNAi干扰c-jun后幼虫表征结果图。
具体实施方式
以下将结合实施例对本发明的构思及产生的技术效果进行清楚、完整地描述，以充分地理解本发明的目的、特征和效果。显然，所描述的实施例只是本发明的一部分实施例，而不是全部实施例，基于本发明的实施例，本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例，均属于本发明保护的范围。
实施例1草地贪夜蛾转录因子c-jun检测
实验方法：
使用Trizol方法从三个不同地点(广州、海南和深圳)的田间采集或毒死蜱、溴氰菊酯汰选品系中至少3只幼虫中提取总RNA。使用GoScript进行RNA逆转录TM反向转录混合物，寡核苷酸(dT)试剂盒(Promega(北京)生物技术有限公司)。实验步骤严格按照说明书进行，一般步骤如下：用4μL不含核酸酶的水制备10μL反向转录混合物，4μL GoScriptTM缓冲液(包括Oligo(dT))和2μL GoScriptTM酶混合物；对于每个反应，将10μL逆转录混合物和2μg总RNA和无核酸酶的水加入到20μL中。在25℃下反应5分钟，42℃下反应60分钟，70℃下反应15分钟，可以获得cDNA的第一链。产物加入80μL无核酸酶的水，在-20℃下保存。荧光定量PCR使用qPCR Master Mix试剂于QuantStudioTM 6 Flex Real-Time PCR System进行。每个反应包含：/>qPCR Master Mix 10μl，cDNA模板2μl，上下游引物各0.4μl(终浓度各0.2μM)和无核酸酶水7.2μl。检测流程为：95℃预变性2min，紧接着40个循环，每个循环包含95℃15秒和60℃40秒，最后进行溶解曲线检测。
蛋白质样品经浓度为12％的SDS-聚丙烯酰胺凝胶电泳分离后，300mA冰上转膜1h；用3％BSA37℃封闭PVDF膜2h；4℃孵育一抗过夜；TBST洗涤3次，37℃孵育二抗2h；TBST洗涤3次，加入化学发光超敏显色试剂于ChemiDoc Touch进行化学发光成像。
通过对田间材料的筛选和鉴定，发现在广州、深圳和海南的田间草地贪夜蛾中转录因子c-Jun的表达远高于室内低抗品系，同时在室内毒死蜱和溴氰菊酯抗性品系中也证明了这点，结果如图1-图3所示。
实施例2
选取体型大小一致、健康状况良好的3龄第一天(L3D1)的毒死蜱品抗性品系(CPF-R)草地贪夜蛾幼虫，称量每只虫子的重量，取体重相近的幼虫进行实验。随机分为为2组，即注射GFP-dsRNA(对照组)和c-jun-dsRNA(处理组)，毒死蜱品系每组均为20只。沿着血液循环流动方向，用微量注射器从草地贪夜蛾幼虫的侧腹部倒数第二腹节处，分别向两种抗性品系的两组草地贪夜蛾幼虫注射1μl，10μg的c-jun-dsRNA(处理组)，和1μl,10μg的GFP-dsRNA(对照组)，其中dsRNA是根据试剂盒T7 RiboMAXTM Express RNAi System(购自Promega公司)的说明书合成。
草地贪夜蛾c-jun的dsRNA正义链为：
TTCTATTCCCGGCCAGCATCGCGCCTACAGAGGAACAAGAAATGTACGCGCGCCCATTCGTCGAGGCGCTGGACAAGTTACACCACTCGGACCCAACGCCACAGATCGGACGTGTCGACCGACGAGTGTACGCTGATTTGGACAGGCCGCTTGACCGCTACCCTACTCCGATGGTGAAGGACGAGCCACAGACTGTTCCAAGTGCTGCAAGTTCACCTCCCATGTCACCTATTGACATGGACACACAAGAAAGAATCAAATTGGAACGCAAACGGCAAAGGAATCGAGTGGCCGCGTCCAAGTGCAGACGGCGTAAAC(SEQ ID NO.1)。
注射后的幼虫继续喂饲含有杀虫剂的培养基，草地贪夜蛾毒死蜱抗性品系喂饲含有毒死蜱浓度为0.003％的培养基。注射后每天观察虫体的形态变化并拍照取证，统计其注射48h后虫子的体重以及死亡率。
结果显示，在毒死蜱抗性品系中RNAi干扰c-jun后，害虫对毒死蜱的敏感性增加。观察发现RNAi干扰c-jun后体重相比与注射GFP-dsRNA(对照组)出现了显著偏低的现象(图4)；且出现死亡的现象，其表型如图5所示，而且处理组的幼虫出现发育不正常和死亡的现象；死亡率显著增加(图4)。
上述具体实施方式对本发明作了详细说明，但是本发明不限于上述实施例，在所属技术领域普通技术人员所具备的知识范围内，还可以在不脱离本发明宗旨的前提下作出各种变化。此外，在不冲突的情况下，本发明的实施例及实施例中的特征可以相互组合。
Claims (3)
1.正义链如SEQ ID NO.1所示的dsRNA在（a1）～（a2）任一项中的应用；
（a1）防治有害昆虫；
（a2）制备防治有害昆虫的产品；
所述昆虫为草地贪夜蛾。
2.一种产品，其特征在于，包括权利要求1中所述的dsRNA；所述产品还包括杀虫剂，所述杀虫剂包括毒死蜱、溴氰菊酯。
3.一种防治昆虫的方法，其特征在于，所述方法包括向昆虫施用权利要求2所述的产品，所述昆虫为草地贪夜蛾。
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| CN105052990A (zh) * | 2015-08-06 | 2015-11-18 | 华南师范大学 | 一种昆虫转录因子在害虫防治中的应用 |
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| CN105052990A (zh) * | 2015-08-06 | 2015-11-18 | 华南师范大学 | 一种昆虫转录因子在害虫防治中的应用 |
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