CN117106039A - 控制斜纹夜蛾害虫的方法 - Google Patents
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Abstract
本发明涉及一种控制斜纹夜蛾害虫的方法,属于害虫控制领域。本发明通过测试发现WBY‑7.06蛋白对斜纹夜蛾有较好的杀虫活性,为防治斜纹夜蛾害虫提供了新的手段。
Description
技术领域
本发明涉及一种控制斜纹夜蛾害虫的方法,属于害虫控制领域。
背景技术
鳞翅目昆虫(Lepidoptera)属有翅亚纲下的一目。全世界已知的种类达到20万种,中国已知的约8000余种,绝大多数种类的幼虫为害各类植物。棉铃虫、菜粉蝶、小菜蛾、豆荚螟、玉米螟等害虫均是为害十分广泛的农田害虫,为农业的正常生产造成了极大的障碍,带来了很高的防治成本。
斜纹夜蛾(学名:Spodoptera litura Fabricius)属鳞翅目夜蛾科斜纹夜蛾属,广泛分布于全国各地,寄主植物广泛,可危害各种农作物及观赏花木。幼虫取食甘薯、棉花、芋、莲、田菁、大豆、烟草、甜菜和十字花科和茄科蔬菜等近300种植物的叶片,间歇性猖獗为害。
除了传统物理和化学防治方法外,利用杀虫蛋白开发生物工程菌或转基因植物也能够达到防治害虫的效果,且防治成本更低、环境更友好。因此找到每种害虫对应的杀虫蛋白就至关重要。
CN202311033251.5、CN202311218202.9、CN202311218205.2专利中的WBY-1、WBY-7、WBY-1.03、WBY-7.06几个蛋白是利用人工智能技术辅助生成的新型杀虫蛋白,它们对草地贪夜蛾均有较高的杀虫活性。但是,这些蛋白对其他一些害虫是否也有活性却并不知道。测试这些蛋白的杀虫谱能够为特定害虫提供新的防治手段。
发明内容
为了解决上述问题,本发明采用如下技术方案:
本发明提供一种控制斜纹夜蛾害虫的方法,其特征在于,包括将斜纹夜蛾害虫至少与WBY-7.06蛋白接触。
在一些实施方案中,所述WBY-7.06蛋白存在于至少产生所述WBY-7.06蛋白的宿主细胞中,所述斜纹夜蛾害虫通过摄食所述宿主细胞至少与所述WBY-7.06蛋白接触。
在一些实施方案中,所述WBY-7.06蛋白存在于至少产生所述WBY-7.06蛋白的细菌或转基因植物中,所述斜纹夜蛾害虫通过摄食所述细菌或所述转基因植物的组织至少与所述WBY-7.06蛋白接触,接触后所述斜纹夜蛾害虫生长受到抑制和/或导致死亡,以实现对斜纹夜蛾危害植物的控制。
在一些实施方案中,所述转基因植物为大豆、小麦、大麦、甘薯、棉花、甜菜、玉米、烟草、水稻、油菜、高粱或向日葵。
在一些实施方案中,所述转基因植物的组织为根、叶片、茎秆、果实、雄穗、雌穗、花药或花丝。
在一些实施方案中,所述WBY-7.06蛋白氨基酸序列如SEQ ID NO. 4所示。
在一些实施方案中,所述WBY-7.06蛋白在细菌中的核苷酸序列如SEQ ID NO. 8所示。
在一些实施方案中,所述转基因植物还包括至少一种不同于编码所述WBY-7.06蛋白的核苷酸的第二种核苷酸。
在一些实施方案中,所述第二种核苷酸编码Cry类杀虫蛋白质、Vip类杀虫蛋白质、蛋白酶抑制剂、凝集素、α-淀粉酶或过氧化物酶。
在一些实施方案中,所述第二种核苷酸为抑制目标昆虫害虫中重要基因的dsRNA。
本发明还提供WBY-7.06蛋白质控制斜纹夜蛾害虫的用途。
本发明还提供一种产生控制斜纹夜蛾害虫的植物的方法,其特征在于,包括向所述植物的基因组中引入编码WBY-7.06蛋白的多核苷酸序列。
本发明还提供一种产生控制斜纹夜蛾害虫的植物种子的方法,其特征在于,包括将由上述方法获得的第一植株与第二植株杂交,从而产生含有编码WBY-7.06蛋白的多核苷酸序列的种子。
本发明还提供一种培养控制斜纹夜蛾害虫的植物的方法,其特征在于,包括:种植至少一粒植物种子,所述植物种子的基因组中包括编码WBY-7.06蛋白的多核苷酸序列;使所述植物种子长成植株;使所述植株在人工接种斜纹夜蛾害虫和/或斜纹夜蛾害虫自然发生危害的条件下生长,收获与其他不具有编码WBY-7.06蛋白的多核苷酸序列的植株相比具有减弱的植物损伤和/或具有增加的植物产量的植株。
本发明的有益效果在于:本发明通过测试WBY-1、WBY-7、WBY-1.03、WBY-7.06蛋白对桃蛀螟、东方粘虫、亚洲玉米螟、棉铃虫、小地老虎、斜纹夜蛾、大豆卷叶螟这几种昆虫的杀虫活性,明确了这4种蛋白的杀虫谱,并发现WBY-7.06蛋白对斜纹夜蛾有较好的杀虫活性,为斜纹夜蛾害虫防治提供了新的手段。
附图说明
图1 斜纹夜蛾蛋白生测结果。左:WBY-1.03;右:WBY-7.06。
具体实施方式
提供以下定义和方法用以更好地界定本申请以及在本申请实践中指导本领域普通技术人员。除非另作说明,术语按照相关领域普通技术人员的常规用法理解。本文所引用的所有专利文献、学术论文、行业标准及其他公开出版物等,其中的全部内容整体并入本文作为参考。
以下实施例用于说明本发明,但不用来限制本发明的范围。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所作的修改或替换,均属于本申请的范围。若无特别指明,实施例按照常规实验条件,如Sambrook等人的分子克隆实验手册(Sambrook J& Russell DW, Molecular cloning: a laboratory manual, 2001),或按照制造厂商说明书建议的条件。若未特别指明,实施例中所用的化学试剂均为常规市售试剂,实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1蛋白的制备合成
使用蛋白表达实验系统合成这些蛋白实物,并测试它们对几种鳞翅目害虫的杀虫效果。
首先根据氨基酸序列设计编码该序列的核酸序列(在如下在线工具中开展http://www.friendbio.com/codon.html),其中密码子设置为大肠杆菌(K12菌株)偏爱性,并避免XhoI和HindIII酶切位点。WBY-1、WBY-7、WBY-1.03、WBY-7.06蛋白的氨基酸序列分别如SEQ ID NO. 1~SEQ ID NO. 4所示,获得的编码蛋白的核酸序列如SEQ ID NO. 5~SEQ IDNO. 8所示。
人工合成上述序列的核酸分子,克隆到载体pET28a表达载体中限制性内切酶XhoI和HindIII的位点之间,获得蛋白表达载体。将该载体转入大肠杆菌BL21细胞系,并进行蛋白表达。具体步骤如下:
单个菌落接种到0.5 mL LB液体培养基,在37℃下培养4 h至培养基浑浊,取100uL菌液加入 IPTG(Isopropyl-β-D-thiogalactoside)至终浓度为0.8 mM,同时取100 uL菌液作为阴性对照,继续培养4 h,在100 μL菌液中加入25 μL上样缓冲液制样电泳,根据阴性对照和加入IPTG诱导的结果对比,判断是否有表达。有表达的取剩余20 μL,接种到2 mL LB液体培养基中37℃下培养12-16 h作为种子液,种子液再接种到250 mL LB液体培养基至OD600=0.5-0.6,然后加IPTG(Isopropyl-β-D-thiogalactoside)至浓度为0.8 mM,并继续在同样的条件下培养4小时。 培养液经过5000 g离心10分钟沉淀大肠杆菌细胞,然后弃上清收集沉淀。沉淀中加30 mL 20mM Tris-50mM NaCl缓冲液,超声破碎。离心后检测上清液是否含有重组蛋白,并进行定量。
实施例2杀虫效果测试
进一步将上述实施例获得的重组蛋白进行杀虫活性测试。具体为:
采用表面涂抹方法进行生物测定,先在24孔板中先加入约1 mL未凝固的人工饲料(约1 g),轻微晃动使饲料铺满孔板底部,待饲料凝固后,再加入不同浓度的蛋白溶液(20 μL/孔),加入后轻轻晃动使药液均匀地平铺在饲料表面上,在通风橱内自然风干1 h。实验设置6个梯度浓度(0.001、0.01、0.1、0.5、1、2 μg/cm2)和一个空白对照(缓冲液),每个处理接24头人工饲养的目标昆虫初孵幼虫(孵化时间为2~12 h),设3次重复,放置在温度26±2℃,光周期14:10(L:D)h,相对湿度50-70%的养虫室培养,7天后调查死亡率。以用毛笔轻触幼虫尾部,幼虫不动视为死亡,幼虫未发育2龄的也视为死亡。
根据如下公式计算死亡率和校正死亡率,并利用graphpad计算LC50值。
(公式1)
(公式2)
测试结果表明,WBY-1对亚洲玉米螟和东方粘虫,WBY-7对东方粘虫,WBY-1.03对斜纹夜蛾和东方粘虫,WBY-7.06对大豆卷叶螟、斜纹夜蛾和东方粘虫的LC50值不到1 μg/g(见表1),杀虫活性较佳,可以满足对害虫防治的要求。因此,WBY-7.06可以用于防治斜纹夜蛾害虫(图1)。
表1靶标昆虫生测结果
昆虫 | 蛋白 | 杀虫活性(LC501) |
桃蛀螟Dichocrocis punctiferalis | WBY-1 | 1.21 |
东方粘虫Mythimna separata | WBY-1 | 0.11 |
亚洲玉米螟Ostrinia furnacalis | WBY-1 | 0.84 |
棉铃虫Helicoverpa armigera | WBY-1 | 1.15 |
小地老虎Agrotis ipsilon | WBY-1 | 1.39 |
斜纹夜蛾Spodoptera litura | WBY-1 | 1.45 |
大豆卷叶螟Lamprosema indicate | WBY-1 | 1.57 |
桃蛀螟Dichocrocis punctiferalis | WBY-7 | 1.12 |
东方粘虫Mythimna separata | WBY-7 | 0.15 |
亚洲玉米螟Ostrinia furnacalis | WBY-7 | 1.29 |
棉铃虫Helicoverpa armigera | WBY-7 | 1.15 |
小地老虎Agrotis ipsilon | WBY-7 | 1.27 |
斜纹夜蛾Spodoptera litura | WBY-7 | 1.30 |
大豆卷叶螟Lamprosema indicate | WBY-7 | 1.28 |
桃蛀螟Dichocrocis punctiferalis | WBY-1.03 | 1.35 |
东方粘虫Mythimna separata | WBY-1.03 | 0.38 |
亚洲玉米螟Ostrinia furnacalis | WBY-1.03 | 1.48 |
棉铃虫Helicoverpa armigera | WBY-1.03 | 1.35 |
小地老虎Agrotis ipsilon | WBY-1.03 | 1.18 |
斜纹夜蛾Spodoptera litura | WBY-1.03 | 0.09 |
大豆卷叶螟Lamprosema indicate | WBY-1.03 | 1.25 |
桃蛀螟Dichocrocis punctiferalis | WBY-7.06 | 1.65 |
东方粘虫Mythimna separata | WBY-7.06 | 0.46 |
亚洲玉米螟Ostrinia furnacalis | WBY-7.06 | 1.27 |
棉铃虫Helicoverpa armigera | WBY-7.06 | 1.57 |
小地老虎Agrotis ipsilon | WBY-7.06 | 1.43 |
斜纹夜蛾Spodoptera litura | WBY-7.06 | 0.11 |
大豆卷叶螟Lamprosema indicate | WBY-7.06 | 0.99 |
1:单位μg/g
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (10)
1.一种控制斜纹夜蛾害虫的方法,其特征在于,包括将斜纹夜蛾害虫至少与WBY-7.06蛋白接触;
优选地,所述WBY-7.06蛋白存在于至少产生所述WBY-7.06蛋白的宿主细胞中,所述斜纹夜蛾害虫通过摄食所述宿主细胞至少与所述WBY-7.06蛋白接触;
更优选地,所述WBY-7.06蛋白存在于至少产生所述WBY-7.06蛋白的细菌或转基因植物中,所述斜纹夜蛾害虫通过摄食所述细菌或所述转基因植物的组织至少与所述WBY-7.06蛋白接触,接触后所述斜纹夜蛾害虫生长受到抑制和/或导致死亡,以实现对斜纹夜蛾危害植物的控制。
2.根据权利要求1所述的控制斜纹夜蛾害虫的方法,其特征在于,所述转基因植物为大豆、小麦、大麦、甘薯、棉花、甜菜、玉米、烟草、水稻、油菜、高粱或向日葵;
优选地,所述转基因植物的组织为根、叶片、茎秆、果实、雄穗、雌穗、花药或花丝。
3.根据权利要求1或2所述的控制斜纹夜蛾害虫的方法,其特征在于,所述WBY-7.06蛋白氨基酸序列如SEQ ID NO. 4所示;
更优选地,所述WBY-7.06蛋白在细菌中的核苷酸序列如SEQ ID NO. 8所示。
4.根据权利要求1至3任一项所述的控制斜纹夜蛾害虫的方法,其特征在于,所述转基因植物还包括至少一种不同于编码所述WBY-7.06蛋白的核苷酸的第二种核苷酸。
5.根据权利要求4所述的控制斜纹夜蛾害虫的方法,其特征在于,所述第二种核苷酸编码Cry类杀虫蛋白质、Vip类杀虫蛋白质、蛋白酶抑制剂、凝集素、α-淀粉酶或过氧化物酶。
6.根据权利要求4所述的控制斜纹夜蛾害虫的方法,其特征在于,所述第二种核苷酸为抑制目标昆虫害虫中重要基因的dsRNA。
7.WBY-7.06蛋白质控制斜纹夜蛾害虫的用途。
8.一种产生控制斜纹夜蛾害虫的植物的方法,其特征在于,包括向所述植物的基因组中引入编码WBY-7.06蛋白的多核苷酸序列。
9.一种产生控制斜纹夜蛾害虫的植物种子的方法,其特征在于,包括将由权利要求8所述方法获得的第一植株与第二植株杂交,从而产生含有编码WBY-7.06蛋白的多核苷酸序列的种子。
10.一种培养控制斜纹夜蛾害虫的植物的方法,其特征在于,包括:种植至少一粒植物种子,所述植物种子的基因组中包括编码WBY-7.06蛋白的多核苷酸序列;使所述植物种子长成植株;使所述植株在人工接种斜纹夜蛾害虫和/或斜纹夜蛾害虫自然发生危害的条件下生长,收获与其他不具有编码WBY-7.06蛋白的多核苷酸序列的植株相比具有减弱的植物损伤和/或具有增加的植物产量的植株。
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