CN116789787A - 蠋蝽多肽Ach1及其应用 - Google Patents
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Abstract
本发明公开了一种蠋蝽多肽Ach1及其应用，属于生物防治技术领域，该蠋蝽多肽Ach1基因序列为FQEQNCIPRGNECVAKELDCCDGMKCSHYANKCMT，具有6个半胱氨酸形成3个二硫键，二硫键的方式为C1‑C4，C2‑C5，C3‑C6。其在制备褐飞虱、斜纹夜蛾和烟蚜杀虫剂中的应用。本发明的蠋蝽多肽Ach1对褐飞虱、斜纹夜蛾和烟蚜具有杀虫活性。
Description
技术领域
本发明属于生物防治技术领域，具体来说涉及一种蠋蝽多肽，同时还涉及该蠋蝽多肽在制备褐飞虱、斜纹夜蛾和烟蚜杀虫剂中的应用。
背景技术
生物农药因其自然资源丰富和生产成本低廉和相对较好的环境相容性是目前农药发展的重点。动物毒素是高效杀虫剂开发的重要来源，尤其是昆虫天敌的毒素，比传统农药更具靶特异性，是高效安全杀虫剂开发的优质资源(King and Hardy,2013；Senji Laxmeet al.,2019)。目前杀虫剂开发研究最多的昆虫天敌毒素是蛛毒(King and Hardy,2013；Luddecke et al.,2022)。在全世界所有现存蜘蛛毒液中就有超过千万种杀虫毒素物质(Luddecke et al.,2022)，但仅有少数蜘蛛毒素具有较高的杀虫选择性。例如目前唯一面市的来源于蛛毒的杀虫剂是基于澳大利亚漏斗形蜘蛛Hadronyche versuta的一个多肽毒素GS-ω/κ-hexatoxin-Hv1a，其对害虫高效的同时对蜜蜂和脊椎动物比较安全。
相对于蜘蛛等其它天敌，捕食性昆虫的食性更窄，其毒素具有更高的选择性。目前毒素研究较多的捕食性昆虫有捕食性蝽、寄生蜂和捕食蝇等。已知捕食性蝽毒液具有很强的杀虫活性，如猎蝽毒液注入昆虫体内导致瘫痪、组织液化和死亡(Walker et al.,2016)；益蝽Picromerusnigrispinus毒腺提取样品对大豆夜蛾的LD50约为2uL/头(Martinez etal.,2016)。
动物毒液中最普遍存在的毒肽是Knottin毒肽(King,2019；Matsubara et al.,2017)。Knottin是指具有3对及以上的二硫键串成的扭结状结构(knottin)的小肽，在生物体内非常稳定。Knottin多肽结构具有非凡的可塑性，不同生物进化出丰富多样的Knottin多肽行使着不同功能(King,2019)。例如已商品化的蛛毒杀虫剂有效成分GS-ω/κ-hexatoxin-Hv1a就是一个Knottin多肽。捕食性蝽猎蝽毒液中的Knottin毒肽，具有抑制电压门控钙离子通道(Cav2.2)的活性，但是尚未发现其具有杀虫活性(Corzo et al.,2001；Rugen et al.,2021)。
同为捕食性蝽科的蠋蝽Arma chinensis可捕食鳞翅目、鞘翅目如斜纹夜蛾、草地贪夜蛾、叶甲等40多种农林害虫，是优良的捕食性天敌，目前已进行人工规模化繁育和应用。然而目前尚无关于蠋蝽毒液及毒素的研究报道。
发明内容
本发明的目的在于克服上述缺点而提供的一种对褐飞虱、斜纹夜蛾和烟蚜具有杀虫活性的蠋蝽多肽Ach1。
本发明的另一目的在于提供该蠋蝽多肽Ach1在制备褐飞虱、斜纹夜蛾和烟蚜杀虫剂中的应用。
本发明的蠋蝽多肽Ach1,其基因序列为FQEQNCIPRGNECVAKELDCCDGMKCSHYANKCMT，具有6个半胱氨酸形成3个二硫键，二硫键的方式为C1-C4，C2-C5，C3-C6。
本发明的蠋蝽多肽Ach1在制备褐飞虱杀虫剂中的应用。
本发明的蠋蝽多肽Ach1在制备斜纹夜蛾杀虫剂中的应用。
本发明的蠋蝽多肽Ach1在制备烟蚜杀虫剂中的应用。
本发明与现有技术相比，具有明显的有益效果，从以上技术方案可知：利用微量注射法进行毒肽对褐飞虱的活性测定，发现蠋蝽多肽Ach1对褐飞虱4龄若虫毒杀效果很快，1-2天内见效，2天后的存活曲线基本稳定，其24h的LD50值为0.316ng/头，多次注射测试的LD50值(0.124-0.316ng/头)；浸苗法发现Ach1多肽80mg/L处理褐飞虱4龄若虫7天后死亡率为56.67％，其LC50值为66.75mg/L，表明，无论是注射法还是浸苗法，Ach1对褐飞虱具有较高的毒杀活性。利用显微注射法进行毒肽对斜纹夜蛾的活性测定，发现Ach1多肽4ng/头(10μmol/L，100nL/头)处理斜纹夜蛾2龄幼虫8天死亡率为83.33％。表明，蠋蝽多肽Ach1测试浓度对斜纹夜蛾毒力较高。利用人工饲料饲喂法测定法进行毒肽对烟蚜的活性测定，发现Ach1多肽40μmol/L处理5天死亡率为76.7％。结果表明蠋蝽多肽Ach1测试浓度对烟蚜毒力较高。采用静水法测定了蠋蝽多肽Ach1对斑马鱼幼鱼的急性毒性，发现Ach1多肽10mg/L(2.5μmol/L)处理96h死亡率为0％。结果表明蠋蝽多肽Ach1测试浓度96h内对斑马鱼幼鱼无急性毒性。采用灌胃法测定了蠋蝽多肽Ach1对小鼠雌成鼠的急性毒性。发现Ach1多肽100μg/只(500mol/L，200nL/只)灌胃处理组小鼠七天内均无死亡、未见异常外观变化、活动状态都很活跃、食欲和排泄情况也正常、口鼻眼处也未见异常分泌物、未见体重异常。表明蠋蝽Ach1多肽测试浓度7天内对小鼠雌成鼠无急性毒性。
附图说明
图1为蠋蝽多肽Ach1、猎蝽毒肽Ptu1和蜘蛛毒肽Hv1a的序列比对分析图(Ptu1：乌黑盗猎蝽，P58606.1、Hv1a：漏斗网蜘蛛，P56207)；
图2为蠋蝽多肽Ach1的三维结构；
图3为蠋蝽多肽Ach1的化学合成制备样品质谱检测图；
图4为蠋蝽多肽Ach1的化学合成制备样品HPLC分析图；
图5为蠋蝽多肽Ach1对褐飞虱的注射毒杀活性图；
图6为蠋蝽多肽Ach1对褐飞虱的浸苗处理毒杀活性图；
图7为蠋蝽多肽Ach1对斜纹夜蛾的注射毒力图；
图8为蠋蝽多肽Ach1对人工饲喂法烟蚜的杀虫活性图；
图9为蠋蝽多肽Ach1对斑马鱼幼鱼急性毒性测定图。
图10为蠋蝽多肽Ach1对小鼠雌成鼠体重的影响图。
具体实施方式
实施例1：蠋蝽多肽Ach1的合成方法，包括以下步骤：
(1)蠋蝽多肽基因的设计和构建方法
解剖蠋蝽成虫毒腺50对，提取总RNA，三个重复样品，进行RNA-seq转录组测序分析，根据注释信息和BLAST来查找蠋蝽多肽基因，提取蠋蝽成虫总RNA，反转录获得cDNA模板，之后通过PCR扩增目的基因，进行测序验证。
在蠋蝽毒腺转录组数据中，将一个富含半胱氨酸的小肽基因命名为蠋蝽多肽Ach1。经基因克隆测序验证，该蠋蝽多肽Ach1基因的编码序列区域(CDS)的全长序列为
ATGTTGCTAATCAACAGAAGAGTAGGGAGAACACTTCTGAGAAGATCAACAAGACTTGAATACATATATATATGGTGGGGGCTCTGCGCTTTACTCAACACAGTCAGCAACATGAAGTTACACCTCGTACTCTTCCTCGTCATCGCAGTCTTGGCTTTCGGAGTCCTGGCTTTTCAAGAGCAGAAGGAACAGAACTGCATACCGAGGGGGAAAGAATGCGTAGGCCAAGATATGAGGTGCTGTGGGGGGTTGACGTGTATGTTCTACGCCAACAGGTGCGTTGGCGCTTGA。蠋蝽多肽Ach1的成熟肽序列为FQEQKEQNCIPRGKECVGQDMRCCGGLTCMFYANRCVGA。其与已知猎蝽毒肽Ptu1和蜘蛛毒肽Hv1a一样，都含有6个半胱氨酸组成的三个二硫键的knottin结构，但是氨基酸序列相似度较低(26％-39％)。(图1-2和表1)。
表1蠋蝽多肽Ach1和其它毒肽的氨基酸序列相似度
注：Hv1a:漏斗网蜘蛛P56207、Ptu1:乌黑盗猎蝽P58606.1、Pr1a：红斑猎蝽QHB21534.1、Ado1：多氏田猎蝽P58608.1、Iob1：褐菱猎蝽P58609.1、Pp1a：食蜂猎蝽AQM58372.1、Pp1b：食蜂猎蝽AQM58373.1
(2)蠋蝽多肽Ach1的化学合成
蠋蝽多肽Ach1(FQEQNCIPRGNECVAKELDCCDGMKCSHYANKCMT)，有效成分含量为94.69％，由强耀生物科技有限公司通过9-芴甲氧羰基(Fmoc)固相化学合成技术生产。保证二硫键定向连接，即N端第一个和第四个、第二个和第五个、第三个和第六个半胱氨酸分别形成二硫键(C1-C4,C2-C5和C3-C6)。
合成步骤如下：将Fmoc保护的C端第一个氨基酸与树脂连接；去除Fmoc保护基，茚三酮检测是否成功连接；加入第二个Fmoc保护氨基酸进行缩合，茚三酮检测；然后依次加入不同氨基酸进行连接直到最后一个氨基酸为止；最后加入切割液切除Fmoc和树脂；加入乙醚后离心和吹干；利用DMSO氧化法进行定向二硫键修改得到粗产物；利用高效液相色谱(HPLC)进行分离纯化，冻干得到成品。最后进行质谱确认(图3)和HPLC纯度鉴定(图4)。
试验例1：蠋蝽多肽Ach1对褐飞虱的毒力测定
(1)显微注射法测定蠋蝽多肽Ach1对褐飞虱的毒力
采用微量注射法测定了蠋蝽多肽Ach1不同浓度对褐飞虱4龄若虫的毒力。用DMSO将毒肽样品配制成母液，再用超纯水稀释成测试浓度溶液。利用显微注射仪(WPI公司)将毒肽溶液注射至褐飞虱4龄若虫的体内，注射部位为褐飞虱若虫第二节和第三节的节间膜。用含等量溶剂的水溶液作为对照。每头注射量为50nL，注射流速20nL/s。每头注射处理试虫转移至一个装有水稻幼苗的一次性培养皿中，每个处理10头，做三组重复。培养皿置在温度27±1℃、湿度80±10％和光暗周期16h:8h的培养室中。注射1d、2d、3d后观察试虫死亡情况。死亡症状判定标准为毛笔轻触无自主反应。
溶剂对照组(0.5％DMSO)5天死亡率为16.67％，Ach1多肽1ng/头(5μmol/L,50nL/头)及以上剂量5天死亡率为100％。以三氟苯嘧啶(TFM)0.2ng/Larva(10μmol/L,50nL/头)为阳性对照，其处理第2天死亡率为100％。该多肽毒杀效果很快速，1-2天内见效，第二天后的存活曲线基本稳定(图5)。Ach1多肽注射处理第一天的LD50值为0.316ng/头。表1展示了多次注射测试的LD50值(0.124-0.316ng/头)。结果表明蠋蝽多肽Ach1对褐飞虱有非常高的毒力。
表1蠋蝽多肽Ach1对褐飞虱4龄若虫的毒力(注射法)
(2)浸苗法测定蠋蝽多肽Ach1对褐飞虱的毒力
采用浸苗法测定了蠋蝽多肽Ach1不同浓度对褐飞虱3龄若虫的毒力。用含0.1％(m/v)乳化剂曲拉通的蒸馏水将母液配成测试浓度药液。用含等量乳化剂的蒸馏水溶液作为对照。选取用营养液培育的2周龄水稻苗(高10cm左右)，浸没到药液中30秒，然后在滤纸上自然晾干，直至无明水。将晾干的稻苗用湿棉花包住根部，放置在一次性塑料杯中。用吸虫管将10头3龄褐飞虱若虫转移到装置内，再放置在温度27±1℃、湿度80±10％和光暗周期16h:8h的培养室中。每个处理重复3次。处理1-7天内观察试虫死亡情况。死亡症状判定标准为毛笔轻触无自主反应。
溶剂对照组(0.5％DMSO)7天死亡率为20％，Ach1多肽80mg/L(20μmol/L)7天死亡率为56.67％。Ach1多肽浸苗处理第7天的LC50值为66.75mg/L。结果表明Ach1浸苗处理对褐飞虱有较高的毒力(见图6)。
表2蠋蝽多肽Ach1对褐飞虱3龄若虫的毒力(浸苗法)
试验例2：蠋蝽多肽Ach1对斜纹夜蛾的毒力测定
采用微量注射法测定了蠋蝽多肽Ach1对斜纹夜蛾2龄幼虫的毒力。利用显微注射仪将毒肽溶液分别注射至2龄斜纹夜蛾幼虫体内，注射部位为斜纹夜蛾幼虫中胸背部。用含等量溶剂的水溶液作为对照。每头注射量为100nL，注射流速20nL/s。每头注射处理试虫转移至一个装有烟草叶碟的培养皿(直径为10cm，倒入2％琼脂)。每个处理10头，三组重复共30头。温度27±1℃、湿度50％±10％和光照周期16:8(L:D)h的培养室中。注射1d、2d、3d后观察试虫的死亡情况。死亡症状判定标准为毛笔轻触无自主反应。
对照组(1％DMSO)8天死亡率为16.67％。Ach1多肽4ng/头(10μmol/L，100nL/头)处理8天死亡率为83.33％。结果表明蠋蝽多肽Ach1测试浓度对斜纹夜蛾毒力较高(见图7)。
试验例3：蠋蝽多肽Ach1对烟蚜的毒力测定
采用人工饲料喂饲法测定了蠋蝽多肽Ach1对烟蚜2龄若虫的毒力。用DMSO将毒肽原样配制成1mmol/L的毒肽母液，再用人工饲料稀释成测试浓度溶液。用1％DMSO的人工饲料溶液作为对照。处理试虫转移至一个装有烟草叶碟的培养皿(直径为10cm，皿底倒入2％琼脂)中。每隔一天更换一次人工饲料，每个重复10头，共3个重复。培养皿置在温度21±1℃、湿度50％±10％和光照周期16:8(L:D)h的培养室中。注射1d、2d、3d、4d和5d后观察试虫的死亡情况。死亡症状判定标准为毛笔轻触无自主反应。
溶剂对照组5天死亡率为30％。Ach1多肽40μmol/L处理5天死亡率为76.7％。结果表明蠋蝽多肽Ach1测试浓度对烟蚜毒力较高(见图8)。
试验例4：蠋蝽多肽Ach1对斑马鱼的急性毒性测定
采用静水法测定了蠋蝽多肽Ach1对斑马鱼幼鱼的急性毒性。首先利用DMSO将蠋蝽多肽Ach1配置成10000mg/L的母液。然后，利用蒸馏水将蠋蝽多肽Ach1母液稀释成测试浓度10mg/L，0.1％DMSO的水溶液为对照。吸取2mL溶液至48孔板的小孔中。挑取2月龄的健康幼鱼放入每个小孔中。每个处理重复10次，每个重复两尾。96h后观察并记录鱼的死亡情况(图9)。
对照组(0.1％DMSO)96h死亡率为0％。Ach1多肽10mg/L(2.5μmol/L)处理96h死亡率为0％。结果表明蠋蝽多肽Ach1测试浓度96h内对斑马鱼幼鱼无急性毒性。
试验例5：蠋蝽多肽Ach1对小鼠的急性毒性测定
采用灌胃法测定了蠋蝽多肽Ach1对小鼠雌成鼠的急性毒性。首先利用DMSO将蠋蝽多肽Ach1配置成10000mg/L的母液。然后，利用玉米油将蠋蝽多肽Ach1母液稀释成测试浓度500mg/L，5％DMSO的玉米油溶液为对照。将小鼠称重后在测试容器内适应三天，于3月13号14：00-15：00给每只小鼠灌胃不同的溶液，多肽处理组和对照组分别灌胃5只，每只小鼠灌胃200μL溶液。连续七天观察小鼠的各项指标，观察的时间在14：00-15：00。发现Ach1多肽100μg/只(500mol/L，200nL/只)灌胃处理组小鼠七天内均无死亡、未见异常外观变化、活动状态都很活跃、食欲和排泄情况也正常、口鼻眼处也未见异常分泌物、未见体重异常(图10)。表明蠋蝽Ach1多肽测试浓度7天内对小鼠雌成鼠无急性毒性。
以上所述，仅是本发明的较佳实施例而已，并非对本发明作任何形式上的限制，任何未脱离本发明技术方案内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰，均仍属于本发明技术方案的范围内。
Claims (5)
1.一种蠋蝽多肽Ach1,其基因序列为FQEQNCIPRGNECVAKELDCCDGMKCSHYANKCMT。
2.一种蠋蝽多肽Ach1，其具有6个半胱氨酸形成3个二硫键，二硫键的方式为C1-C4，C2-C5，C3-C6。
3.一种蠋蝽多肽Ach1在制备褐飞虱杀虫剂中的应用。
4.一种蠋蝽多肽Ach1在制备斜纹夜蛾杀虫剂中的应用。
5.一种蠋蝽多肽Ach1在制备烟蚜杀虫剂中的应用。
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