CN110754471B - Amep412蛋白对白粉虱的杀虫活性及其在防治白粉虱中的应用 - Google Patents
Amep412蛋白对白粉虱的杀虫活性及其在防治白粉虱中的应用 Download PDFInfo
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Abstract
本发明涉及一种枯草芽孢杆菌蛋白AMEP412在防治白粉虱中的应用。本发明发现了AMEP412蛋白对白粉虱的杀虫活性及其在防治白粉虱中的应用，该蛋白能够以低浓度杀伤白粉虱，且具有耐热和耐自然降解的稳定性，能够被白粉虱摄入肠道内，但并不抑制肠道内蛋白酶活性；此外，在温室内喷施AMEP412对白粉虱也取得了良好的防效；本发明为白粉虱的生物防治工作积累了新的材料。
Description
技术领域
本发明属于植物保护与生物防治领域，涉及一种AMEP412蛋白对白粉虱的杀虫活性及其在防治白粉虱中的应用。
背景技术
白粉虱(Bemisia tabaci)是蔬菜、棉花、观赏植物等多种农作物的重要害虫。它通过取食植物茎叶汁液和传播植物病毒来损害作物，导致农业生产和国民经济的巨大损失。防治白粉病的主要策略是以化学杀虫剂为主。然而，白粉虱对那些过度和频繁使用的杀虫剂产生了抗药性。有鉴于此，探索新型杀虫剂应成为新的研究热点。
与化学杀虫剂相比，具有杀虫活性的蛋白质因为作用方式的不同，导致抗虫的概率相对较低，成为了目前杀虫剂研究的热点。关于杀虫蛋白在害虫防治中的应用已有许多报道。最著名的例子是Bacillus thuringiensis(Bt)的Cry毒素，它被开发成Bt转基因棉花并有效地控制鳞翅目害虫幼虫。转Bt基因棉花的广泛种植大大减少了化学农药的使用。然而，Cry毒素对白粉虱无效。近年来，一些研究者致力于从很少被白粉虱侵染的植物中筛选杀虫蛋白。Das等人于2009年报道了一种从大蒜叶中提取的甘露糖结合凝集素，能够有效地抑制白粉虱。Jin等人于2012年将半夏的凝集素在叶绿体中表达，并证明其具有抗白粉虱的能力。Shukla等人于2016年从一种食用蕨类植物中鉴定出一种杀虫蛋白(Tma12)，并在转基因棉花中表达，该蛋白对白粉虱表现出一定的抗性。尽管这些来自于植物的杀虫蛋白具有一定的防治白粉虱的潜力，但由于提取率低和转基因的应用受限，目前并没有广泛应用。
在之前的研究中，我们从枯草芽孢杆菌BU412中分离鉴定了蛋白AMEP412，能够激发植物的防卫反应，提高植物的抗病性，还能够对疮痂链霉菌产生拮抗作用，是开发为生物农药的理想候选。在本发明中，我们旨在提供AMEP412蛋白的新用途。
发明内容
本发明的目的是提供一种枯草芽孢杆菌蛋白AMEP412的新用途，克服目前针对白粉虱的杀虫蛋白太少的问题，以及解决白粉虱的绿色防控的问题。
本发明通过以下技术方案来实现：一种枯草芽孢杆菌蛋白AMEP412在防治白粉虱中的应用。
上述的枯草芽孢杆菌蛋白AMEP412，其氨基酸序列如SEQ ID NO:1所示。
进一步的，所述的AMEP412蛋白饲喂白粉虱成虫后，达到半数致死量所需蛋白浓度为15.57μg/ml，达到90％的致死率所需蛋白浓度为63.27μg/ml。
进一步的，所述的AMEP412蛋白能够被白粉虱成虫摄食进入体内，集中定位于肠道内。
进一步的，所述的AMEP412蛋白具有耐热稳定性。
进一步的，所述的AMEP412蛋白具有对自然降解的稳定性。
进一步的，所述的AMEP412蛋白在温室内喷施浓度为100μg/ml，2天后白粉虱的致死率为56％。
首先使用不同浓度梯度的AMEP412蛋白对白粉虱成虫进行人工饲喂，计算半数致死率(LC50)和90％致死率所需的蛋白浓度。随后将AMEP412蛋白进行热处理和自然降解处理，检测处理后蛋白样品对白粉虱成虫的杀虫活性，确定AMEP412蛋白的稳定性。使用FITC标记AMEP412蛋白，饲喂白粉虱成虫后，使用荧光显微镜观察白粉虱体内荧光分布，确定AMEP412在白粉虱体内的定位。测试AMEP412对胰蛋白酶的抑制作用，排除其通过抑制白粉虱肠道内蛋白酶活性来杀伤白粉虱的可能。
采用上述技术方案的积极效果：本发明发现了AMEP412蛋白对白粉虱的杀虫活性及其在防治白粉虱中的应用，该蛋白能够以低浓度杀伤白粉虱，且具有耐热和耐自然降解的稳定性，能够被白粉虱摄入肠道内，但并不抑制肠道内蛋白酶活性；此外，在温室内喷施AMEP412对白粉虱也取得了良好的防效；本发明为白粉虱的生物防治工作积累了新的材料。
附图说明
图1为不同浓度的AMEP412蛋白对白粉虱成虫的致死率，相同的字母表示差异不显著，不同的字母表示处理间差异显著(α＝5％)；
图2为经过热处理和自然降解的AMEP412蛋白对白粉虱成虫的致死率，CK表示未添加AMEP412蛋白，Untreated表示未进行处理的AMEP412蛋白，相同的字母表示差异不显著，不同的字母表示处理间差异显著(α＝5％)；
图3为FITC标记的AMEP412在白粉虱成虫体内的荧光定位，腹腔内的亮斑为激发的绿色荧光，显示AMEP412定位在肠道内。
具体实施方式
以下通过实施例来进一步描述本发明，应该理解的是，这些实施例仅用于例证的目的，绝不限制本发明的范围。
本发明中生物材料的来源：
1、所用的枯草芽孢杆菌(Bacillus subtilis)BU412于2016年3月30日在中国典型培养物保藏中心进行保藏，保藏编号为CCTCC M 2016142；
2、AMEP412蛋白：专利申请号201810928176.1，专利名称“一种枯草芽孢杆菌AMEP412蛋白的新功能及其抗菌肽的鉴定”，申请日为20180814，公开日为20181228，公开号为CN109096379A。
实施例1
本实施例说明枯草芽孢杆菌AMEP412蛋白人工饲喂白粉虱的杀虫效果。
饲喂用的液体人工饵料由5％酵母提取物和30％蔗糖溶于蒸馏水后灭菌，添加不同浓度的AMEP412(1、5、10、20、40和80μg/ml)，混合备用。以不含AMEP412的人工饵料为对照。将1-2日龄的白粉虱成虫从植物叶片上驱赶至50ml的试管中，每个试管中至少取50只白粉虱成虫。在试管管帽上覆盖两层拉伸的封口膜，将饵料(100μl)添加至两层膜之间，然后将管帽反向盖住管帽，使双层膜结构保持在试管内侧，并向上垂直放置。每组实验重复三次。饲喂2天后进行死亡率的测定，通过在试管底部计数已死亡的白粉虱成虫来记录死亡率。采用单因素方差分析(ANOVA)对数据进行分析，并采用Tukey's-HSD试验(α＝5％)时进行比较。通过SPSS程序的probit分析计算半数致死浓度(LC50)和90％致死浓度。
饲喂2天后统计白粉虱死亡率，如图1所示。随着AMEP412蛋白浓度的增加，白粉虱死亡率逐渐提升，呈正相关趋势。AMEP412蛋白浓度为0、1、5、10、20、40、80mg/ml时，所对应的白粉虱死亡率分别为7.2％、17.4％、31.0％、47.9％、65.5％、82.7％和95.6％。经软件计算，达到半数致死率所需的AMEP412蛋白浓度为15.57μg/ml(表1)。此外，达到90％致死率所需的AMEP412蛋白浓度为63.27μg/ml。这些结果说明AMEP412蛋白具有对白粉虱成虫的杀虫活性，且其所需的半数致死浓度较低，说明其杀虫活性较强。
表1 AMEP412蛋白的半数致死浓度计算结果
实施例2
本实施例说明枯草芽孢杆菌AMEP412蛋白的稳定性实验。
为了测试AMEP412蛋白的耐热性，将蛋白样品在95℃分别处理15分钟和30分钟。冷却后的蛋白样品按照实施例1中方法制备人工饵料，调整终浓度为60μg/ml进行白粉虱的杀虫效果测试。以未处理的蛋白样品作为对照，每个处理重复三次。为了测试AMEP412蛋白对自然降解的稳定性，将蛋白样品放置在1.5ml离心管中在室温25℃分别静置处理24小时和48小时。之后蛋白样品按照实施例1中方法制备人工饵料，调整终浓度为60μg/ml进行白粉虱的杀虫效果测试。以未处理的蛋白样品作为对照，每个处理重复三次。以上白粉虱的致死率数据使用单因素方差分析，使用Tukey’s HSD检验(α＝5％)进行比较。
结果显示(图2)，与未进行热处理的AMEP412蛋白相比，AMEP412蛋白经95℃处理15分钟和30分钟，对白粉虱的致死率分别下降了0.57％和0.49％，差异不显著。说明AMEP412具有良好的热稳定性。与未进行自然放置处理的AMEP412蛋白相比，AMEP412蛋白经过25℃自然放置24小时后，对白粉虱的致死率下降了2.73％，差异不显著。而AMEP412蛋白经过25℃自然放置48小时后，对白粉虱的致死率下降了16.38％。虽然达到了显著差异，但是致死率仍然有70％，故认为AMEP412蛋白具有良好的对自然降解的稳定性。这些结果说明AMEP412具有对热的稳定性和对自然降解的稳定性，有利于在高温环境施用，并延长其作用时间。
实施例3
本实施例说明枯草芽孢杆菌AMEP412蛋白在白粉虱体内的荧光定位。
将AMEP412蛋白与荧光素FITC进行孵育，通过分子筛色谱层析依据分子体积差异将未与AMEP412蛋白结合的FITC除去，得到FITC标记的AMEP412蛋白。随后将FITC标记的AMEP412蛋白饲喂白粉虱成虫。取死亡的白粉虱成虫用蒸馏水清洗后使用Olympus BX60荧光显微镜在495nm的波长下激发绿色荧光，进行荧光显微观察。结果显示(图3)，绿色荧光集中在白粉虱成虫腹腔的肠道内，说明AMEP412蛋白能够被白粉虱成虫摄入体内，并定位于肠道内。这个结果提示我们AMEP412发挥杀虫作用的部位在肠道内。
实施例4
本实施例说明枯草芽孢杆菌AMEP412蛋白对胰蛋白酶活性的影响。
一些杀虫蛋白的作用机制是作为蛋白酶抑制剂，通过抑制昆虫肠道内的丝氨酸蛋白酶(如胰蛋白酶)的活性，使昆虫无法消化摄入的食物导致死亡。为了检测枯草芽孢杆菌AMEP412蛋白是否作为蛋白酶抑制剂达到杀虫效果，本实施例检测了AMEP412蛋白对胰蛋白酶活性的影响。
胰蛋白酶的活性试验是在检测胰蛋白酶对底物N-α-苯甲酰-DL-精氨酸-对硝基苯胺(BAPNA)进行催化产生产物的基础上进行的。BAPNA在405nm处有特殊的吸收。对胰蛋白酶的活性影响可以通过测定吸光度的变化来确定。将0.2ml胰蛋白酶溶液(0.1mg/ml)、0.2mlAMEP412蛋白(0.1mg/ml)和1.6ml缓冲液(0.2M Tris HCl，pH7.8)在反应杯中孵育3分钟。酶促反应通过添加1ml底物(1mg/ml BAPNA)开始。然后立即混合溶液，每分钟记录405nm处吸光度的增加，持续5分钟。以不加AMEP蛋白作为对照。每次处理重复三次。抑制率的计算如下：抑制率(％)＝(AMEP412处理ΔA405nm/分钟-对照ΔA405nm/分钟：)*100/对照ΔA405nm/分钟。
结果显示(表2)，AMEP412蛋白对胰蛋白酶的抑制率为2.97％，无显著差异。说明AMEP412蛋白对胰蛋白酶活性无抑制作用，提示AMEP412的杀虫活性不是蛋白酶抑制剂的作用机制。
表2 AMEP412蛋白对胰蛋白酶活性的影响
实施例5
本实施例说明枯草芽孢杆菌AMEP412蛋白在温室中对白粉虱的防控效果。
在温室内种植番茄植株至约50cm高，将三株番茄作为一个处理，使用纱网罩住，每个网内至少接入200只1-2日龄的白粉虱成虫。将纯化后的激发子AMEP412蛋白样品稀释至100μg/ml进行喷施处理，以缓冲液作为对照，每个处理重复3次。喷施后2天统计白粉虱死亡率。
结果显示(表3)，100μg/ml的AMEP412蛋白喷施两天后，白粉虱死亡率为56.2％，具有极显著差异(α＝1％)。虽然与饲喂实验相比较，死亡率有所下降，但考虑在实际环境中的各种因素影响，能达到50％以上的抑制率是比较理想的。因此，枯草芽孢杆菌AMEP412蛋白在温室中对白粉虱起到了良好的防控效果。
表3 AMEP412蛋白对温室内白粉虱的防治效果
本发明发现了枯草芽孢杆菌蛋白AMEP412的对白粉虱成虫的杀虫活性，并进一步确认了该蛋白对热处理和自然降解的稳定性，检测了该蛋白在白粉虱体内的定位，确认了其对昆虫细胞的裂解作用。进一步的，本发明检测了该蛋白在温室内对白粉虱的防控效果，为生物防治工作积累了新的材料和经验。
序列表
<110> 黑龙江八一农垦大学
<120> AMEP412蛋白对白粉虱的杀虫活性及其在防治白粉虱中的应用
<141> 2019-12-02
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 76
<212> PRT
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 1
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Pro Trp Gly Lys Val Ala Ser Phe Leu Lys Trp Ala Gly Asn Leu Ala
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Ala Ala Ala Ala Lys Tyr Ser Tyr Thr Ser Gly Lys Lys Ile Leu Ala
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Tyr Ile Gln Lys His Pro Gly Lys Ile Val Asp Trp Phe Leu Lys Gly
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Tyr Ser Val Tyr Asp Val Ile Lys Met Ile Leu Gly
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Claims (6)
1.一种氨基酸序列如SEQ ID NO:1所示的枯草芽孢杆菌蛋白AMEP412在防治白粉虱中的应用。
2.根据权利要求1所述的应用，其特征在于：所述的AMEP412蛋白饲喂白粉虱成虫后，达到半数致死量所需蛋白浓度为15.57 µg/ml，达到90%的致死率所需蛋白浓度为63.27 µg/ml。
3.根据权利要求1所述的应用，其特征在于：所述的AMEP412蛋白能够被白粉虱成虫摄食进入体内，集中定位于肠道内。
4.根据权利要求1所述的应用，其特征在于：所述的AMEP412蛋白具有耐热稳定性。
5.根据权利要求1所述的应用，其特征在于：所述的AMEP412蛋白具有对自然降解的稳定性。
6.根据权利要求1所述的应用，其特征在于：所述的AMEP412蛋白在温室内喷施浓度为100µg/ml，2天后白粉虱的致死率为56%。
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