CN117089547B - 一种dsRNA在防治溴氰虫酰胺抗性烟粉虱中的应用 - Google Patents
一种dsRNA在防治溴氰虫酰胺抗性烟粉虱中的应用 Download PDFInfo
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Abstract
本发明提供了一种dsRNA在防治溴氰虫酰胺抗性烟粉虱中的应用，属于农药技术领域，所述dsRNA为dsDrosha、dsDicer1或dsAgo2A中的一种或多种；本发明发现并证实了miRNA合成路径中的关键基因drosha、dicer1和Ago2A与烟粉虱对溴氰虫酰胺的抗药性有关，当取食dsDrosha、dsDicer1或dsAgo2A后，烟粉虱体内的drosha、dicer1或Ago2A表达量下降，从而抑制烟粉虱体内miRNA的合成，降低了烟粉虱对溴氰虫酰胺的抗性。
Description
技术领域
本发明涉及农药技术领域，具体是一种dsRNA在防治溴氰虫酰胺抗性烟粉虱中的应用。
背景技术
烟粉虱属于半翅目粉虱科，是一种世界性农业害虫，可危害400多种植物，传播200多种植物病毒。溴氰虫酰胺作为第一个广谱性的双酰胺类杀虫剂，可以通过结合并激活昆虫体内的鱼尼丁受体，引起内质网Ca2+大量释放，致使细胞内外钙离子失衡，最终导致昆虫死亡，但目前烟粉虱已经对溴氰虫酰胺产生了明显的抗药性。
miRNA(microRNA)是内源性的非编码单链小RNA，主要通过参与基因的转录后调控，来实现对靶基因的表达调节。miRNA的功能具有多样性，可参与动植物的生长发育、机体疾病的发生和发展等。miRNA的生物合成是一个复杂的过程，miRNA基因在RNA聚合酶的作用下转录生成长度约为几千个碱基的初级转录本(pri-miRNA)，随后在drosha的作用下进一步被加工成长度为60-80个碱基且具有茎环结构的前体miRNA(pre-miRNA)，pre-miRNA被转运到细胞质中，细胞质中的dicer识别pre-miRNA，通过对茎环结构进行剪切和修饰，形成长度为20-24个碱基的双链miRNA；双链miRNA中的其中一条链被转载到RNA诱导沉默复合体(RISC)，最终生成成熟的、具有功能的单链miRNA，RISC中的Argonaute(Ago)将复合物引导至信使RNA(mRNA)并发挥其调节功能。
为了治理烟粉虱对溴氰虫酰胺杀虫剂的抗药性，本发明拟选择miRNA生物合成途径中的上述三个关键蛋白drosha、dicer和Ago来评估其在杀虫剂抗性中的作用，寻找新的溴氰虫酰胺抗性烟粉虱防治方法。
发明内容
针对上述问题，本发明发现并证实了miRNA合成路径中的关键基因drosha、dicer1和Ago2A与烟粉虱对溴氰虫酰胺的抗药性有关，当烟粉虱取食drosha、dicer1或Ago2A的dsRNA后，三个基因的表达水平降低，烟粉虱对溴氰虫酰胺抗性水平下降，为研发新的溴氰虫酰胺抗性烟粉虱防治方法奠定了良好的理论和应用基础。
本发明技术方案如下：
一种dsRNA在防治溴氰虫酰胺抗性烟粉虱中的应用，所述dsRNA为dsDrosha、dsDicer1或dsAgo2A中的一种或多种；其中，所述dsDrosha的核苷酸序列如SEQ ID NO.1所示，所述dsDicer1的核苷酸序列如SEQ ID NO.2所示，所述dsAgo2A的核苷酸序列如SEQ IDNO.3所示。
优选的，应用方法是通过对烟粉虱喂食dsDrosha、dsDicer1或dsAgo2A调控烟粉虱体内drosha、dicer1或Ago2A的表达量，从而抑制miRNA的合成。
优选的，所述喂食dsDrosha、dsDicer1或dsAgo2A的方法为：用含有dsDrosha、dsDicer1或dsAgo2A的蔗糖水喂食烟粉虱。
优选的，所述蔗糖水中蔗糖的浓度为0.17～0.23mg/L。
优选的，所述蔗糖水中蔗糖的浓度为0.2mg/L。
优选的，所述dsDrosha、dsDicer1或dsAgo2A在蔗糖水中的浓度为250ng/μL。
一种抑制生物体内miRNA合成的方法，是用含有dsDrosha、dsDicer1或dsAgo2A的蔗糖水对生物体进行喂食。
优选的，所述蔗糖水中蔗糖的浓度为0.17～0.23mg/L。
优选的，所述蔗糖水中蔗糖的浓度为0.2mg/L。
优选的，所述dsDrosha、dsDicer1或dsAgo2A在蔗糖水中的浓度为250ng/μL。
有益效果：
本发明发现并证实了miRNA合成路径中的关键基因drosha、dicer1和Ago2A与烟粉虱对溴氰虫酰胺的抗药性有关，当烟粉虱取食drosha、dicer1或Ago2A的dsRNA后，可降低烟粉虱体内drosha、dicer1或Ago2A的表达水平，进而抑制烟粉虱体内miRNA的合成，最终降低烟粉虱对溴氰虫酰胺的抗性，为研发新的溴氰虫酰胺抗性烟粉虱防治方法奠定了良好的理论和应用基础。
附图说明
图1为dsDrosha、dsDicer1和dsAgo2A产物的琼脂糖凝胶电泳图；
图2为喂食dsDrosha、dsDicer1、dsAgo2A后烟粉虱体内相应基因的表达量变化；
图3为喂食dsDrosha、dsDicer1、dsAgo2A后烟粉虱体内miR-14-3p、miR-100-5p、miR-277-3p和miR-305-5p的表达量变化。
具体实施方式
实验材料来源：
Trizol：购自赛默飞世尔科技公司；
PrimeScriptTMII 1st Strand cDNA Synthesis Kit：购自宝日医生物技术公司；
ApexHF HSDNA Polymerase：购自艾科瑞生物公司；
SteadyPure DNA凝胶回收试剂盒：购自艾科瑞生物公司；
TranscriptAid T7 High Yield Transcription Kit：购自赛默飞世尔科技公司；
Trizol：购自赛默飞世尔科技公司；
PrimeScriptTMRT reagent Kit with gDNA eraser：购自宝日医生物技术公司；
Mir-XTMmiRNA First-Strand Synthesis Kit：购自宝日医生物技术公司；
溴氰虫酰胺(94％原药)：购自富美实公司；
荧光定量PCR仪：购自伯乐公司，型号184-5096；
实施例中涉及的引物均委托擎科生物公司合成。
实施例中涉及的溴氰虫酰胺汰选抗性种群SG19抗性水平和实验室敏感种群QS抗性水平如下表1所示：
表1.烟粉虱种群抗性水平
下面结合具体实施例进行说明：
实施例1
一、合成dsRNA
(1)采用Trizol法提取SG19抗性种群总RNA，使用PrimeScriptTMII 1st StrandcDNA Synthesis Kit合成第一链cDNA；使用ApexHF HSDNA Polymerase分别扩增drosha、dicer1和Ago2A的基因片段作为合成dsDrosha、dsDicer1和dsAgo2A的DNA模板，使用SteadyPure DNA凝胶回收试剂盒回收目的片段并对回收产物克隆测序验证；
其中，扩增drosha基因片段所用的引物分别如SEQ ID NO.4～5所示，扩增dicer1基因片段所用的引物分别如SEQ ID NO.6～7所示，扩增Ago2A基因片段所用的引物分别如SEQ ID NO.8～9所示；
其中，PCR扩增体系(50μL)为：ApexHF HSDNA Polymerase：25μL；Primer-F：1μL；Primer-R：1μL；模板(drosha、dicer1或Ago2A)：2μL；ddH2O：19μL；
PCR扩增程序为：94℃预变性1min；94℃变性15s，55℃退火15s，72℃延伸10s，共35个循环；72℃，1min。
(2)利用步骤(1)合成的DNA模板，使用TranscriptAid T7 High YieldTranscription Kit分别合成drosha、dicer1和Ago2A的双链RNA(dsRNA)，得dsDrosha、dsDicer1和dsAgo2A；
其中，dsRNA合成体系为：5×TranscriptAid Reaction Buffer 5μL，核糖核苷酸(A\G\C\U,100mM)各2μL，DNA模板1μg，无酶水补足20μL，充分混匀；
将上述dsRNA合成体系在37℃孵育4h，孵育结束后，加入2μL DNase I，在37℃水浴中孵育15min；加入2μL 0.5M EDTA，在65℃孵育10min；加无酶水补足到500μL，再加入200μL氯仿轻轻混匀，静置10min；12000g，4℃离心15min，取上清；重复加氯仿，离心，取上清；加入上清液1/10体积的醋酸钠(3M，PH 5.2)和上清液2.5倍体积的乙醇，在-80℃中放置2.5h；12000g，4℃离心30min，弃上清，加入75％乙醇1mL，重悬沉淀；10000g，4℃离心10min，弃上清，待管中剩余的乙醇干燥后，加入200μL无酶水溶解，得dsDrosha、dsDicer1和dsAgo2A产物，对产物进行琼脂糖凝胶电泳，结果如图1所示；
同时，合成绿色荧光蛋白(GFP)的dsRNA(dsGFP)作为对照组，用于后续的RNA干扰(RNAi)实验。
二、RNAi
将dsDrosha、dsDicer1、dsAgo2A和dsGFP分别溶解于20％(0.2mg/L)蔗糖水中，使各dsRNA的终浓度为250ng/μL，得饲喂液；将饲喂液分别加入饲喂装置，所述饲喂装置由50mL离心管制成，管的四周及底部被防虫网封闭，顶端由离心管盖密封；通过聚四氟乙烯膜将饲喂液与约500只三日龄SG19种群的烟粉虱成虫分离，烟粉虱口器可刺穿聚四氟乙烯膜，吸取膜内侧的饲喂液；饲喂72小时后，收集存活下来的烟粉虱进行后续的实时荧光定量PCR(qPCR)实验和生物测定实验。
三、qPCR
(1)通过qPCR法检测存活烟粉虱体内Drosha、Dicer1和Ago2A的表达水平：
分别用Trizol法提取存活烟粉虱总RNA，然后用PrimeScriptTMRT reagent Kitwith gDNA eraser反转录mRNA，反转录按照试剂盒说明书进行；利用琥珀酸脱氢酶复合物A(SDHA)和热休克蛋白40(HSP40)作为mRNA表达分析的两个内参基因；每个处理进行三次生物学重复，采用2-ΔΔCt法分析RNAi后烟粉虱体内相关基因的相对表达情况；
其中，qPCR反应体系(20μL)如下：TB Green Advantage Premix：10μL；Primer-F：0.5μL；Primer-R：0.5μL；cDNA：2μL；ddH2O：7μL；
qPCR扩增程序如下：95℃预变性30s；95℃变性5s，60℃退火及延伸34s，40个循环；
其中，检测drosha所用的上、下游引物分别如SEQ ID NO.10～11所示；检测dicer1所用的上、下游引物分别如SEQ ID NO.12～13所示，检测dicer2所用的上、下游引物分别如SEQ ID NO.14～15所示；检测Ago2A所用的上、下游引物分别如SEQ ID NO.16～17所示，检测Ago2B所用的上、下游引物分别如SEQ ID NO.18～19所示，检测Ago1所用的上、下游引物分别如SEQ ID NO.20～21所示；
各基因的qPCR检测结果如图2所示；由图2中的A图可得，当烟粉虱取食dsDrosha72小时后，烟粉虱体内drosha的表达量显著降低至对照组的0.76倍；由图2中的B图可得，当烟粉虱取食dsDicer1后，dicer1的表达量显著降低至对照组的0.67倍，而dicer2的表达量与对照组相比变化不大；由图2中的C图可得，当烟粉虱取食dsAgo2A后，Ago2A的表达量显著降低至对照组的0.63倍，而Ago2B和Ago1的表达量与对照组相比变化不大；上述结果表明，饲喂靶标基因dsRNA 72h后成功敲低了烟粉虱体内drosha、dicer1和Ago2A的表达水平，且没有发生脱靶效应。
(2)以4条烟粉虱体内高表达的miRNA(miR-14-3p、miR-100-5p、miR-277-3p和miR-305-5p)为例，验证miRNA生物合成路径关键基因敲低后，miRNA表达水平的变化：
将上述获得的烟粉虱总RNA用Mir-XTMmiRNA First-Strand Synthesis Kit反转录miRNA，反转录按照试剂盒说明书进行；利用U6基因作为miRNA表达分析的内参基因；每个处理进行三次生物学重复，采用2-ΔΔCt法分析drosha、dicer1和Ago2A被敲低后烟粉虱体内miR-14-3p、miR-100-5p、miR-277-3p和miR-305-5p的相对表达情况；
其中，qPCR反应体系(20μL)如下：TB Green Advantage Premix：10μL；Primer-F：0.5μL；Primer-R：0.5μL；cDNA：2μL；ddH2O：7μL；
qPCR扩增程序如下：95℃预变性10s；95℃变性5s，60℃退火及延伸20s，40个循环；
其中，检测miR-14-3p、miR-100-5p、miR-277-3p和miR-305-5p所用的上游引物序列分别如SEQ ID NO.22～25所示，下游引物采用Mir-XTMmiRNA First-Strand SynthesisKit中自带的通用引物mRQ3’Primer；
qPCR检测结果如图3所示，当烟粉虱体内的drosha、dicer1和Ago2A分别被敲低后，烟粉虱体内miR-14-3p的表达水平降低至对照组的0.67-0.73倍，miR-100-5p的表达水平降低至对照组的0.48-0.77倍，miR-277-3p的表达水平降低至对照组的0.62-0.71倍，miR-305-5p的表达水平降低至对照组的0.67-0.73倍；上述结果表明，drosha、dicer1和Ago2A基因参与烟粉虱体内miRNA的合成，当三条基因表达水平被敲低后，明显抑制了烟粉虱体内miRNA的合成。
四、验证miRNA合成路径关键基因敲低后，烟粉虱对溴氰虫酰胺的抗性水平变化
通过琼脂保湿浸叶法获得RNAi后溴氰虫酰胺对烟粉虱抗性种群(SG19)的致死中浓度：首先将1.0638g的溴氰虫酰胺(94％原液)溶解在二甲基亚砜(DMSO)中配制成20mL、5×104的母液，然后用0.5‰的Triton X-100溶液(溶剂为去离子水)将母液梯度稀释至五个浓度，稀释后的浓度分别为120mg/L、60mg/L、30mg/L、15mg/L、7.5mg/L，并设置0.5‰的Triton X-100溶液作为空白对照组；将棉花真叶切成直径为2.5cm的圆叶片，将圆叶片分别在上述不同浓度的溴氰虫酰胺和空白对照组中浸泡10s；浸泡后将圆叶片拿出在空气中干燥15min，然后背面朝下放置在50mL离心管盖内的1.0％(0.01g/mL)琼脂床上，用于喂食烟粉虱；离心管的一侧由防虫网封闭，另一侧由上述含有圆叶片的离心管盖密封，在离心管中放置15头饲喂dsRNA的存活下来的SG19烟粉虱成虫；48h后统计烟粉虱死亡率，将浓度与死亡率输入PoloPlus分析数据，得LC50及其95％置信区间；
RNAi后的溴氰虫酰胺对烟粉虱的毒力测定结果如表2所示，与饲喂dsGFP组(对照组)相比，SG19种群取食dsDrosha、dsDicer1和dsAgo2A后，抗性水平均出现明显下降；其中，dsGFP组的LC50值分别是饲喂dsDrosha组、饲喂dsDicer1组和饲喂dsAgo2A组的3.7倍、1.8倍和2.6倍；由于饲喂dsGFP组的95％置信区间与dsdrosha或dsAgo2A之间没有重叠，表明与对照组的LC50值相比，敲低drosha或Ago2A后SG19烟粉虱种群对溴氰虫酰胺的LC50显著降低，即对溴氰虫酰胺更敏感。
表2.烟粉虱对溴氰虫酰胺的抗性水平测定结果
综上，本发明发现，当烟粉虱取食dsDrosha、dsDicer1或dsAgo2A后，dsDrosha、dsDicer1或dsAgo2A能够与烟粉虱体内的基因drosha、dicer1或Ago2A结合，导致drosha、dicer1或Ago2A表达量下降，从而抑制烟粉虱体内miRNA的合成，降低了烟粉虱对溴氰虫酰胺的抗性。
Claims (5)
1.一种dsRNA在防治溴氰虫酰胺抗性烟粉虱中的应用，其特征在于，所述dsRNA为dsDrosha、dsDicer1或dsAgo2A中的一种或多种；其中，所述dsDrosha的核苷酸序列如SEQID NO.1所示，所述dsDicer1的核苷酸序列如SEQ ID NO.2所示，所述dsAgo2A的核苷酸序列如SEQ ID NO.3所示；
其中，应用方法是通过对烟粉虱喂食dsDrosha、dsDicer1或dsAgo2A抑制烟粉虱体内drosha、dicer1或Ago2A的表达量，从而抑制miRNA的合成。
2.如权利要求1所述的应用，其特征在于，所述喂食dsDrosha、dsDicer1或dsAgo2A的方法为：用含有dsDrosha、dsDicer1或dsAgo2A的蔗糖水喂食烟粉虱。
3.如权利要求2所述的应用，其特征在于，所述蔗糖水中蔗糖的浓度为0.17～0.23mg/L。
4.如权利要求2所述的应用，其特征在于，所述蔗糖水中蔗糖的浓度为0.2mg/L。
5.如权利要求2所述的应用，其特征在于，所述dsDrosha、dsDicer1或dsAgo2A在蔗糖水中的浓度为250ng/μL。
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