CN106282183B - 一种miRNA模拟物及几丁质合成抑制类生物农药及其应用 - Google Patents
一种miRNA模拟物及几丁质合成抑制类生物农药及其应用 Download PDFInfo
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Abstract
本发明提供了一种miRNA模拟物，以及几丁质合成抑制类生物农药，所述miRNA模拟物的核苷酸序列如SEQ ID NO：1所示。本发明的miRNA模拟物及几丁质合成抑制类生物农药具有低剂量高效率的优点，杀虫效果显著。
Description
技术领域
本发明属于昆虫控制领域，具体涉及一种miRNA模拟物及几丁质合成抑制类生物农药，以及该生物农药的应用。
背景技术
几丁质是N-乙酰氨基葡萄糖的多聚糖，广泛存在于真菌和节肢动物中，是昆虫外骨骼、气管与中肠围食膜(peritrophic matrix，PM)的重要组分(Cohen,2001；Merzendorfer,2006)。昆虫几丁质的生物合成通路以海藻糖为起点，经过8个酶的参与形成几丁质(Candy等，1962)。目前研究最多的是海藻糖酶(Trehalase,Tre)和几丁质合成酶(Chitin synthase，CHS)，即几丁质合成的第一个和最后一个酶(Zhu等，2016)。昆虫通常存在2个几丁质合成酶基因，分别为几丁质合成酶A基因(chitin synthetase A，CHSA)和几丁质合成酶B基因(chitin synthetase B，CHSB)(Zhu等，2016)。几丁质合成酶A基因主要在表皮和气管中表达，负责表皮和气管几丁质的合成；而几丁质合成酶B基因仅在中肠中表达，负责中肠围食膜几丁质的合成(Arakane等，2004；Merzendorfer和Zimoch，2003；Qu和Yang，2011)。在半翅目昆虫中，如褐飞虱(Nilaparvata lugens)、灰飞虱(Laodelphaxstriatellus)、长红猎蝽(Rhodnius prolixus)仅克隆得到几丁质合成酶A(Mansur等，2014；Wang等，2012b)。因此几丁质合成酶A对于半翅目昆虫的生长发育和繁殖有着不可或缺的作用。
通过基因沉默技术抑制昆虫几丁质合成相关基因的表达，有望对害虫几丁质合成进行调控，进而控制其危害。RNAi技术指通过导入dsRNA或者siRNA，使得内源的靶标mRNA被切割降解，在细胞、组织或者活体中实现转录后水平基因沉默的手段(Flores-Jasso等，2004；Sharp，1999；Valdes等，2003)。通过RNAi干涉几丁质合成酶A的研究已经在冈比亚蚊(Anopheles gambiae)、亚洲玉米螟(Ostrinia furnacalis)、甜菜夜蛾(Spodopteraexigua)、赤拟谷盗(Tribolium castaneum)、褐飞虱等9种昆虫中有所报道。完全变态昆虫中，通过RNAi沉默CHSA，会导致幼虫-幼虫、幼虫-蛹和蛹-成虫的转变障碍；半变态昆虫中，沉默几丁质合成酶A会导致若虫-若虫和若虫-成虫的蜕皮障碍，不仅显著降低虫体的几丁质含量，也会导致其死亡率显著升高(Chen等，2008；Wang等，2012b)。
除了外源dsRNA介导的RNAi之外，microRNA(miRNA)介导的基因沉默也有作为生物农药的潜力。miRNA是一类生物内源的单链非编码小RNA(non-coding small RNA)，长度大约为21-24nt。在动植物中，miRNA在转录后水平对基因表达起到关键调控作用，其主要的作用方式是与靶基因mRNA靶标区域的配对，引导沉默复合体(RISC)降解靶标mRNA或阻止其翻译(Carrington和Ambros，2003；Smibert和Lai，2008)。miRNA参与了动植物的生理活动，在细胞分化、凋亡、增殖，生长发育调节和免疫应答等方面均具有重要功能(Carrington和Ambros，2003)。
由于CHSA在昆虫生长发育中的重要性和几丁质存在的特殊性，以几丁质合成酶为靶标的农药非常适合应用到害虫控制中。此类化学农药不仅有核苷肽类化合物，如尼克霉素(nikkomycins)类化合物，还有苯甲酰基脲类化合物(benzoylphenylurea，BPU)，如已成功商品化的Diflubenzuron(DFB)和Lufenuron(LFN)(Gangishetti等，2009)。这些杀虫剂的重要优点是对环境安全友好。但由于昆虫抗药性的增加，以及新型结构研发难度大，这极大的限制了此类杀虫剂的使用。目前的褐飞虱防治方法包括农业防治、化学防治和生物防治等。农业防治方法效率低下，防治效果差，而化学药物往往导致昆虫的抗性作用以及环境污染等问题，使得褐飞虱防治工作难以得到持续性发展。
发明内容
本发明的目的在于克服现有技术的缺点和不足，提供一种能够有效防治昆虫(特别是褐飞虱)的几丁质合成抑制类生物农药。
本发明的另一个目的是提供所述几丁质合成抑制类生物农药在制备用于防治昆虫的药物中的应用。
本发明的又一个目的是提供含有所述几丁质合成抑制类杀虫剂的组合物。
为了实现上述目的，本发明提供了一种miRNA模拟物，其核苷酸序列如SEQ ID NO：1所示。优选地，合成所述miRNA模拟物的正向引物的核苷酸序列如SEQ ID NO：2所示，合成所述miRNA模拟物的反向引物的核苷酸序列如SEQ ID NO：3所示。
本发明还提供了一种几丁质合成抑制类生物农药，包括作为活性成分的miRNA模拟物，所述miRNA模拟物的核苷酸序列如SEQ ID NO：1所示。优选地，合成所述miRNA模拟物的正向引物的核苷酸序列如SEQ ID NO：2所示，反向引物的核苷酸序列如SEQ ID NO：3所示。
本发明还提供了所述miRNA模拟物或所述几丁质合成抑制类生物农药在制备用于防治昆虫的药物中的应用。优选地，所述昆虫为褐飞虱。
本发明还提供了含有所述miRNA模拟物或所述几丁质合成抑制类生物农药的组合物。特别地，该组合物为昆虫饲料。
优选地，所述miRNA模拟物在所述组合物中的浓度为0.02-0.1μg/μL，最优选为0.1μg/μL。
与现有防治昆虫(特别是褐飞虱)的主要办法相比，本发明通过利用miRNA模拟物作为活性成分，进行昆虫(特别是褐飞虱)的生物防治，具有低剂量高效率的优点，杀虫效果显著。
附图说明
图1是饲喂miRNA模拟物后褐飞虱的存活率图。
图2是饲喂miRNA模拟物后褐飞虱的表型变化图。
图3是饲喂miRNA模拟物后褐飞虱的几丁质含量变化图。
图4是注射miRNA模拟物后褐飞虱的后代孵化率图。
图5是注射miRNA模拟物后褐飞虱的卵巢变化图。
图6是注射miRNA模拟物后褐飞虱的CHSA蛋白含量变化图。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。如未特别指出，本发明实施例中所使用的试剂均可通过商业渠道获得。如未特别说明，本发明实施例中所使用的实验技术手段均为本领域技术人员所知晓，故不在此一一赘述其细节。
为便于说明，本发明将所涉及的miRNA模拟物命名为“miR-2703”(下文以该名称指代)，其核苷酸序列如SEQ ID NO：1所示。
实施例1
步骤1：根据核苷酸序列(SEQ ID NO：1)合成miR-2703的模拟物(委托广州锐博公司合成)。
miR-2703模拟物为化学合成的成熟miRNA双链，能增强内源性miRNA功能，包括一条与目标miRNA成熟体序列一致的序列(如SEQ ID NO：1所示，5’-TAGCTTTGATCTGTAGCTAGC-3’)，以及一条与上述miRNA成熟体序列互补的序列。将合成的miRNA模拟物干粉用RNase-free水稀释成储存1μg/μL，分装保存于-20℃。
合成miR-2703模拟物的正向引物和反向引物的核苷酸序列如下：
正向引物：5’-UAGCUUUGAUCUGUAGCUAGC-3’；(SEQ ID NO：2)
反向引物：5’-GCUAGCUACAGAUCAAAGCUA-3’。(SEQ ID NO：3)
步骤2：将miR-2703单独混在褐飞虱(Nilaparvata lugens)人工饲料中，对褐飞虱进行饲喂。
采用miR-2703在人工饲料中终浓度为0.02μg/μL、0.06μg/μL和0.1μg/μL三个梯度，持续饲喂7天，以negative control模拟物(来源于C.elegans的miRNA，与所有miRNA同源性最小)为阴性对照(NC)，CK为空白对照(仅饲喂空白人工饲料)。
步骤3：在步骤2中的饲喂过程中，统计褐飞虱每一天的存活率和观察表型变化。
结果如图1和图2所示。图1是饲喂miR-2703模拟物后褐飞虱的存活率图。图2是饲喂miR-2703模拟物后褐飞虱的表型变化图，其中，从左至右依次分别为阴性对照、0.02μg/μL miR-2703、0.06μg/μL miR-2703和0.1μg/μL miR-2703的结果。结果表明，无论是0.02μg/μL、0.06μg/μL或0.1μg/μL浓度下，miR-2703模拟物对褐飞虱的生长发育均有显著影响，其存活率均与阴性对照组和空白对照组存在显著差异，并出现褐飞虱蜕皮障碍以及腹部干瘪的表型。
步骤4：通过几丁质含量测定的方法进行褐飞虱几丁质含量的检测。
检测方法如下：取褐飞虱处理虫样称重，置于1.5mL Eppendorf管中，加入500μL6％KOH进行研磨并剧烈震荡5min；80℃加热90min；4℃，12000g离心20min，弃上清，重置于1mL PBS溶液中；4℃，12000g离心20min，弃上清，重置于200μL的Mcllvaine’s buffer(Mcllvaine’s Buffer：称取0.1mol的柠檬酸和0.2mol的NaH2PO4，加入80mL ddH2O溶解，调节pH值到6.0左右，定容至100mL；)；加入50μL 5mg/mL的几丁质酶溶液(Streptomycesplicatus chitinase-63溶于Mcllvaine’s buffer)，37℃温浴96h；取60μL 0.27M硼酸钠和60μL上述样品上清，混合；12000g离心1min；99℃处理10min；快速降至室温，加入600μLDMAB溶液；37℃温浴20min；分光光度计检测585nm处吸光值。标准曲线根据0.075-2.0mMGlcNAc的585nm读数制定。
结果如图3所示。图3是饲喂miR-2703模拟物后褐飞虱的几丁质含量变化图。可以发现，通过饲喂含有0.1μg/μL浓度的miR-2703模拟物的人工饲料，在96h时观测到褐飞虱几丁质含量下降。结果表明，通过对褐飞虱持续饲喂miR-2703模拟物，能够有效阻碍几丁质的合成。
实施例2
步骤1：统计卵粒的孵化率。
选取羽化一天的正常短翅型雌虫，每头注射50ng的miR-2703模拟物，然后与未处理的单雄配对后产卵，统计卵粒的孵化率。
结果如图4所示。图4是注射miRNA模拟物后褐飞虱的后代孵化率图。与对照(未注射miR-2703模拟物)相比，注射miR-2703模拟物后，褐飞虱的后代孵化率有显著降低。
解剖处理后的雌虫卵巢，结果如图5所示。图5是注射miRNA模拟物后褐飞虱的卵巢变化图，左图为经miR-2703模拟物处理后的雌虫卵巢形态，右图为未经处理的雌虫卵巢形态。由图5可见，注射miR-2703模拟物后的卵巢形态正常，但是卵泡的发育程度较低，虽然卵粒总数目并未发现显著减少，但是其大多为不成熟的卵粒，这说明miR-2703阻碍了褐飞虱的正常发育和繁殖。
步骤2：检测褐飞虱几丁质合成酶A基因的蛋白水平变化。
选取发育龄期一致的4龄末期褐飞虱和刚羽化的短翅型雌虫若干头，分别注射50ng miRNA模拟物，注射后48H随机取样5头，通过Western Blot的方法检测褐飞虱几丁质合成酶A基因(CHSA)的蛋白水平变化。未注射miRNA模拟物为阴性对照(NC)。
取5头已处理48h褐飞虱虫样于1.5mL的Eppedorf管中，于液氮中充分研磨，加入200μL的RIPA缓冲液、2μL的蛋白酶抑制剂和2μL的100X EDTA溶液，重悬混匀，4℃摇床孵育4h；4℃，12000g离心20min，取上清，使用BAC蛋白浓度测定试剂盒检测蛋白浓度。取20μg提取的褐飞虱蛋白，加入5×蛋白上样缓冲液，混匀，于100℃变性5min。制备8％分离胶和5％浓缩胶，将SDS-PAGE凝胶放入Bio-Rad小型蛋白电泳仪，加入蛋白电泳缓冲液。上样完毕后，进行电泳。80V，30min；120V，1h，等溴酚蓝迁移至凝胶底部边缘，停止电泳。切除浓缩胶，将分离胶上的蛋白转到PVDF膜上，电泳条件为100V，90min。转膜结束后，使用PBST溶液洗膜5min，5％封闭液封闭PVDF膜2h，更换新的5％封闭液，按1:200的比例加入Anti-Nl CHSA抗体，4℃过夜，PBST洗涤3次，每次10min。加入PBST，按1:20000的比例加入HRP标记的羊抗兔抗体，孵育45min。使用ECL试剂盒(Millipore公司)显色：200μL A液+200μL B液，滴到膜上抹匀。通过Image Lab软件拍照。
结果如图6所示。图6是注射miRNA模拟物后褐飞虱的CHSA蛋白含量变化图，图中，左侧为若虫期的蛋白质表达量变化结果，右侧为成虫期的蛋白质表达量变化结果。由结果可见，无论是成虫期还是若虫期，注射miR-2703模拟物均显著降低了褐飞虱CHSA(Nl CHSA)的蛋白表达量，表明miR-2703干扰了靶标基因CHSA的表达。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他任何在未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均因为等效的置换方式，都包含在本发明的保护范围之内。
Claims (5)
1.一种miRNA模拟物，所述miRNA模拟物为化学合成的成熟miRNA双链，包括一条与目标miRNA成熟体序列一致的如SEQ ID NO：1所示的序列，以及一条与所述miRNA成熟体序列互补的序列。
2.一种几丁质合成抑制类生物农药，包括作为活性成分的miRNA模拟物，所述miRNA模拟物为化学合成的成熟miRNA双链，包括一条与目标miRNA成熟体序列一致的如SEQ ID NO：1所示的序列，以及一条与所述miRNA成熟体序列互补的序列。
3.权利要求1所述的miRNA模拟物或权利要求2所述的几丁质合成抑制类生物农药在制备用于防治褐飞虱的药物中的应用。
4.一种组合物，其含有权利要求1所述的miRNA模拟物或权利要求2所述的几丁质合成抑制类生物农药。
5.根据权利要求4所述的组合物，其特征在于，所述miRNA模拟物在所述组合物中的浓度为0.02-0.1μg/μL。
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Non-Patent Citations (3)
| Title |
|---|
| Conserved microRNAs miR-8-5p and miR-2a-3p modulate chitin biosynthesis in response to 20-hydroxyecdysone signaling in the brown planthopper, Nilaparvata lugens;Jie Chen等;《Insect Biochemistry and Molecular Biology》;20131231;第43卷;第839-848页 |
| Functional screen for microRNAs of Nilaparvata lugens reveals that targeting of glutamine synthase by miR-4868b regulates fecundity;Xian Fu等;《Journal of Insect Physiology》;20151104;第83卷;第22-29页 |
| Genome-wide screening for components of small interfering RNA (siRNA) and micro-RNA (miRNA) pathways in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae);H-J. Xu等;《Insect Molecular Biology》;20130813;第22卷(第6期);第635–647页 |
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