CN116411028A - 桔小实蝇嗅觉受体OR43a-1和OR63a-2的应用及其突变体的构建方法 - Google Patents
桔小实蝇嗅觉受体OR43a-1和OR63a-2的应用及其突变体的构建方法 Download PDFInfo
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Abstract
本发明涉及昆虫基因工程技术领域,公开了桔小实蝇嗅觉受体或受体基因BdorOR43a‑1或/和BdorOR63a‑2作为靶标在以下任一中的应用:(1)调控桔小实蝇的行为;(2)制备桔小实蝇行为调节剂;(3)制备桔小实蝇雌虫引诱剂或雌虫产卵引诱剂;(4)调控桔小实蝇对刺激气味分子的敏感性。还公开了一种调控桔小实蝇对苯并噻唑的敏感性的基因编辑方法和桔小实蝇嗅觉受体基因突变体的构建方法。本发明揭示了桔小实蝇感受产卵引诱物质苯并噻唑的关键嗅觉受体OR43a‑1和OR63a‑2,为开发桔小实蝇新型高效雌虫引诱剂提供可靠分子靶标。
Description
技术领域
本发明涉及昆虫基因工程技术领域,具体涉及桔小实蝇嗅觉受体OR43a-1和OR63a-2的应用及其突变体的构建方法。
背景技术
桔小实蝇Bactroceradorsalis(Hendel)是一种世界范围内分布的危险性害虫,可危害多种果树的果实。除柑桔外,还能为害芒果、番石榴、番荔枝、杨桃、枇杷等。成虫产卵于寄主果实,幼虫在果实中取食果肉并发育成长,幼虫成熟后从果实中跳出并入土化蛹,成虫在土壤中羽化。果实受桔小实蝇幼虫的危害后,可造成落果或使果实失去经济价值,该虫严重发生的地区可造成产量的巨大损失。由于其寄主范围广、入侵扩散能力强、抗药性发展速度快等特点,加之其幼虫在果实内取食危害的特性,对桔小实蝇的有效防控较为困难。目前,基于引诱剂的诱杀法是防治桔小实蝇最为有效的方法,特别在疫区对桔小实蝇种群动态监测、扩散前沿带的快速扑灭等均依赖于诱剂的使用。其中,甲基丁香酚(Methyleugenol,ME)是目前常用商业化引诱剂的主要成分,其仅对实蝇类害虫的雄虫具有引诱力。然而在田间桔小实蝇一旦交配,防治雄虫就无济于事。
昆虫嗅觉器官感知外界环境的刺激气味之后,位于嗅觉感受器神经元的嗅觉受体(Odorant Receptors,ORs)“编码”外界气味分子,将刺激物的化学信号转化为电信号,最终传递至脑。目前,有多个研究团队试图对桔小实蝇嗅觉受体进行注释,但往往受限于目前已公布基因组的质量。相比于模式昆虫果蝇Drosophila melanogaster,桔小实蝇嗅觉受体的研究较为缓慢,目前仅有少量嗅觉受体基因的功能的已验证。例如,BdorOR13a参与感受1-辛烯-3-醇,BdorOR82a参与感受乙酸香叶酯(Miyazaki et al.,2018),BdorOR88a参与感受甲基丁香酚(Liu et al.,2018)。
桔小实蝇主要通过交配后雌虫产卵于寄主果实内部,卵孵化后幼虫取食危害。产卵引诱剂能够引诱交配后的雌虫过去产卵。因此,发掘靶向雌虫产卵的引诱剂,研发诱集雌虫的方法,对桔小实蝇的防控更具有现实意义。芒果中的苯并噻唑(benzothiazole)对桔小实蝇具有强烈的产卵引诱力,且其对交配后的桔小实蝇引诱力更强,触角电生理反应也更敏感,被认为是极具应用潜力的桔小实蝇产卵引诱物质。目前,为靶向关键嗅觉受体创制害虫引诱剂,发掘重要化学信号物质的嗅觉受体已成为反向化学生态学的一个热点领域。开发稳定高效、环境友好型的行为调控方法,已成为桔小实蝇防控新思路。
然而,桔小实蝇感受苯并噻唑的嗅觉受体及其结合的分子机制尚不清楚,严重制约了苯并噻唑在桔小实蝇产卵引诱剂研发中的应用。因此,挖掘桔小实蝇感受苯并噻唑的关键受体及其与苯并噻唑结合的关键位点,将为桔小实蝇新型雌虫引诱剂研发提供极具潜力的分子靶标;获得的关键受体结构及其与苯并噻唑结合能力相互关系的基础数据,将为靶向关键嗅觉受体的桔小实蝇特异性引诱剂的改造、效力提升提供重要理论依据。
发明内容
本发明针对上述问题,综合利用行为学观察、qPCR、异源表达以及基于爪蟾卵母细胞表达系统的电压钳技术在离体水平挖掘可与苯并噻唑结合的关键嗅觉受体;利用基因编辑CRISPR/Cas9技术在活体水平探究该嗅觉受体的生理功能;利用蛋白三维结构模拟和分子对接技术揭示嗅觉受体与苯并噻唑结合的关键位点。基于此,本发明提供了如下技术方案:
本发明提供了桔小实蝇嗅觉受体BdorOR43a-1或/和BdorOR63a-2,或者桔小实蝇嗅觉受体基因BdorOR43a-1或/和BdorOR63a-2作为靶标在以下任一中的应用:
(1)调控桔小实蝇的行为;
(2)制备桔小实蝇行为调节剂;
(3)制备桔小实蝇雌虫引诱剂或雌虫产卵引诱剂;
(4)调控桔小实蝇对刺激气味分子的敏感性。
在上述应用技术方案中,以BdorOR43a-1或/和BdorOR63a-2作为靶标,调节桔小实蝇雌虫对刺激气味分子的嗅觉敏感性。
在上述应用技术方案中,所述刺激气味分子为雌虫引诱剂或雌虫产卵引诱剂。
在上述应用技术方案中,所述刺激气味分子为苯并噻唑。
在上述应用技术方案中,BdorOR43a-1受体与苯并噻唑结合的关键位点是氨基酸SER69,BdorOR63a-2受体与苯并噻唑结合的关键位点是氨基酸TYR324。
本发明还提供了一种调控桔小实蝇对苯并噻唑的敏感性的基因编辑方法,首先针对桔小实蝇BdorOR43a-1基因或者BdorOR63a-2基因设计gRNA,然后向桔小实蝇中注射入gRNA和Cas9蛋白的混合物进行基因编辑;
对于BdorOR43a-1基因,gRNA在BdorOR43a-1基因E1外显子的gRNA靶点的核苷酸序列为:CCTTGCTGCGTACGTGCCTTATT;
对于BdorOR63a-2基因,gRNA在BdorOR63a-2基因E3外显子的gRNA靶点的核苷酸序列为:GAAAACTATGAACTACTGCAAGG。
优选地,将gRNA和Cas9蛋白的混合物注射入桔小实蝇新鲜胚胎中。
本发明最后还提供了一种桔小实蝇嗅觉受体基因突变体的构建方法,采用上述的方法对桔小实蝇进行基因编辑,然后将胚胎注射后存活至成虫的G0代个体与野生型个体杂交、传代,筛选突变体。
所述筛选突变体的具体方法为:将胚胎注射后存活至成虫的G0代个体与野生型个体单对杂交,获得G1代后,检测G0代的基因编辑情况,保留成功编辑的G0代与野生型杂交所产生的G1代个体,检测G1代基因编辑情况,并将成功编辑的G1代个体与野生型杂交产生G2代,将基因型一致的G2代个体自交,在G3代中可筛选到纯合突变体;
优选地,为避免脱靶效应,G1代与野生型交配至少5代后,再将杂合突变体自交产生纯合突变体。
上述突变体的构建方法,获得了BdorOR43a-1-/-突变体、BdorOR63a-2-/-突变体,
所述BdorOR43a-1-/-突变体是指SEQ ID NO.55所示的野生型序列的第224位核苷酸缺失突变;
所述BdorOR63a-2-/-突变体是在SEQ ID NO.56所示的野生型的序列基础上,第340位后增加了7个碱基AAGGAAA;同时野生型序列的第341-343位的三个碱基“TGC”对应突变体的第348-350位变成了“TTA”。
本发明的有益效果是:
本发明揭示了桔小实蝇感受产卵引诱物质苯并噻唑的关键嗅觉受体OR43a-1和OR63a-2,为开发桔小实蝇新型高效雌虫引诱剂提供可靠分子靶标。同时,OR43a-1和OR63a-2的蛋白结构及其与苯并噻唑结合能力相互关系的基础数据,可为靶向关键嗅觉受体的桔小实蝇特异性引诱剂的改造、效力提升提供重要理论依据。
本发明挖掘到的桔小实蝇感受苯并噻唑的关键受体OR43a-1和OR63a-2及其与苯并噻唑结合的关键位点,为桔小实蝇新型雌虫引诱剂研发提供极具潜力的分子靶标;获得的关键受体结构及其与苯并噻唑结合能力相互关系的基础数据,为靶向关键嗅觉受体的桔小实蝇特异性引诱剂的改造、效力提升提供重要理论依据。
附图说明
图1是苯并噻唑对交配前后桔小实蝇的引诱力实验结果。
图2是妊娠桔小实蝇在苯并噻唑介导下的产卵行为实验结果。
图3是嗅觉受体基因在桔小实蝇交配前后的表达量检测结果。
图4是BdorOR43a-1和BdorOR63a-2与苯并噻唑的体外结合能力检测结果。
图5是对获得的BdorOR43a-1-/-和BdorOR63a-2-/-突变体的分析结果。
图6是BdorOR43a-1-/-和BdorOR63a-2-/-突变体在苯并噻唑刺激下的触角电位反应实验结果。
图7是BdorOR43a-1-/-和BdorOR63a-2-/-突变体在苯并噻唑刺激下的产卵量结果。
图8是BdorOR43a-1和BdorOR63a-2与苯并噻唑的结合位点。
具体实施方式
下面结合实施例对本发明作进一步说明,但并不因此而限制本发明。
下述实施例中的实验方法,如无特别说明,均为常规方法;所用生物、化学试剂、材料,如无特殊说明,均为本领域常规试剂和材料,均可商购获得。
矿物油(Mineraloil):品牌Sigma-aldrich;
pT7Ts表达载体:大肠杆菌表达载体。
实施例1桔小实蝇对苯并噻唑产卵偏好性检测
一、苯并噻唑对交配和未交配桔小实蝇的引诱力检测
利用双陷阱检测苯并噻唑(CAS编号:95-16-9)对桔小实蝇的引诱力。取30头15日龄未交配或交配后的雌虫放入20cm×20cm×20cm的透明养虫笼中,用矿物油将苯并噻唑稀释为浓度10%(V/V),对照组为矿物油。将20μL10%苯并噻唑和20μL矿物油分别加入到改造后的50mL离心管中并放置在养虫笼的对角线上。
50mL离心管改造方法为:将离心管倒置,在锥形顶部钻孔,小孔直径约0.5cm,并于离心管顶部锥形斜面上钻两个直径约0.5cm的小孔。在离心管盖内加入苯并噻唑或矿物油后将离心管的管盖扣合,然后锥形顶部朝上地放置在养虫笼的对角线上。
每隔2h统计两个离心管的中引诱到的桔小实蝇的数量,连续统计24h。产卵偏好性计算公式:(苯并噻唑引诱的虫量-矿物油引诱的虫量)/总虫量。如图1所示,苯并噻唑对交配后桔小实蝇雌虫引诱力显著强于对未交配的桔小实蝇雌虫。
二、桔小实蝇对苯并噻唑的产卵偏好性检测
取8头15日龄交配后的雌虫放入10cm×10cm×10cm的透明养虫笼中,用矿物油将苯并噻唑稀释为浓度10%(V/V),对照组为矿物油。准备诱卵装置:将1%琼脂(取1g的琼脂糖溶解在100mL的超纯水中)分装至直径为9cm的培养皿中,将凝固后的琼脂划分为两部分,左侧加入20μL10%苯并噻唑,右侧加入20μL矿物油(如图2A)。然后用扎了孔的保鲜膜覆盖培养皿,并将该诱卵装置放入养虫笼中。用摄像机监测桔小实蝇在苯并噻唑诱导下的产卵行为,并在24h后统计产卵量,同时用Ethovision软件(动物运动轨迹跟踪系统)分析桔小实蝇的运动轨迹。
结果如图2B所示,10%和1%苯并噻唑均对桔小实蝇雌虫具有较强的产卵引诱力,实蝇轨迹热图也显示轨迹主要集中在10%和1%苯并噻唑区域,其中10%的苯并噻唑的效果更为显著,表明苯并噻唑对于桔小实蝇雌虫引诱具有较强的应用潜力。而0.1%苯并噻唑引诱效果不好,在0.1%苯并噻唑诱导下,桔小实蝇轨迹及产卵地点随机。
实施例2桔小实蝇感受苯并噻唑的嗅觉受体的鉴定
一、嗅觉受体在桔小实蝇交配前后的表达量检测
将初羽化的桔小实蝇雌、雄分开,在正常饲养条件下饲养至15日龄后,在冰上剪下雌虫头部置于1.5mL无核酶离心管中,用液氮迅速冷冻后提取RNA,作为未交配的桔小实蝇雌虫样品。将刚羽化的桔小实蝇雌、雄虫混合饲养,9日龄时观察其交配情况,将未交配雌虫去除。用同样的方法获得雌虫头部模板,作为交配后的桔小实蝇15日龄雌虫样品。两组样品均包含4个生物重复,每个生物重复包含8-10头虫。所有样品均使用TRIzol试剂提取其总RNA,并反转录合成第一链cDNA,置于-80℃超低温冰箱待用。
qPCR扩增条件如下:95℃预变性反应2min;随后,95℃变性15s,60℃退火和延伸30s,循环40次;最后60℃进行30s,95℃进行15s形成溶解曲线。10uL的反应体系包含5μLSYBRqPCRSuperMixPlus,上、下游引物(10μM)各0.3μL,0.5μL桔小实蝇cDNA模版和2.9μLNuclease-freewater。
如图3所示,有20个嗅觉受体基因(其qPCR扩增引物如表1中所示)在桔小实蝇交配后表达量显著上调。由于苯并噻唑对交配后桔小实蝇引诱力更强,因此将在交配后表达量显著上调的嗅觉受体基因作为参与桔小实蝇感受苯并噻唑的候选嗅觉受体。
表1.qPCR引物
二、候选嗅觉受体与苯并噻唑结合能力的检测
利用限制性内切酶NotI,将候选嗅觉受体构建至pT7Ts载体上。利用限制性内切酶XbaI线性化处理构建到pT7Ts载体上的重组质粒,并利用体外转录试剂盒mMESSAGET7Kit试剂盒合成候选嗅觉受体的cRNA,纯化后暂存于-80℃备用。
提前配置下列储备液及培养基:四环素贮存液(50mg/mL)、链霉素贮存液(100mg/mL)、庆大霉素贮存液(10mg/mL)、丙酮酸钠贮存液(275mg/mL)、100×CaCl2贮存液(60mM);10×Ringer溶液:56.1gNaCl、1.5gKCl、MgCl2和11.9gHEPEs加1LddH2O;1×Ringer溶液(无Ca2+):10×Ringer溶液100mL,加ddH2O至1L,调节pH至7.6;1×Ringer溶液(有Ca2+):1×Ringer溶液(无Ca2+),100×CaCl2溶液10mL,加ddH2O至1L,调节pH至7.6;洗液:1mL10mg/mL庆大霉素加入到1L1×Ringer溶液(无Ca2+)中;营养液:50mL马血清、1mL四环素(50mg/mL)、1mL链霉素(100mg/mL)、2mL丙酮酸钠(275mg/mL)加入到1×Ringer溶液(有Ca2+)中;麻醉剂:1.5g麻醉剂加入到1LddH2O中。以上溶液均需抽滤后保存。
取健康成熟的雌性非洲爪蟾,放入1L预先配制好的麻醉剂中30min,待其四肢麻痹后,在其下腹部左侧或右侧开一个0.5-1cm小口,拉出宫瓣,剪出研究所需量的爪蟾卵母细胞,置于含有预先配制的洗液的无菌培养皿中待用。缝合爪蟾的肌肉及皮肤,置于浅水中,使其头部露于水面上方,让其自然苏醒;将培养皿中成串的细胞剪成小块,用洗液反复清洗直至洗液变得澄清为止;将剪成小块并清洗干净的卵母细胞在1mg/mL的胶原酶中于室温轻柔振荡消化30min-1h;用洗液洗涤消化成单个的卵母细胞,直至洗液变得澄清为止;将清洗干净的卵母细胞转移至含有预先配制好的营养液的培养皿中,在18℃培养箱中过夜培养;挑出黑白分明、较大且饱满的卵母细胞进行下一步试验。
将挑选好的细胞整齐排列,用Nano显微注射器注射合成好的cRNA(每个候选OR和Orco混合注射),注射的位置为卵母细胞黑白相间的位置或者是卵母细胞的黑色部分。每个卵母细胞注射60nL,同时注射等量的无核酶水作为对照。注射后的细胞在18℃培养箱中培养2-3d,进行双电极电压钳检测。用DMSO溶解苯并噻唑饼配置成1M的储备液,并用Ringer溶液将其分别稀释到10-1、10-2、10-3、10-4和10-5M。检测时,由低浓度到高浓度依次检测,每次加入5μL稀释好的气味物质到50mL预先配置好的灌流液中,充分混匀。对照组为不同浓度的DMSO溶液,每个浓度检测6-8个细胞。卵母细胞的钳制电压设置为-80mV,利用pClamp15软件记录灌流时气味物质引起的内向电流,计算二者之间差异。
结果显示(图4),在不同浓度苯并噻唑(10-6M至10-3M)刺激下(图4A),表达了BdorOR43a-1和BdorOR63a-2的卵母细胞电流呈明显的浓度依赖性,而其它18个嗅觉受体在苯并噻唑刺激下无电流响应,表明这18个嗅觉受体不能与苯并噻唑结合;证明BdorOR43a-1和BdorOR63a-2两个嗅觉受体可与苯并噻唑结合,其有效中浓度(50%effectiveconcentration,EC50)分别为69μM(图4B)和12.6μM(图4C)。
用于BdorOR43a-1和BdorOR63a-2两个嗅觉受体的在爪蟾卵母细胞中异源表达的引物序列如下:
表2.BdorOR43a-1和BdorOR63a-2用于爪蟾卵母细胞表达系统的引物
实施例3关键嗅觉受体的基因编辑及表型分析
一.BdorOR43a-1和BdorOR63a-2的基因编辑
基于桔小实蝇基因组数据,分析BdorOR43a-1和BdorOR63a-2外显子和内含子结构,在其外显子上选择符合gRNA结构的潜在靶点。gRNA靶点一般包含20个任意碱基,紧邻靶点3’端的PAM区包含三个碱基且序列为NGG(N为任意碱基)。由于该体系的启动子为T7启动子,为保证后续合成的gRNA高效表达,靶点5’端的碱基应为GG或者G。gRNA靶点可选在靶标基因的正义链或负义链上,并以全基因组为参考序列评估靶点的潜在脱靶风险。
表3.gRNA合成及突变体筛选引物序列信息
利用上述合成的gRNA引物,参照GeneArtTMPrecisiongRNASynthesisKit说明书体外合成并纯化gRNA。OR43a-1和OR63a-2的gRNA靶点序列分别是:CCTTGCTGCGTACGTGCCTTATT(SEQ ID NO.53)和GAAAACTATGAACTACTGCAAGG(SEQ ID NO.54)。Cas9蛋白购于ThermoFisherScientific公司。将gRNA与Cas9蛋白混合至终浓度均为500-600ng/μL后注射经次氯酸钠处理后的桔小实蝇新鲜胚胎。将胚胎注射后存活至成虫的G0代个体与野生型(WT)个体单对杂交,获得足够数量的G1代后,提取G0代个体整虫的基因组DNA,检测G0代的编辑情况。保留成功编辑的G0代与WT杂交所产生的G1代个体,提取G1代个体单个足的DNA,用同样的方法检测,保留成功编辑的G1代个体,并将其进一步与WT型个体单对杂交产生G2代,由同一母本产生的基因型一致的G2代个体自交,在G3代中可筛选到纯合突变个体,为了避免潜在脱靶风险,将G2代个体至少与野生型交配10代后再自交以产生纯合突变体。基因编辑的具体参数和对应实验结果如表4所示:
表4 BdorOR43a-1和BdorOR63a-2的胚胎注射情况及G0突变体检测情况
结果如图5,经基因编辑和突变体筛选获得了稳定缺失1个碱基的BdorOR43a-1-/-突变体(图5A),其编码的氨基酸序列在第75个氨基酸发生改变并在第80个氨基酸后翻译终止,而正常WT可编码378个氨基酸。同时,获得了在靶点处插入7个碱基的BdorOR63a-2-/-突变体(图5B),且有3个碱基在PAM区附近发生改变,其所编码的氨基酸序列在第114个氨基酸处发生改变并在第116个氨基酸后翻译终止,而正常WT可编码417个氨基酸。BdorOR43a-1的野生型的编码基因的核苷酸序列(SEQ ID NO.55)如下:
ATGGTCACCGCGGTCGTAGACAACCCGATGCTCTCGGTCAATGTGAAGCTGTGGCAGTTCCTCTCCGTGCTCTTTGCACGCGATTGGCGGCGCTGTGTAGCTTTAGTGGCACCCGTCTGCCTAATGAATGCAATGCAATTCGTTTATTTGTATCAACAGTGGGGTGATTTGTCCACTTTCATATTGAATACCTTCTTTGCGGTCTCCGTTTTCAACGCCTTGCTGCGTACGTGCCTTATTATTAAGAATCGAGATAAATTTGAAGCGCTGATGGAAGAATTGGTAACACTATACGACGATATACAAGATTCGGATGATGATTATGCGAAGAGCGTGCTGGCTGCAGCCACAAAAAGCGCACGAAATATTTCCATTTTCAATTTGTCAGCTTCGTTCTCCGATTTAATTGTGGCAATGGCATATCCACTTTTTCAGCAACAGAGAGTTCATCCTTTTGGCGTTGCATTACCCGGGATCGATGTCACACGCTCTCCACTCTATGAACTCATCTATATCAGTCAATTATCTTTCCCGTTCACTCTGTCCAGCATGTATATGCCCTACGTAAGTTCATTCGCCACTTTCTCGATGTTCGGGAAGGCAGCACTACAAATATTACAAAATAACCTCAGAAATTTATGCGATAATATGAAAAGTAAAACTGAAGAAGAGCTCTTCGAAATACTACGAAAGAATATCGCCTATCATGCGAGAATCGCTAGATATGTGAGTGACTTCAATGAATTGGTTACCTATATGGTACTCATCGAATTTCTGCTCTTCAGTTGCGTTATTTGCTCGCTGCTCTTTTGCATCAATATTACAACCTCTACGGCAGAGAAGATTTCCATTGTCATGTACATTGGCACAATGCTGTATGTGCTCTTCACCTATTACTGGCAAGCCAATGGAGTTTTAGAAATGAGCCTCCTCGTGTCAGATGCGGCTTACGAAATGCAATGGTACAATTGTAGTCCACATTTTAAGAGAACTCTACTCATATTCATTGCACGCACGCAAAATCCTTTACAGATCCGCGTTGGTCAGATGCACCCAATGACAATGGAAGTATTTCAATCACTGCTTAATAACGCGTACTCCTATTTTACGCTTTTGCATAATCTTTATAATGATTAA。
BdorOR43a-1-/-突变体是指SEQ ID NO.55所示的野生型序列的第224位核苷酸缺失突变。BdorOR63a-2的野生型的编码基因的核苷酸序列(SEQ ID NO.56)如下:ATGTACAACGCAGCAGAGTTTGCAGAATTGAAAAACAACAATCGCTTCAAAATAAGAGAACTTAGAAATGTGTCATACATTTTAGGCATAAACTATGGCTCAGAAACGTCATTGAAGAGATTTCTCCGAGTGCTTAATCTATTTCTTATCATCATTTGTGCCATATCATTATATCCACGATGGTTGATGCTAGAAAGGGCTGATGGCAATGTGCCGCTAATTGCAGAGACCATCACTACCATGTTACAAACAACTACAAGCATGGTTAAAATGACATTCTGCCTGTTTATGCAAGGTCAGTGTCGTGCATTGCTTAAGAAGGCTGAAAACTATGAACTACTGCAAGGAATTAAAATCTTCCTGACTGATATGGACATCAAAGCTGAGTTGAAAGTGGAGATTAACGCCATTATGGCAACTATATGGAAGGAATCAAGGCGACAGCTTTTAAGCTGTCTCATAACTTGTTCGTGTATTCTTAGCAACTACTTTCTCTACGCCTTCTTCACTAACTTGTATCATCAAATAAAGAAGACGCCGAACTATGTGCATATATTACCTTTCACTGGTTACCCCATGTTTCTGGACAAAGGCATGGCCTCGCCTTATTATGCAGTGGAAATGTTCATCGGTGGCTGTTCACTCCTCACCTGTGGCATGTGTTCCGTCAGCTTTCATTGCATTTTTATGATCCTCTGCAAACATGCTTGCGGTCTAGTTAAGGTCCTTTGCGTCCTTCTGATGCGATCCACCTCACTCCAAGTGCCAGCACACCGGCGTGATGAATATTTGCGTTATTGTGTTATCCAACATCAACAGACTTTGCGGTTTATAAATGACATCAATGACCTTTTCAAGCACATTACCCTTTCACATTTCCTTCACAGCTTGGCAATATATGGACTTGTGCTTTTCGAAATGAACTTTGGACTAGAAACAGATAAAACAACATTTGTTCGTATGCTTATGTACATCGGAGCTGCACTCACTGTCGATTCCATGTATTATGTAAATGGGCAATTTTTGGCCACAGAGTTGGAAAAGATTCCATTCGTTTGCTACAGCTGCGATTGGTTTAACGAATCAGAGGATTTCAAGAGAACATTGAAGATGATAATTATGCGATCCAATAAAGATTTCTGTTTTCAAATTTCGTGGTTCGGCATAATGTCCTTGACCACATTAATGGGTATATTAAAAGCCAGCTTTTCGTATTTTTTGATTCTTAGAGATATGACGGATGAGACAAACTAA。
BdorOR63a-2-/-突变体是在SEQ ID NO.56所示的野生型的序列基础上,第340位后增加了7个碱基,序列为AAGGAAA;同时野生型序列的第341-343位的三个碱基“TGC”对应突变体的第348-350位变成了“TTA”。
二、BdorOR43a-1-/-和BdorOR63a-2-/-突变体在苯并噻唑诱导下的表型分析
利用SYNTECH触角电位仪记录BdorOR43a-1-/-和BdorOR63a-2-/-突变体在苯并噻唑刺激下的触角电位反应。在毛细管中注入导电液,并分别连接在触角电位仪中参比电极和记录电极上;取桔小实蝇头部,用刀片划破其触角末端,将桔小实蝇头部基部连接在充满导电液的参比电极上,并调节参比电极的位置将触角末端连接到充满导电液记录电极上;用5.0cm×1.0cm的滤纸条作为承载测试化合物的载体,每张滤纸条上加20μL待测化合物;测试系统的载气流量为100mL/min,气味刺激持续时间为1s,待测气味的间隔时间为30s;不同浓度的苯并噻唑测试顺序由低浓度到高浓度依次进行,最低浓度化合物测试之前和最高浓度化合物测试之后均用石蜡油作为对照来校准;突变体和野生型分别测试20头15日龄交配后的雌虫,所有EAG信号均由软件GcEAD记录。
如图6所示,BdorOR43a-1-/-和BdorOR63a-2-/-突变体在苯并噻唑刺激下的触角电位反应显著下降。
同时,利用实施例1中的实验方案,检测15日龄BdorOR43a-1-/-和BdorOR63a-2-/-雌虫在苯并噻唑介导下的产卵行为。结果如图7所示,BdorOR43a-1-/-和BdorOR63a-2-/-突变体在苯并噻唑诱导下的产卵量显著下降。说明BdorOR43a-1-/-和BdorOR63a-2-/-突变体对苯并噻唑的产卵偏好性显著降低。上述结果证明BdorOR43a-1和BdorOR63a-2是桔小实蝇感受苯并噻唑的关键嗅觉受体,并在苯并噻唑的诱导下调控桔小实蝇的产卵行为。
实施例4关键OR43a-1和OR63a-2与苯并噻唑结合位点的解析
利用人工智能程序AlphaFold2预测关键嗅觉受体的蛋白结构,采用深度神经网络算法,从具有进化渊源的蛋白质分子群中提取氨基酸残基多序列比对信息和残基对的特征作为初始输入,构建出蛋白质的三维结构。通过分子动力学模拟的简单优化,得到最终预测的蛋白质三维结构,并利用蛋白结构在线评估网站SAVES6.0(https://saves.mbi.ucla.edu/)中PROCHECKRamachandranplot(拉氏图)模块评估蛋白结构构象的质量和合理性。利用AutoDockVina1.1.2软件进行对接分析。根据蛋白结构和活性位点设置格点及对接参数,将对接模型导入Pymol和DiscoveryStudio2016Client软件分析结果并处理图像,根据Affinity(Kcal/mol)选取最优对接模型并分析嗅觉受体与苯并噻唑的关键亲和位点。
如图8所示,分子对接结果表明OR43a-1的氨基酸SER69和OR63a-2的氨基酸TYR324分别是OR43a-1和OR63a-2与苯并噻唑结合的关键位点。
基于桔小实蝇产卵引诱物质苯并噻唑与多个嗅觉受体结合的动力学测定,获得的关键受体结构及其与苯并噻唑结合能力相互关系的基础数据,将为靶向关键嗅觉受体的桔小实蝇特异性引诱剂的改造、效力提升提供重要理论依据,进而提升桔小实蝇防控体系的控制力、特异性、有效性和环境友好性。
Claims (10)
1.桔小实蝇嗅觉受体BdorOR43a-1或/和BdorOR63a-2,或者桔小实蝇嗅觉受体基因BdorOR43a-1或/和BdorOR63a-2作为靶标在以下任一中的应用:
(1)调控桔小实蝇的行为;
(2)制备桔小实蝇行为调节剂;
(3)制备桔小实蝇雌虫引诱剂或雌虫产卵引诱剂;
(4)调控桔小实蝇对刺激气味分子的敏感性。
2.根据权利要求1所述的应用,其特征在于:以BdorOR43a-1或/和BdorOR63a-2作为靶标,调节桔小实蝇雌虫对刺激气味分子的嗅觉敏感性。
3.根据权利要求2所述的应用,其特征在于:所述刺激气味分子为雌虫引诱剂或雌虫产卵引诱剂。
4.根据权利要求3所述的应用,其特征在于:所述刺激气味分子为苯并噻唑。
5.根据权利要求4所述的应用,其特征在于:BdorOR43a-1受体与苯并噻唑结合的关键位点是氨基酸SER69,BdorOR63a-2受体与苯并噻唑结合的关键位点是氨基酸TYR324。
6.一种调控桔小实蝇对苯并噻唑的敏感性的基因编辑方法,其特征在于:首先针对桔小实蝇BdorOR43a-1基因或者BdorOR63a-2基因设计gRNA,然后向桔小实蝇中注射入gRNA和Cas9蛋白的混合物进行基因编辑;
对于BdorOR43a-1基因,gRNA在BdorOR43a-1基因E1外显子的gRNA靶点的核苷酸序列为:CCTTGCTGCGTACGTGCCTTATT;
对于BdorOR63a-2基因,gRNA在BdorOR63a-2基因E3外显子的gRNA靶点的核苷酸序列为:GAAAACTATGAACTACTGCAAGG。
7.根据权利要求6所述的方法,其特征在于:
将gRNA和Cas9蛋白的混合物注射入桔小实蝇新鲜胚胎中。
8.一种桔小实蝇嗅觉受体基因突变体的构建方法,其特征在于:采用权利要求7所述的方法对桔小实蝇进行基因编辑,然后将胚胎注射后存活至成虫的G0代个体与野生型个体杂交、传代,筛选突变体。
9.根据权利要求8所述的方法,其特征在于:所述筛选突变体的具体方法为:
将胚胎注射后存活至成虫的G0代个体与野生型个体单对杂交,获得G1代后,检测G0代的基因编辑情况,保留成功编辑的G0代与野生型杂交所产生的G1代个体,检测G1代基因编辑情况,并将成功编辑的G1代个体与野生型杂交产生G2代,将基因型一致的G2代个体自交,在G3代中可筛选到纯合突变体;
优选地,为避免脱靶效应,G1代与野生型交配至少5代后,再将杂合突变体自交产生纯合突变体。
10.根据权利要求8所述的方法,其特征在于:获得了BdorOR43a-1-/-突变体、BdorOR63a-2-/-突变体,
所述BdorOR43a-1-/-突变体是指SEQ ID NO.55所示的野生型序列的第224位核苷酸缺失突变;
所述BdorOR63a-2-/-突变体是在SEQ ID NO.56所示的野生型的序列基础上,第340位后增加了7个碱基AAGGAAA;同时野生型序列的第341-343位的三个碱基“TGC”对应突变体的第348-350位变成了“TTA”。
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