CN114540506B - 调节obp4基因表达量的制剂在制备调节蜜蜂糖反应性和花粉选择行为药剂中的应用 - Google Patents
调节obp4基因表达量的制剂在制备调节蜜蜂糖反应性和花粉选择行为药剂中的应用 Download PDFInfo
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Abstract
本发明公开了调节OBP4基因表达量的制剂在制备调节蜜蜂糖反应性和花粉选择行为药剂中的应用。本发明通过实验发现，经口饲喂蜜蜂dsOBP4 24h后检测蜜蜂头部OBP4的表达情况，结果显示饲喂dsOBP4的蜜蜂头部OBP4的表达量显著低于饲喂ddH2O的蜜蜂(p<0.05)。经口饲喂蜜蜂dsOBP4 24h后检测蜜蜂对不同浓度糖溶液的反应，结果显示，饲喂dsOBP4的蜜蜂糖反应性极显著低于对照组蜜蜂(P<0.0001)。由此得出OBP4调节蜜蜂的糖反应性行为。经口饲喂dsOBP4的蜜蜂24h后，蜜蜂对花粉挥发物的选择显著低于对照组蜜蜂(P<0.05)，而对正己烷的选择则显著高于对照组蜜蜂(P<0.05)。由此得出OBP4能影响蜜蜂对花粉的选择。因此可将调节气味结合蛋白基因OBP4表达量的制剂在制备调节蜜蜂的糖反应性和花粉选择行为药剂中应用。
Description
技术领域：
本发明属于昆虫领域，具体涉及调节气味结合蛋白基因OBP4表达量的制剂在制备调节蜜蜂的糖反应性和花粉选择行为药剂中的应用。
背景技术：
昆虫是地球上种类最多、分布最广的动物类群，与人类关系非常密切。在长期的进化过程中，昆虫与外界环境相互作用，形成了复杂且灵敏的化学感受系统(如嗅觉和味觉系统)。在参与昆虫嗅觉识别的多种蛋白中，包含一类小分子量的亲水性转运蛋白气味结合蛋白(odorant binding proteins，OBPs)。这类蛋白存在于昆虫嗅觉感受器的淋巴液中，能够结合和运输外界疏水性的气味分子到达嗅觉神经元树突状膜上，并激活膜上的气味受体ORs(odorant receptors，ORs)，实现信号转导，最终通过中枢神经系统控制行为反应(Lealet al.，2013；Pelosi et al.，2018)。因此，从嗅觉发生的过程可以看出，OBPs在识别气味分子和引发生理反应的途径中起到了十分重要的作用。意大利蜜蜂(Apis mellifera)基因组中共鉴定到21个OBPs，其中的9个基因在触角中特异性表达，其余基因在各个组织或特异的组织中表达。其后赵慧婷(2021)对21个OBPs在意大利蜂头部表达模式进行研究表明，13个OBPs在头部高表达，且大多数OBPs在4日龄表达量最高。有报道意大利蜜蜂OBP1和OBP21能结合蜂王信息素，OBP2对大多数气味能结合，OBP13能特异性结合油酸及与它结构类似的化合物等。但是目前为止，关于意蜂OBP4的功能未见报道。利用RNAi技术沉默蜜蜂头部OBP4，发现蜜蜂对糖反应显著性下降，同时对花粉挥发物亚麻酸乙酯的选择显著低于对照蜜蜂。
发明内容
本发明的第一个目的是提供调节气味结合蛋白基因OBP4表达量的制剂在制备调节蜜蜂的糖反应性和花粉选择行为药剂中的应用。
本发明的第二个目的是提供调节蜜蜂的糖反应性和花粉选择行为药剂，其含有调节气味结合蛋白基因OBP4表达量的制剂。
优选，所述的蜜蜂为意大利蜜蜂。
优选，所述的调节气味结合蛋白基因OBP4表达量的制剂是气味结合蛋白基因OBP4过表达剂或气味结合蛋白基因OBP4的抑制剂。
所述的气味结合蛋白基因OBP4的抑制剂是dsRNA，其核苷酸序列为：
agcaattctatgctcgcaaaaagcaggcttcgatctttccgatctgaaaagcatgtacgaaagtaacagcgaggaacaaatgaagaagcttggatgtttcgaagcctgtgtattccaaaagcttcatttcatggacggtaatactttgaacgtagagaaactcgagtctggaactcgagaactaacacccgacgactttacggaggatg，具体如SEQID NO.1所示。
本发明通过实验发现，经口饲喂蜜蜂dsOBP4 24h后检测蜜蜂头部OBP4的表达情况，结果显示饲喂dsOBP4的蜜蜂头部OBP4的表达量显著低于饲喂ddH2O的蜜蜂(p<0.05)。经口饲喂蜜蜂dsOBP4 24h后检测蜜蜂对不同浓度糖溶液的反应，结果显示，饲喂dsOBP4的蜜蜂糖反应性极显著低于对照组蜜蜂(P<0.0001)。由此得出OBP4调节蜜蜂的糖反应性行为。经口饲喂dsOBP4的蜜蜂24h后，蜜蜂对花粉挥发物的选择显著低于对照组蜜蜂(P<0.05)，而对正己烷的选择则显著高于对照组蜜蜂(P<0.05)。由此得出OBP4能影响蜜蜂对花粉的选择。因此可将调节气味结合蛋白基因OBP4表达量的制剂在制备调节蜜蜂的糖反应性和花粉选择行为药剂中应用。
附图说明：
图1是蜜蜂饲喂dsOBP4后，头部OBP4基因表达量检测；
图2是蜜蜂头部OBP4沉默后的蜜蜂糖反应性检测；
图3是蜜蜂头部OBP4沉默后的蜜蜂Y管行为检测。
具体实施方式：
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
实施例1：
1材料与方法
1.1意大利蜜蜂采集
实验用蜜蜂均采自广东省科学院动物研究所综合试验站蜂场。
1.2RNA提取和cDNA合成
按照Trizol试剂盒(Invitrogen，USA)说明书提取各样品的总RNA。经核酸测定仪检测RNA的完整性及其纯度和浓度后,使用反转录试剂盒(AG，中国大连)进行cDNA第一链合成。合成过程分两步:①基因组DNA的去除。反应总体系为10μL:5×gDNA Eraser Buffer2.0μL，总RNA 500ng，RNase Free ddH2O补足10μL。42℃孵育2min；②反转录。反应总体系为20μL:5×Evo M-MLV RT Reaction Mix 4.0μL，RNase Free ddH2O 6μL步骤①的反应液10μL。37℃15min，85℃5s，4℃，2min.
1.3引物设计
在NCBI数据库中搜索中华蜜蜂OBP4(NM_001011589.1)基因的核苷酸序列，采用Primer 5.0软件设计引物(F:5’gagtctggaactcgagaactaacacc3’；R:5’caaccatgcattcgtcttcgtctg3’)，用qPCR试验。
1.4dsOBP4合成
从生工合成dsOBP4的上下游引物(F:5’agcaattctatgctcgcaaaa3’；R:5’catcctccgtaaagtcgtcg3’)，用蜜蜂头部cDNA进行PCR扩增，使用Vazyme试剂盒。反应体系为：cDNA模板2.5μL，上、下游引物1.5μL，ddH2O 19.5μL，2ⅹ Taq plus Master Mix 25μL,总反应体系为50μL。反应程序：95℃预变形1min；95℃变形30s；60℃退火30s；72℃延伸30s；35个循环；72℃终延伸10min，12℃保存。PCR产物经1％琼脂糖凝胶电泳检测(120V，25min)后，在凝胶成像系统中检测并拍照。割取含有目的DNA条带的凝胶置于干净的1.5ml离心管中。胶回收使用Omega试剂盒按照说明书进行。
取胶回收产物DNA作为模板，进行第二次PCR，反应程序和反应体系同上。PCR产物使用Omega试剂盒按照说明书进行液体回收。液体回收所得DNA用于下一步的dsOBP4(5’agcaattctatgctcgcaaaaagcaggcttcgatctttccgatctgaaaagcatgtacgaaagtaacagcgaggaacaaatgaagaagcttggatgtttcgaagcctgtgtattccaaaagcttcatttcatggacggtaatactttgaacgtagagaaactcgagtctggaactcgagaactaacacccgacgactttacggaggatg3’)的合成。dsRNA的合成使用Promega试剂盒按照说明书进行。
1.5ds OBP4的饲喂
从一个蜂群中采集采粉蜜蜂60只(采粉蜂是以后足花粉筐携带花粉为标准)，分成两组。一组蜜蜂每只喂dsOBP4 2μg(dsRNA溶解于30μL蔗糖溶液)，另一组蜜蜂每只喂等量的ddH2O。本次试验重复三个蜂群。
1.6沉默效率的检测
在喂完dsOBP4 24h之后分别取实验组和对照组蜜蜂头部提取总RNA(方法同上)，反转录，用OBP4为上下游引物进行qPCR(反应体系和反应程序同上)。
1.7行为试验
(1)蜜蜂糖反应性检测
试验所需的蔗糖溶液浓度依次为：0.1％，0.3％，1％，3％，10％和30％(w/w)，此外，还有水。10％(w/w)蔗糖溶液配制方法：称量10g蔗糖，加入到90g水中。
蜜蜂排成一排，依次接受检测，糖水浓度由低到高。例如，先检测所有的蜜蜂对0.1％糖水的反应性，然后再检测所有的蜜蜂对0.3％糖水的反应性，以此类推。在用0.1％糖水检测之前，用一滴水接触蜜蜂的每个触角。同样的，在用后续的糖水刺激蜜蜂触角前，先用一滴清水接触蜜蜂的每个触角(即，水，0.1％；水，0.3％；等等)。不同糖水刺激的间隔在2至3min。一只蜜蜂表现伸吻反应的标准是当一滴糖水依次、轮流地接触被检测蜜蜂的每个触角时，该只蜜蜂能够完全伸出它的吻，该只蜜蜂则对该浓度糖水表现出反应。若吻只是很轻微的动，没有完全伸展出来，则认为该蜜蜂未表现出反应。当所有浓度检测结束后，用50％的糖水检测蜜蜂，若蜜蜂伸吻，表示该蜜蜂正常。若不伸吻，表示蜜蜂不正常，对之前检测该蜜蜂的所有数据剔除。
(2)Y管行为
蜜蜂选择试验在Y管中进行，气流300ml/min，气温25℃。LED等置于Y管上方以刺激蜜蜂在管内行动。Y管中央管长21cm，两臂长15cm，两臂夹角为60度。Y管两臂各接一个100ml的磨口抽滤瓶。10μL亚麻酸乙酯(Ethyl)(经正己烷n-hexane稀释100倍后)放在一小片滤纸上，滤纸放于其中一个抽滤瓶，另一个抽滤瓶则放等量的正己烷。在Y管中央管的终端释放蜜蜂，当蜜蜂进入管臂的1/2之一位置时，我们判定它做出选择，并记录数据。
1.8荧光定量PCR
以各样品的cDNA(反转录之后进行4倍稀释)为模板，使用Premix ExTaqTM II(Tli RNaseH Plus)试剂盒进行荧光定量PCR。反应体系(20μL):SYBR Premix ExTaqTMII10μL，上下游引物(10μmol/L)各0.8μL，cDNA模板3μL，DEPC水5.4μL。反应条件：95℃预变性30s；接着进行40个循环：95℃30s，60℃30s。测定熔解曲线的反应条件：以0.5℃的升温速度从60℃升至95℃。
1.9数据处理
对于qPCR检测数据，根据扩增曲线获得的Ct值，在Excel中采用2－△CT法进行数据分析。利用SPSS25.0软件进行单因素方差分析，Duncan法进行多重比较，并利用GraphPadPrism 9.1软件作图。
2试验结果
2.1RNAi沉默效率验证
如图1所示，经口饲喂蜜蜂dsOBP4 24h后检测蜜蜂头部OBP4的表达情况，结果显示饲喂dsOBP4的蜜蜂头部OBP4的表达量显著低于饲喂ddH2O的蜜蜂(p<0.05)。
2.2蜜蜂头部OBP4沉默后对糖反应的影响
如图2所示，经口饲喂蜜蜂dsOBP4 24h后检测蜜蜂对不同浓度糖溶液的反应，结果显示，饲喂dsOBP4的蜜蜂糖反应性极显著低于对照组蜜蜂(P<0.0001)。由此得出OBP4调节蜜蜂的糖反应性行为。
2.3蜜蜂头部OBP4沉默后对花粉挥发物选择的影响
如图3所示，经口饲喂dsOBP4的蜜蜂24h后，蜜蜂对花粉挥发物的选择显著低于对照组蜜蜂(P<0.05)，而对正己烷的选择则显著高于对照组蜜蜂(P<0.05)。由此得出OBP4能影响蜜蜂对花粉的选择。
序列表
<110> 广东省科学院动物研究所
<120> 调节OBP4基因表达量的制剂在制备调节蜜蜂糖反应性和花粉选择行为药剂中的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 209
<212> DNA
<213> 中华蜜蜂(Apis cerana)
<400> 1
agcaattcta tgctcgcaaa aagcaggctt cgatctttcc gatctgaaaa gcatgtacga 60
aagtaacagc gaggaacaaa tgaagaagct tggatgtttc gaagcctgtg tattccaaaa 120
gcttcatttc atggacggta atactttgaa cgtagagaaa ctcgagtctg gaactcgaga 180
actaacaccc gacgacttta cggaggatg 209
Claims (1)
1.调节气味结合蛋白基因OBP4表达量的制剂在制备调节蜜蜂的糖反应性和花粉选择行为药剂中的应用，所述的蜜蜂为意大利蜜蜂，所述的调节气味结合蛋白基因OBP4表达量的制剂是气味结合蛋白基因OBP4的抑制剂，所述的气味结合蛋白基因OBP4的抑制剂是dsRNA，其核苷酸序列为：agcaattctatgctcgcaaaaagcaggcttcgatctttccgatctgaaaagcatgtacgaaagtaacagcgaggaacaaatgaagaagcttggatgtttcgaagcctgtgtattccaaaagcttcatttcatggacggtaatactttgaacgtagagaaactcgagtctggaactcgagaactaacacccgacgactttacggaggatg。
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| CN112359040A (zh) * | 2020-11-02 | 2021-02-12 | 广东省科学院动物研究所 | 调节let-7基因表达量的制剂在制备调节蜜蜂糖反应性行为制剂中的应用 |
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| CN106636363A (zh) * | 2016-11-16 | 2017-05-10 | 中国农业科学院植物保护研究所 | 利用特异性挥发物筛选昆虫相关嗅觉蛋白的方法 |
| WO2022023572A2 (en) * | 2020-07-31 | 2022-02-03 | Rothamsted Research Limited | Compounds with semiochemical properties and biosensors |
| CN112359040A (zh) * | 2020-11-02 | 2021-02-12 | 广东省科学院动物研究所 | 调节let-7基因表达量的制剂在制备调节蜜蜂糖反应性行为制剂中的应用 |
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