CN110144351A - 一种飞蝗脂肪酸合成酶基因LmFAS2的dsRNA及其制备方法和应用 - Google Patents
一种飞蝗脂肪酸合成酶基因LmFAS2的dsRNA及其制备方法和应用 Download PDFInfo
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Abstract
本发明属于基因工程技术领域，具体涉及一种飞蝗脂肪酸合成酶基因LmFAS2的dsRNA及其制备方法和应用。本发明飞蝗脂肪酸合成酶基因LmFAS2的dsRNA的制备方法，包括以下步骤：a，设计上游引物序列和下游引物序列，并合成；b，提取RNA反转录成第一链cDNA，以此做为模板，结合设计飞蝗脂肪酸合成酶基因LmFAS2的上下游引物，通过PCR扩增获得脂肪酸延伸酶基因全长片段，将得到的产物进行纯化，克隆转化至大肠杆菌中，并测序；c，基于飞蝗脂肪酸合成酶基因序列SEQ ID NO：1，设计dsRNA上下游引物，其序列分别为SEQ ID NO：3和SEQ ID NO：4，上下游引物均携带T7启动子序列；d，转录合成dsRNA。
Description
技术领域
本发明属于基因工程技术领域，具体涉及一种飞蝗脂肪酸合成酶基因LmFAS2的dsRNA及其制备方法和应用。
背景技术
飞蝗是一种危害较大的农业害虫，分布范围极广并且迁移能力很强，破坏力大，因此对农业造成严重危害。目前常用于防控飞蝗的办法还是依靠化学防治，即化学杀虫剂。然而长期使用化学农药不止会使害虫产生抗药性降低防治效率，同时还会破坏生态环境，对其他非靶标生物造成危害。因此，迫切需要开发新型的害虫防治方法。
2006年获诺贝尔奖的RNA干扰(RNAi)技术，是一种由双链RNA分子引起的特异性转录后基因沉默技术。该技术为基因功能、人类疾病治疗和作物害虫防治等方面的研究都开辟了新途径。通过RNA干扰进行害虫防治具有如下特点：1、杀虫具有专一性，对非靶标生物无杀伤作用；2、RNA在自然界极易降解，无残留；3、对环境无毒无害，相对安全。研究表明，RNA干扰技术可有效控制特定的农作物害虫，在害虫防治领域具有非常重要的应用前景。而实现基于该技术进行害虫防控的关键是筛选出对害虫高效致死的特异dsRNA。
昆虫表皮主要化学成分是几丁质、蛋白质、酚类和脂类物质。昆虫表皮是作为一个双向的屏障行使作用，既可以防止昆虫体内水分的蒸发，还能够反向的防止外界病原微生物的入侵。而表皮脂类物质在这一过程中发挥着重要功能。此外，表皮脂类物质还可以作为信息素发挥作用，在昆虫发育生殖以及通讯等行为中起着重要作用。昆虫表皮脂类物质的合成涉及多种酶类的参与，如脂肪酸合成酶，其是昆虫表皮脂类合成通路中的关键酶之一，参与催化由乙酰辅酶A和丙二酸单酰辅酶A合成软脂酸的整个过程。目前已知的有关功能研究主要集中在其时空表达、滞育以及生殖方面的影响。此外，在模式生物线虫中发现沉默脂肪酸合成酶基因会影响表皮形成及其结构。因此，脂肪酸合成酶对于昆虫生存具有十分重要的意义。
发明内容
本发明针对上述问题提供了一种飞蝗脂肪酸合成酶基因LmFAS2的dsRNA及其制备方法和应用。
为达到上述目的本发明采用了以下技术方案：
一种飞蝗脂肪酸合成酶基因LmFAS2的dsRNA，该dsRNA的核苷酸序列为SEQ ID NO：2。
一种飞蝗脂肪酸合成酶基因LmFAS2的dsRNA的制备方法，包括以下步骤：
a，基于飞蝗的转录组数据库，采用生物信息学方法对飞蝗脂肪酸合成酶基因进行搜索，经过序列分析，拼接及比对后，获得飞蝗脂肪酸合成酶基因LmFAS2序列，设计上游引物序列和下游引物序列，并合成；
b，选取生长健康，大小一致雌雄各半的五龄飞蝗若虫，将其表皮快速解剖下来，并提取RNA，将所提RNA反转录成第一链cDNA，以此做为模板，结合设计飞蝗脂肪酸合成酶基因LmFAS2的上下游引物，通过PCR扩增获得脂肪酸延伸酶基因全长片段，将得到的产物进行纯化，克隆转化至大肠杆菌中，并测序，测序结果与转录组搜索结果比对，验证并获得该基因的核苷酸序列全长，其核苷酸序列为SEQ ID NO：1；
c，基于飞蝗脂肪酸合成酶基因序列SEQ ID NO：1，设计dsRNA上下游引物，其序列分别为SEQ ID NO：3和SEQ ID NO：4，上下游引物均携带T7启动子序列；
d，以上述脂肪酸合成酶基因克隆载体大肠杆菌为模板，SEQ ID NO：3和SEQ IDNO：4为上下游引物，进行PCR扩增，将扩增的PCR产物，纯化后转录合成dsRNA。
一种飞蝗脂肪酸合成酶基因LmFAS2的dsRNA的应用，用于防治害虫。
与现有技术相比本发明具有以下优点：
1、本发明提供的飞蝗脂肪酸合成酶基因LmFAS2的dsRNA，可以显著抑制飞蝗脂肪酸合成酶基因LmFAS2的表达，从而导致飞蝗蜕皮困难死亡或蜕皮后脱水死亡；
2、本发明提供的飞蝗脂肪酸合成酶基因LmFAS2的dsRNA用于飞蝗害虫防治具有专一性和高效性，不存在害虫产生抗药性导致防治效率降低的情况，也不会破坏生态环境，不会对其它非靶标生物造成危害。
附图说明
图1为本发明实施例试验中四龄第1天的飞蝗若虫分别注射核苷酸序列为SEQ IDNO：2的dsLmFAS2和dsGFP，96h后对飞蝗脂肪酸延伸酶基因的转录影响的结果对照图；
图2为本发明实施例试验中飞蝗若虫分别注射核苷酸序列为SEQ ID NO：2的dsRNA和后对飞蝗发育影响的结果对照图；
图3为本发明实施例试验中飞蝗若虫注射核苷酸序列为SEQ ID NO：2的dsRNA后对飞蝗表皮渗透性影响的结果对照图。
具体实施方式
为了进一步阐述本发明的技术方案，下面结合附图及实施例对本发明进行进一步说明。
一种飞蝗脂肪酸合成酶基因LmFAS2的dsRNA，该dsRNA的核苷酸序列为SEQ ID NO：2。
一种飞蝗脂肪酸合成酶基因LmFAS2的dsRNA的制备方法，包括以下步骤：
a，基于飞蝗的转录组数据库，采用生物信息学方法对飞蝗脂肪酸合成酶基因进行搜索，经过序列分析，拼接及比对后，获得飞蝗脂肪酸合成酶基因LmFAS2序列，采用primerpremier5.0软件设计上游引物序列和下游引物序列，并送往生工生物工程(上海)有限公司合成；
b，选取生长健康，大小一致雌雄各半的五龄飞蝗若虫，在体式显微镜下将其表皮快速解剖下来，并冷冻于液氮中，3头一个生物学重复，设置四个生物学重复，依照TaKaRa公司Trizol试剂盒提取RNA，采用M-MLV反转录酶将所提RNA反转录成第一链cDNA，以此做为模板，结合设计飞蝗脂肪酸合成酶基因LmFAS2的上下游引物，通过PCR扩增获得脂肪酸延伸酶基因全长片段，将得到的产物进行纯化，克隆转化至大肠杆菌中，并送往华大生物科技有限公司测序，测序结果与转录组搜索结果比对，验证并获得该基因的核苷酸序列全长，其核苷酸序列为SEQ ID NO：1；
c，基于飞蝗脂肪酸合成酶基因序列SEQ ID NO：1，采用primerpremier5.0软件设计dsRNA上下游引物，其序列分别为SEQ ID NO：3和SEQ ID NO：4，上下游引物均携带T7启动子序列，所有引物均由上海生物工程有限公司合成；
d，以上述脂肪酸合成酶基因克隆载体大肠杆菌为模板，SEQ ID NO：3和SEQ IDNO：4为上下游引物，进行PCR扩增，将扩增的PCR产物，经Gel Extraction Kit(Sigma)试剂盒纯化后按照T7RiboMAXTMExpress RNAi System(Promega)试剂盒说明体外转录合成dsRNA，以绿色荧光蛋白基因GFP为对照，合成dsGFP，使用NaNoDrop 2000(Thermoscientific)进行定量，使其终浓度达到2.5μg/μl，保存于-80℃超级低温冰箱备用。
一种飞蝗脂肪酸合成酶基因LmFAS2的dsRNA的应用，其特征在于：用于防治害虫。
飞蝗脂肪酸合成酶基因dsRNA致死飞蝗试验：
1、飞蝗脂肪酸合成酶基因dsRNA注射
选取生长健康、大小一致、雌雄各半30头四龄第1天若虫进行试验。将合成的dsLmFAS2使用25μl规格微量注射器将4μl顺着血流方法轻轻注射进入若虫侧腹部的二、三腹节之间。同时选取30头若虫设立为对照组。注射相同体积和浓度的dsGFP至对照组体内。分别将注射后飞蝗置于30℃恒温生化培养箱中饲养(光照：黑暗时间＝14h:10h，温度30±2℃，湿度60％)，每天饲喂新鲜小麦幼苗和麦麸。
2、飞蝗脂肪酸合成酶基因沉默检测
各收集9头注射dsGFP和dsLmFAS2，96h后若虫虫体进行总RNA提取，并反转录成第一链cDNA，每组设置3个生物学重复，每个生物学重复3头若虫。采用Real-time PCR方法分别检测目的基因LmFAS2和管家基因Rpl-32的相对表达量，对其沉默效率进行计算，Rpl-32为内参基因，其中***，P<0.001。如图1所示，结果表明，与对照组比较，注射dsLmFAS2后，处理组脂肪酸延伸酶基因表达显著降低，其沉默效率达到91.4％。
3、注射dsRNA后五龄若虫表型观察
如图2所示，四龄若虫注射dsRNA后，对照组在六天后全部成功蜕皮至五龄若虫，且蜕皮后生长发育状况良好。而实验组注射dsLmFAS2后，有13头无法成功蜕皮直至死亡，同时有10头若虫可以成功蜕皮至下一龄期，但在蜕皮至五龄若虫后会因为失水而死亡，两种表型死亡率达到79.4％。
4、注射dsRNA后若虫的伊红染色试验
四龄若虫注射dsRNA后，选取蜕皮困难的实验组以及相同数量正在蜕皮的对照组若虫，置于0.5％伊红溶液，于45℃水浴锅中加热30min，后将附着的旧表皮移除，然后进行多次清洗，观察新表皮的着色情况。如图3所示，结果显示，与对照相比，实验组飞蝗体壁着色更为明显，表明其渗透性增强。
以上显示和描述了本发明的主要特征和优点,对于本领域技术人员而言，显然本发明不限于上述示范性实施例的细节，而且在不背离本发明的精神或基本特征的情况下，能够以其他的具体形式实现本发明。因此，无论从哪一点来看，均应将实施例看作是示范性的，而且是非限制性的，本发明的范围由所附权利要求而不是上述说明限定，因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。不应将权利要求中的任何附图标记视为限制所涉及的权利要求。
此外，应当理解，虽然本说明书按照实施方式加以描述，但并非每个实施方式仅包含一个独立的技术方案，说明书的这种叙述方式仅仅是为清楚起见，本领域技术人员应当将说明书作为一个整体，各实施例中的技术方案也可以经适当组合，形成本领域技术人员可以理解的其他实施方式。
序列表
SEQ ID NO：1
ggggagtgtaatttgttctgtggttttgtgtctgaaatccactggaggcacaatggcaaatgaaaaagttgaagaagttgtgatttctggagtggcaggcgtttttccatacagtgacaatatagatgagttggctgataatcttttctctaagacccatcttgtgaggagtgatgattgccgctggaaagttgcagagaaatccattgtgcctccagctacaggaaaaataaaatctcctgcagtatttgatacagtacactttggaatccatagaaaacagacggagtcgatggatcctctttgttgtcaagtgttagaaagagcatttgaagcagttatcgatgcaggtgtaaacccaaatgaaataagagggacaaatactgctgtatatgtggcaacagcaatgagtgatgcagacaatgtatttgattatgaggccactggtgatggctttggtgtaatggggcacaaccgttcaatgattgcctcaagagttgcttattattttgatgccaaaggtcctacctacgttgttatttctgcttgggcgggaggattacaaactttagaattagcatacaaagcaataagtgaaggaagatgcaaagcagctattattggatccacaaatcttgtgcttttcccttatacaagttatcagatggccaaaatgggtcttcttagcccagatggtgtcactcgctcttttgatgatgcagggaatgggtatggaagaagtgaagcatcagttgccttatttcttcagcgtaaaagagatgccaaacggatttactgtgaagttcttggttctgaagtactctgcatgggagatagaaaaagtttctttccaggcacacaactaaatttctcacgtatcctgagagaattttataataagtttcctgttaacccttcagagctttcctatttggaggctgatggttcaacatttatgggaaatgatcgagaggaactcaatgcagtggacgaggttctccttaaagataggaagactcctttacttattggttctgtgaagtcaaacattggaaatactgaagcatcttcctcactgtgttcaattgtaaaacttattatagcaatgacaagaaaatgcattcccccaaatcttcactacaataagcccatatcaggtgttcctgctctcacaaatgggaaattacaggtggtgacagaaagtacaccatgggaaggtggactggcagctgtaacaagtctgggaattgatggaaattatggccatgcagtgctaaaatattatgaccaaaagaatcctgtaattgaagatgatttaccacgacttgtgacaatgtccagcagaacagaagagggacttaataaaatactgtcaaaagtggagtcaatgcctctggacaaagaatatattagattattacatgagatttatggaacaaatgtccaaaattatatgcacagaggctacacaattttaacacaaggagaaaagaaaagggaaattgagttctgcacagcagcaaaccgtccagtatggttcatattctctggcatgggcagccagtggccacagatgggtacacaactaatgcaactgccagtatttgcagagtcaattaggaagtgtcacgctactctagaaccactgggtgttgatctaatcaatataataacaacagaagataaatcggtatttgataatatccttaacagttttgttggaattgctgccatccagattggattagttgatgttctacgtaaacttgaaatacatccagatgggttaattggccactctgtgggtgaactgggttgtgcatatgcagatggttgtctcacagcagaacaaatgatactttcatcttactatcgaggaaaggcttcgcttgaagcagaacttattaaaggaatgatggctgccattggtttgggttatgaacagataaaagacctactcccaccagggattgaagttgcatgccataattctcgtggcagctgcacactttctggtccaacacaagctgttgaagattttgtggcagtacttacagcaaaaggcatatttgcaaaggcagtgaatgtttcaaatattgcatatcatagtacatacatcaaaccagctgctccactccttcttgattatctgaaaaagatcattccaaacccacagccaagatcatcaaaatggatttgtacatcaatacaacaaaaagactgggaatctgatttgggaaaaactgtgtcagctgaatacttaaccaacaatttactgagctatgtttattttgaagaaggaagtgctcatataccaaagaatgctatatgtattgaaatagctccccatggtctcttacaagctatcctacgaaaatctcttagtgcagattgtactaatattcctctcacaaggcgaggagaaaagcaaggcctggaataccttcttgcagcaattggcaaattgtatctttgtgggctggatcctaaggtggcagaaatttatccgcctgtaccatttccagtatcagcaggaacaccatttatatctccaatttgtcaatgggaccattcggaagagtggccaattatttcagctcgagaggagactatttctggtgaagtaactgtcgacatcagtatggtcaaagaagaaataagagattacagtgaaaatgttttaggtggaaatattgttttgccacctatggcatttctgaaaatagcgctccagcaactagcacatattttggagcggccaagtgagaatatgcctgcagtctttgaaaacgtcttcattcatcgaattgttgttgtaccaagaacaggagaattgaaattcatacttctagttaaaagaggaagtggaaaatttgagtttattgagaatgggcagctaattgtaacaggccaaattattttaccagaggacattgataagcagttcagttctttgccaaaaatagccaacactggtatcaaactgactgaagatgatatttatagtgaatttagacaccgtgatcatcagtacagtggtaaattccgagcaataagctcagcaacagtgaatgatacaggatgtgttgctacaataaaaccatgtggaaaatggacactactggcagaacatttgttgcagttagtagtcttacacaatgcagaagcaagtcaagatcttcactatatttcatcaattagaaggatagtttttgatccatcaaaattgccacagaatttggaagagacagaagctatgttttatgcacactcaaatattgtgaaatgtgatggaatagaaatacatggaactgacataaatgtgcctcctaacggtttttcaaaaaggacagtattgaagattggaactaagaagttcctaccacacaacagctctacttttaagaatataaatgaatttgtggatgtttcaatccaacttatgttagaaagcaaagctgctgaaataaaatccaaaaccaaaggaattgtgaaagacatctgcagtgacagtagtttgtctgaagctgtgaagtcatcattactcaattttccacaaacacagattgatgttgatggctctaatgatttaaaagttggacaagctataggaaaaatatcatcagactgtgtgttgatagtcgcagatgagtcctctttaacaactgctgaatcattacctcaagaaaatgatgtgtttttgcttgctgctctttctgatggaagttcatggaaaccaaaaacaaacattgaaattgtgaaccagtgttatattaatggaaagaagtacttactgttgaagaaggaaagaaaaattgatctgaatatgagcagtgtggtaaacgtaatacctaagacagagaaatggattcagaatctgaagttggctgttgaaaaatgtaaaagctcttctcaaaggctatacgtagtttctaaaacattacctcagtgtggagtggaaaaatttgtctccatcattgataagctctcagacttgaacaacatacggtacttttttattctggatgattctgcaccagaattttccttatctgacaatttctacaagcagcacattcttctagacctgaccattaatgtttatgctgaacgttcgtgggggaccattaggagtgtgttaccatcaagtgaaaatacagttatcgaggaagcatctttacatttaagggccaatgatattgacctgagttacattgggtttaacaaaatggatccatctatcatcactggaacggaaacgaaagaattgcctatgaaactgggcgtagtagattttgctggatgtaacaagaaaggtcaacgaatcatgggtatgatacagccaaacaccctcaagactagtgaacatcttatgtgggacattcctgacagctggagtttagaagatgctgctactgtaccctttgcatttgctatggcatattactcagtaaagatgttagttactcaccagaatcaaggaagattaatacttattcattctggttgctcatcacttggtgtagctgccataactgtggccttaaaagaaggctacacagtatggacaactgtgggatctgatgaggaacgtactgtcctgagacaaacatttccacagataaatgatgaccatatataccactacgatgatcacaaactatacagtaacatcctcatttcaaccaaaggaaatggtctctcattggtaataaacacactcacaggagacaagctggatcattcactaatgtgtattgaatactttgggtcttttgtccagctagcacaagcagaaatggataaatctcacaaactaggactgaacccatttctacgaagtatttcatttcatgctgtatcatgtaacaagttatttgacttacatgatgacgtaaaatcagaattacatatgcatgtcaaagaaggtataagaaaaggtgttgtcaaaccactgaacaggatagttcttaaccagaatgaaacacaaaaagcatttaagctcctgtttgacaagaaaaattaccaaaaagtgctcctgaagtcccattctacacagaagaaacctctagcagtagtaaaggacaatttaaaggtgctgtataactgtgaacctgaaagttcttatttaataataggtggcaaaaatgaagaatggctgagcatgacagaatggcttattaaacatggagcaaagaaggtagttgttgctcatcagtcagacaaaggaaacagctacataaccagaagactcaatttatttaaaaagtattacaattcagagatagtattagagtcagcgtccaaggctagtacaccaaatggtgccacaaatctcttgaaacaaacaaacagcagggggacattgtcagcaatatttttgttgccagtggatcagacagaaaattcaagattaaccgattcaggaaacacgacagcacagatggttaataatatcgatgttgcctctcgctattctccagacttattgtttgtttgcatgttctgtgatggaggagacagttcttgcttggcaagacaaaaaatgggtctcaggggtctctcaataaattggtcccaacaaaatggaagcatttttgattttaaacaatttgtgcagcagctggatgatatactgctttcatttaaagatcccattctaagtgttaccaacattacacctgacttcaaaaatagagataaagcaatagttgaaagattagattacttgttaccaacttccacaaaagaaattgctgaaattggatggaatctgaggaatcaacactacatttatgaaatgtccacactgagtccagaatggaatgaggttaaggattcagtacctgtcttcttagtgtcaggaatttattcacgttacaaacgccacagagagcttgccaacaatattatgtttccattatttaatattcatgcaacaccaattaaatctacaattgaagaatgtgctgaagaaattgttaaggaaatcaaaaaaaatctggccaaagggtgtttatttactaattggggactcatatggaggtgtgctggccctagagatagcagcaaaattagagaagaaaggagaaacagtgcacacatttcttcttgatggtgcacccaaaggaataaaaacggctgtgaaaacccttggagaaggaccacagttgcacagtaacatcatccgcagattcttaggagtttcatcagaaaaggaattgttacagttaccaaactgggatgctcgtctgcagtatgcactatctgtcatagactttccaacagcattagaaaagcaacttgtagctgaaggaatgactgcaatattgaagtatttgactctattactttcatatgaaccaaattatgaactttctgcaaatacttcacttgtagtacctgagtatttagaggaagaaaacagtggactttatgaggttagtggagtgtattacatgtgataatcttcttatatctggctagataatatgtccataaaatatctgtttggaaaataaccctataaaaatataggttacagacttgtcatcattacttaactatattttttttaaaaaaagaaaaaaatctggacctctaaagtgaagaatcttagtatgttcagaatgaaatatataaaaccttcatctgtctgcttttaatagaaaatgagacgaaatttaatggacaggagacaccaaccaaaatatttttttggaaacctgaagtctgtgaatggctgcaagctacctctgagaagccatacataaccagtttaagccaataatcagttttggtgacactggaaggtaccagagtgatagactctgaccatagattagatgtccttcttttccaattgatgccaaaacaacataataaataaatataattttagtgatctagacagtttttattgttattgaaaaaaatgaagatcgtggacttgtacaggaaattcatttcctttacataaaaaatttactgttagttgttatctagcctccgttattgaaattccaatctggagtttcccttgtcaaatattaaaataggcagcacacagtaggaagagagacatgaattggaaggagatggataatggagggaggttgtggggctatctaagcttgtggccagaatggttatgggactgactccccttcgtcatcagctcttcagaactgcaccaatggaagttggccttatgaaacattcaagggatactataaccaccgtgccctcgaactgagataactatcattcctgcaacacatgcctaacagtttccacctccctctattctgtatctccttctaattgatgtctcccttccaactgtgtactgcctaatttaatatttcaattttttaaatattccaatatttttaattcatttttattaatttttttttacatttttttgacacacacacacacctcccccttctcacttccccaccccttccctttacttcattcctaactacatctcacatcttccctttactcttcaatatgctaccacctccactcccctctgcatctcttctctctcactgtcaagatgcttgtcatggtggtcatgtgtgtttgaattatgtctgagtgattgtgtgaaagtgtgtgtactttttctgatgaaggctgtggcaaaacatttaagttgaccactagtgccaatactccttgtgaatgtggttgttttttctacttgcctagctgcagctcagcaactgtctttagagtctgttgctatgtatcctttattattgacattccaacctgaactttccattgttagacatttgtaatccaactgaaggattcttgataggtagcatagtgcatattattttaactgtagaatgtaaagcactcgggtataccaccaaaatgtgttacgatctgtgctggtcatattgtttatttgtgacctggcaaccaataatgacctcagacattttacagatggtgtgattctttgacacataacagcaaacttcttgaatttgagtatcatagggagtatagtttactagttccaatagaagtggggctccagggaaatcatgctgctgtataggctaccctgcagaacaaggatctcaagtgggatcaatgggccttaagccactctcacacagagcatggttcacaagaagtttcctggatggcgcgctcgtcagtcggctgtcgagtaggaatgcggctgaaacacactgcttctctctctcacggtgcaacattgggtgcggaagtgtgcaggcctggcttcgccgggtggacgccaagatgcacaacatggccaagctgctttatttatcaaacaattgtgaagaaactattccgtaaaaaaaaattgattctcacacatcttatagctgcattcttcagcttcatgaagatctatctactattttgtttgtggttatagtttttgtgatattaatggattcagtatagtactgcaagaaatccaaaaattttgcattgaaaaatagaggtcactatgaatttgcgtttggtgcaagttaggtcatatgttgctgcggatgaaatgtaactggaatgttgaattgttcttcggggttggaggggt
SEQ ID NO：2
aaaatgggtcttcttagcccagatggtgtcactcgctcttttgatgatgcagggaatgggtatggaagaagtgaagcatcagttgccttatttcttcagcgtaaaagagatgccaaacggatttactgtgaagttcttggttctgaagtactctgcatgggagatagaaaaagtttctttccaggcacacaactaaatttctcacgtatcctgagagaattttataataagtttcctgttaacccttcagagctttcctatttggaggctgatggttcaacatttatgggaaatgatcgagaggaactcaatgcagtggacgaggttctccttaaagataggaagactcctttacttattggttctgtgaagtcaaacattggaaatactgaagcatcttcctcactgtgttcaattgtaaaacttattatagcaatgacaagaaaatgcattcccccaaatcttcactacaataagccca
SEQ ID NO：3
taatacgactcactatagggaaaatgggtcttcttag
SEQ ID NO：4
taatacgactcactatagggtgggcttattgtagtg。
序列表
<110> 山西大学
<120> 一种飞蝗脂肪酸合成酶基因LmFAS2的dsRNA及其制备方法和应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8605
<212> DNA
<213> 飞蝗(Locusta migratoria)
<400> 1
ggggagtgta atttgttctg tggttttgtg tctgaaatcc actggaggca caatggcaaa 60
tgaaaaagtt gaagaagttg tgatttctgg agtggcaggc gtttttccat acagtgacaa 120
tatagatgag ttggctgata atcttttctc taagacccat cttgtgagga gtgatgattg 180
ccgctggaaa gttgcagaga aatccattgt gcctccagct acaggaaaaa taaaatctcc 240
tgcagtattt gatacagtac actttggaat ccatagaaaa cagacggagt cgatggatcc 300
tctttgttgt caagtgttag aaagagcatt tgaagcagtt atcgatgcag gtgtaaaccc 360
aaatgaaata agagggacaa atactgctgt atatgtggca acagcaatga gtgatgcaga 420
caatgtattt gattatgagg ccactggtga tggctttggt gtaatggggc acaaccgttc 480
aatgattgcc tcaagagttg cttattattt tgatgccaaa ggtcctacct acgttgttat 540
ttctgcttgg gcgggaggat tacaaacttt agaattagca tacaaagcaa taagtgaagg 600
aagatgcaaa gcagctatta ttggatccac aaatcttgtg cttttccctt atacaagtta 660
tcagatggcc aaaatgggtc ttcttagccc agatggtgtc actcgctctt ttgatgatgc 720
agggaatggg tatggaagaa gtgaagcatc agttgcctta tttcttcagc gtaaaagaga 780
tgccaaacgg atttactgtg aagttcttgg ttctgaagta ctctgcatgg gagatagaaa 840
aagtttcttt ccaggcacac aactaaattt ctcacgtatc ctgagagaat tttataataa 900
gtttcctgtt aacccttcag agctttccta tttggaggct gatggttcaa catttatggg 960
aaatgatcga gaggaactca atgcagtgga cgaggttctc cttaaagata ggaagactcc 1020
tttacttatt ggttctgtga agtcaaacat tggaaatact gaagcatctt cctcactgtg 1080
ttcaattgta aaacttatta tagcaatgac aagaaaatgc attcccccaa atcttcacta 1140
caataagccc atatcaggtg ttcctgctct cacaaatggg aaattacagg tggtgacaga 1200
aagtacacca tgggaaggtg gactggcagc tgtaacaagt ctgggaattg atggaaatta 1260
tggccatgca gtgctaaaat attatgacca aaagaatcct gtaattgaag atgatttacc 1320
acgacttgtg acaatgtcca gcagaacaga agagggactt aataaaatac tgtcaaaagt 1380
ggagtcaatg cctctggaca aagaatatat tagattatta catgagattt atggaacaaa 1440
tgtccaaaat tatatgcaca gaggctacac aattttaaca caaggagaaa agaaaaggga 1500
aattgagttc tgcacagcag caaaccgtcc agtatggttc atattctctg gcatgggcag 1560
ccagtggcca cagatgggta cacaactaat gcaactgcca gtatttgcag agtcaattag 1620
gaagtgtcac gctactctag aaccactggg tgttgatcta atcaatataa taacaacaga 1680
agataaatcg gtatttgata atatccttaa cagttttgtt ggaattgctg ccatccagat 1740
tggattagtt gatgttctac gtaaacttga aatacatcca gatgggttaa ttggccactc 1800
tgtgggtgaa ctgggttgtg catatgcaga tggttgtctc acagcagaac aaatgatact 1860
ttcatcttac tatcgaggaa aggcttcgct tgaagcagaa cttattaaag gaatgatggc 1920
tgccattggt ttgggttatg aacagataaa agacctactc ccaccaggga ttgaagttgc 1980
atgccataat tctcgtggca gctgcacact ttctggtcca acacaagctg ttgaagattt 2040
tgtggcagta cttacagcaa aaggcatatt tgcaaaggca gtgaatgttt caaatattgc 2100
atatcatagt acatacatca aaccagctgc tccactcctt cttgattatc tgaaaaagat 2160
cattccaaac ccacagccaa gatcatcaaa atggatttgt acatcaatac aacaaaaaga 2220
ctgggaatct gatttgggaa aaactgtgtc agctgaatac ttaaccaaca atttactgag 2280
ctatgtttat tttgaagaag gaagtgctca tataccaaag aatgctatat gtattgaaat 2340
agctccccat ggtctcttac aagctatcct acgaaaatct cttagtgcag attgtactaa 2400
tattcctctc acaaggcgag gagaaaagca aggcctggaa taccttcttg cagcaattgg 2460
caaattgtat ctttgtgggc tggatcctaa ggtggcagaa atttatccgc ctgtaccatt 2520
tccagtatca gcaggaacac catttatatc tccaatttgt caatgggacc attcggaaga 2580
gtggccaatt atttcagctc gagaggagac tatttctggt gaagtaactg tcgacatcag 2640
tatggtcaaa gaagaaataa gagattacag tgaaaatgtt ttaggtggaa atattgtttt 2700
gccacctatg gcatttctga aaatagcgct ccagcaacta gcacatattt tggagcggcc 2760
aagtgagaat atgcctgcag tctttgaaaa cgtcttcatt catcgaattg ttgttgtacc 2820
aagaacagga gaattgaaat tcatacttct agttaaaaga ggaagtggaa aatttgagtt 2880
tattgagaat gggcagctaa ttgtaacagg ccaaattatt ttaccagagg acattgataa 2940
gcagttcagt tctttgccaa aaatagccaa cactggtatc aaactgactg aagatgatat 3000
ttatagtgaa tttagacacc gtgatcatca gtacagtggt aaattccgag caataagctc 3060
agcaacagtg aatgatacag gatgtgttgc tacaataaaa ccatgtggaa aatggacact 3120
actggcagaa catttgttgc agttagtagt cttacacaat gcagaagcaa gtcaagatct 3180
tcactatatt tcatcaatta gaaggatagt ttttgatcca tcaaaattgc cacagaattt 3240
ggaagagaca gaagctatgt tttatgcaca ctcaaatatt gtgaaatgtg atggaataga 3300
aatacatgga actgacataa atgtgcctcc taacggtttt tcaaaaagga cagtattgaa 3360
gattggaact aagaagttcc taccacacaa cagctctact tttaagaata taaatgaatt 3420
tgtggatgtt tcaatccaac ttatgttaga aagcaaagct gctgaaataa aatccaaaac 3480
caaaggaatt gtgaaagaca tctgcagtga cagtagtttg tctgaagctg tgaagtcatc 3540
attactcaat tttccacaaa cacagattga tgttgatggc tctaatgatt taaaagttgg 3600
acaagctata ggaaaaatat catcagactg tgtgttgata gtcgcagatg agtcctcttt 3660
aacaactgct gaatcattac ctcaagaaaa tgatgtgttt ttgcttgctg ctctttctga 3720
tggaagttca tggaaaccaa aaacaaacat tgaaattgtg aaccagtgtt atattaatgg 3780
aaagaagtac ttactgttga agaaggaaag aaaaattgat ctgaatatga gcagtgtggt 3840
aaacgtaata cctaagacag agaaatggat tcagaatctg aagttggctg ttgaaaaatg 3900
taaaagctct tctcaaaggc tatacgtagt ttctaaaaca ttacctcagt gtggagtgga 3960
aaaatttgtc tccatcattg ataagctctc agacttgaac aacatacggt acttttttat 4020
tctggatgat tctgcaccag aattttcctt atctgacaat ttctacaagc agcacattct 4080
tctagacctg accattaatg tttatgctga acgttcgtgg gggaccatta ggagtgtgtt 4140
accatcaagt gaaaatacag ttatcgagga agcatcttta catttaaggg ccaatgatat 4200
tgacctgagt tacattgggt ttaacaaaat ggatccatct atcatcactg gaacggaaac 4260
gaaagaattg cctatgaaac tgggcgtagt agattttgct ggatgtaaca agaaaggtca 4320
acgaatcatg ggtatgatac agccaaacac cctcaagact agtgaacatc ttatgtggga 4380
cattcctgac agctggagtt tagaagatgc tgctactgta ccctttgcat ttgctatggc 4440
atattactca gtaaagatgt tagttactca ccagaatcaa ggaagattaa tacttattca 4500
ttctggttgc tcatcacttg gtgtagctgc cataactgtg gccttaaaag aaggctacac 4560
agtatggaca actgtgggat ctgatgagga acgtactgtc ctgagacaaa catttccaca 4620
gataaatgat gaccatatat accactacga tgatcacaaa ctatacagta acatcctcat 4680
ttcaaccaaa ggaaatggtc tctcattggt aataaacaca ctcacaggag acaagctgga 4740
tcattcacta atgtgtattg aatactttgg gtcttttgtc cagctagcac aagcagaaat 4800
ggataaatct cacaaactag gactgaaccc atttctacga agtatttcat ttcatgctgt 4860
atcatgtaac aagttatttg acttacatga tgacgtaaaa tcagaattac atatgcatgt 4920
caaagaaggt ataagaaaag gtgttgtcaa accactgaac aggatagttc ttaaccagaa 4980
tgaaacacaa aaagcattta agctcctgtt tgacaagaaa aattaccaaa aagtgctcct 5040
gaagtcccat tctacacaga agaaacctct agcagtagta aaggacaatt taaaggtgct 5100
gtataactgt gaacctgaaa gttcttattt aataataggt ggcaaaaatg aagaatggct 5160
gagcatgaca gaatggctta ttaaacatgg agcaaagaag gtagttgttg ctcatcagtc 5220
agacaaagga aacagctaca taaccagaag actcaattta tttaaaaagt attacaattc 5280
agagatagta ttagagtcag cgtccaaggc tagtacacca aatggtgcca caaatctctt 5340
gaaacaaaca aacagcaggg ggacattgtc agcaatattt ttgttgccag tggatcagac 5400
agaaaattca agattaaccg attcaggaaa cacgacagca cagatggtta ataatatcga 5460
tgttgcctct cgctattctc cagacttatt gtttgtttgc atgttctgtg atggaggaga 5520
cagttcttgc ttggcaagac aaaaaatggg tctcaggggt ctctcaataa attggtccca 5580
acaaaatgga agcatttttg attttaaaca atttgtgcag cagctggatg atatactgct 5640
ttcatttaaa gatcccattc taagtgttac caacattaca cctgacttca aaaatagaga 5700
taaagcaata gttgaaagat tagattactt gttaccaact tccacaaaag aaattgctga 5760
aattggatgg aatctgagga atcaacacta catttatgaa atgtccacac tgagtccaga 5820
atggaatgag gttaaggatt cagtacctgt cttcttagtg tcaggaattt attcacgtta 5880
caaacgccac agagagcttg ccaacaatat tatgtttcca ttatttaata ttcatgcaac 5940
accaattaaa tctacaattg aagaatgtgc tgaagaaatt gttaaggaaa tcaaaaaaaa 6000
tctggccaaa gggtgtttat ttactaattg gggactcata tggaggtgtg ctggccctag 6060
agatagcagc aaaattagag aagaaaggag aaacagtgca cacatttctt cttgatggtg 6120
cacccaaagg aataaaaacg gctgtgaaaa cccttggaga aggaccacag ttgcacagta 6180
acatcatccg cagattctta ggagtttcat cagaaaagga attgttacag ttaccaaact 6240
gggatgctcg tctgcagtat gcactatctg tcatagactt tccaacagca ttagaaaagc 6300
aacttgtagc tgaaggaatg actgcaatat tgaagtattt gactctatta ctttcatatg 6360
aaccaaatta tgaactttct gcaaatactt cacttgtagt acctgagtat ttagaggaag 6420
aaaacagtgg actttatgag gttagtggag tgtattacat gtgataatct tcttatatct 6480
ggctagataa tatgtccata aaatatctgt ttggaaaata accctataaa aatataggtt 6540
acagacttgt catcattact taactatatt ttttttaaaa aaagaaaaaa atctggacct 6600
ctaaagtgaa gaatcttagt atgttcagaa tgaaatatat aaaaccttca tctgtctgct 6660
tttaatagaa aatgagacga aatttaatgg acaggagaca ccaaccaaaa tatttttttg 6720
gaaacctgaa gtctgtgaat ggctgcaagc tacctctgag aagccataca taaccagttt 6780
aagccaataa tcagttttgg tgacactgga aggtaccaga gtgatagact ctgaccatag 6840
attagatgtc cttcttttcc aattgatgcc aaaacaacat aataaataaa tataatttta 6900
gtgatctaga cagtttttat tgttattgaa aaaaatgaag atcgtggact tgtacaggaa 6960
attcatttcc tttacataaa aaatttactg ttagttgtta tctagcctcc gttattgaaa 7020
ttccaatctg gagtttccct tgtcaaatat taaaataggc agcacacagt aggaagagag 7080
acatgaattg gaaggagatg gataatggag ggaggttgtg gggctatcta agcttgtggc 7140
cagaatggtt atgggactga ctccccttcg tcatcagctc ttcagaactg caccaatgga 7200
agttggcctt atgaaacatt caagggatac tataaccacc gtgccctcga actgagataa 7260
ctatcattcc tgcaacacat gcctaacagt ttccacctcc ctctattctg tatctccttc 7320
taattgatgt ctcccttcca actgtgtact gcctaattta atatttcaat tttttaaata 7380
ttccaatatt tttaattcat ttttattaat ttttttttac atttttttga cacacacaca 7440
cacctccccc ttctcacttc cccacccctt ccctttactt cattcctaac tacatctcac 7500
atcttccctt tactcttcaa tatgctacca cctccactcc cctctgcatc tcttctctct 7560
cactgtcaag atgcttgtca tggtggtcat gtgtgtttga attatgtctg agtgattgtg 7620
tgaaagtgtg tgtacttttt ctgatgaagg ctgtggcaaa acatttaagt tgaccactag 7680
tgccaatact ccttgtgaat gtggttgttt tttctacttg cctagctgca gctcagcaac 7740
tgtctttaga gtctgttgct atgtatcctt tattattgac attccaacct gaactttcca 7800
ttgttagaca tttgtaatcc aactgaagga ttcttgatag gtagcatagt gcatattatt 7860
ttaactgtag aatgtaaagc actcgggtat accaccaaaa tgtgttacga tctgtgctgg 7920
tcatattgtt tatttgtgac ctggcaacca ataatgacct cagacatttt acagatggtg 7980
tgattctttg acacataaca gcaaacttct tgaatttgag tatcataggg agtatagttt 8040
actagttcca atagaagtgg ggctccaggg aaatcatgct gctgtatagg ctaccctgca 8100
gaacaaggat ctcaagtggg atcaatgggc cttaagccac tctcacacag agcatggttc 8160
acaagaagtt tcctggatgg cgcgctcgtc agtcggctgt cgagtaggaa tgcggctgaa 8220
acacactgct tctctctctc acggtgcaac attgggtgcg gaagtgtgca ggcctggctt 8280
cgccgggtgg acgccaagat gcacaacatg gccaagctgc tttatttatc aaacaattgt 8340
gaagaaacta ttccgtaaaa aaaaattgat tctcacacat cttatagctg cattcttcag 8400
cttcatgaag atctatctac tattttgttt gtggttatag tttttgtgat attaatggat 8460
tcagtatagt actgcaagaa atccaaaaat tttgcattga aaaatagagg tcactatgaa 8520
tttgcgtttg gtgcaagtta ggtcatatgt tgctgcggat gaaatgtaac tggaatgttg 8580
aattgttctt cggggttgga ggggt 8605
<210> 2
<211> 481
<212> DNA
<213> 飞蝗(Locusta migratoria)
<400> 2
aaaatgggtc ttcttagccc agatggtgtc actcgctctt ttgatgatgc agggaatggg 60
tatggaagaa gtgaagcatc agttgcctta tttcttcagc gtaaaagaga tgccaaacgg 120
atttactgtg aagttcttgg ttctgaagta ctctgcatgg gagatagaaa aagtttcttt 180
ccaggcacac aactaaattt ctcacgtatc ctgagagaat tttataataa gtttcctgtt 240
aacccttcag agctttccta tttggaggct gatggttcaa catttatggg aaatgatcga 300
gaggaactca atgcagtgga cgaggttctc cttaaagata ggaagactcc tttacttatt 360
ggttctgtga agtcaaacat tggaaatact gaagcatctt cctcactgtg ttcaattgta 420
aaacttatta tagcaatgac aagaaaatgc attcccccaa atcttcacta caataagccc 480
a 481
<210> 3
<211> 37
<212> DNA
<213> 飞蝗(Locusta migratoria)
<400> 3
taatacgact cactataggg aaaatgggtc ttcttag 37
<210> 4
<211> 36
<212> DNA
<213> 飞蝗(Locusta migratoria)
<400> 4
taatacgact cactataggg tgggcttatt gtagtg 36
Claims (4)
1.一种飞蝗脂肪酸合成酶基因LmFAS2的dsRNA，其特征在于：该dsRNA的核苷酸序列为SEQ ID NO：2。
2.一种飞蝗脂肪酸合成酶基因LmFAS2的dsRNA的制备方法，其特征在于：包括以下步骤：
a，基于飞蝗的转录组数据库，采用生物信息学方法对飞蝗脂肪酸合成酶基因进行搜索，经过序列分析，拼接及比对后，获得飞蝗脂肪酸合成酶基因LmFAS2序列，设计上游引物序列和下游引物序列，并合成；
b，选取生长健康，大小一致雌雄各半的五龄飞蝗若虫，将其表皮快速解剖下来，并提取RNA，将所提RNA反转录成第一链cDNA，以此做为模板，结合设计飞蝗脂肪酸合成酶基因LmFAS2的上下游引物，通过PCR扩增获得脂肪酸延伸酶基因全长片段，将得到的产物进行纯化，克隆转化至大肠杆菌中，并测序，测序结果与转录组搜索结果比对，验证并获得该基因的核苷酸序列全长，其核苷酸序列为SEQ ID NO：1；
c，基于飞蝗脂肪酸合成酶基因序列SEQ ID NO：1，设计dsRNA上下游引物，其序列分别为SEQ ID NO：3和SEQ ID NO：4，上下游引物均携带T7启动子序列；
d，以上述脂肪酸合成酶基因克隆载体大肠杆菌为模板，SEQ ID NO：3和SEQ ID NO：4为上下游引物，进行PCR扩增，将扩增的PCR产物，纯化后转录合成dsRNA。
3.根据权利要求2所述的一种飞蝗脂肪酸延伸酶基因LmElo的dsRNA的制备方法，其特征在于：所述步骤d中纯化后转录合成dsRNA具体操作为经Gel Extraction Kit(Sigma)试剂盒纯化后按照T7 RiboMAXTMExpress RNAi System(Promega)试剂盒说明体外转录合成dsRNA。
4.一种飞蝗脂肪酸合成酶基因LmFAS2的dsRNA的应用，其特征在于：用于防治害虫。
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| CN105755006A (zh) * | 2016-03-29 | 2016-07-13 | 山西大学 | 飞蝗翅特异表皮蛋白基因及其dsRNA的应用 |
| CN106636148A (zh) * | 2017-03-01 | 2017-05-10 | 山西大学 | 一种飞蝗细胞色素P450还原酶基因dsRNA及其应用 |
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| CN106636148A (zh) * | 2017-03-01 | 2017-05-10 | 山西大学 | 一种飞蝗细胞色素P450还原酶基因dsRNA及其应用 |
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