CN1409766A - 转基因昆虫 - Google Patents
转基因昆虫 Download PDFInfo
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- CN1409766A CN1409766A CN00817133A CN00817133A CN1409766A CN 1409766 A CN1409766 A CN 1409766A CN 00817133 A CN00817133 A CN 00817133A CN 00817133 A CN00817133 A CN 00817133A CN 1409766 A CN1409766 A CN 1409766A
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Abstract
一种遗传修饰昆虫胚的方法,包括:首先在防止或延迟昆虫卵绒毛膜硬化的条件下处理昆虫卵,然后向卵中注射一种转座因子,使该因子能整合到胚基因组中。该方法允许对蚊子进行修饰,这可预防宿主寄生虫的传播。
Description
发明领域
本发明涉及昆虫的遗传操作。特别是,本发明涉及蚊子的遗传操作。
发明背景
疟疾是当今世界上最重要的寄生虫病,是非洲的主要健康威胁之一,在那里,世界人口的10%患有世界上90%以上的疟疾感染。
疟疾是由疟原虫(Plasmodium)属的原生动物寄生虫引起的。在公认的四种人寄生虫(恶性疟原虫(P.falciparum)、间日疟原虫(P.vivax)、卵形疟原虫(P.ovale)和三日疟原虫(P.malariae))中,恶性疟原虫是最危险的,是死亡率的主要原因。
人疟疾寄生虫由按蚊属(Anopheles)的蚊子传播。在接近500种已知的按蚊类型中至少有20种与疟疾传播有关。在亚撒哈拉非洲,传播主要由3种按蚊种引起,即冈比亚按蚊(A.gamibiae)、***按蚊(A.arabiensis)和催命按蚊(A.funestus)。这3个种代表恶性疟原虫世界中最有效的媒介***。它们的分布受干燥环境、咸水、低温的限制,对于冈比亚按蚊和***按蚊,受天然森林和湿润热带草原地区的厚植被的限制。这3种非洲蚊子作为疟疾载体是最有效的,因为它们明显地偏爱人作为宿主,以及它们适应人类引起的环境改变的能力。在亚洲,最有效的疟疾载体是斯氏按蚊(A.stephensi)。
基于应用杀虫剂的控制措施不能控制极高的恶性疟原虫接种率。而且,杀虫剂抗性的同时发生,结合与它们的应用有关的生态学成本,产生了对控制寄生虫的备选方法的需要。通过大量使用抗疟药进行的尝试未获成功,这部分上是由于恶性疟原虫的多药耐药株的快速扩散。
已经提出了生物学控制措施作为应用杀虫剂控制疟疾扩散的备选方法。产生对寄生虫的发育有抗性(refractory)、因而不能传播感染的宿主昆虫是控制疟疾的一种可能的方法。昆虫宿主支持寄生虫发育和传播的能力被称为载体能力。库蚊属(Culex)和伊蚊属(Aedes)的蚊子包括经常以人为食但不能传播疟疾的种。其机制不同,通常是种特异的。生理学基础和遗传学基础不完全了解。不能传播疟疾能力可能是由于在蚊子中缺乏寄生虫正常发育所需的某些关键因素,或者可能是抑制寄生虫发育的其它某些因素的作用的结果。
Rosenburg等人,Insect Mol.Biol.,1985;7:1-10表明,弗氏按蚊(A.freeborni)对猿寄生虫诺氏疟原虫(P.knowlesi)有抗性,因为子孢子不能识别并穿透蚊子的唾液腺。
负责蚊子对特定寄生虫抗性的基因的鉴定和作图是分子生物学家的一个主要目的。一旦向蚊子基因组中导入DNA的技术可以应用,决定特定种的易感性或抗性的基因的操作对于预防疟疾传播可能是极其重要的,于是能发展向野生群体中导入抗性基因的方法(Curtis和Graves,J.Trop.Med.Hyg.,1988;91:43-48)。而且,昆虫具有多种防御机制,包括多种肽在体内的产生,保护它们免于细菌和真菌感染。抗细菌肽中有昆虫防卫素和杀菌肽,而drosomycin是研究得最多的抗真菌肽。已经显示,这些肽具有干扰疟疾寄生虫发育的能力。
缺乏建立的转化蚊子DNA的技术已经严重阻碍了通过遗传操作控制载体携带疾病的努力。不育雄性的产生作为一种遗传控制方法已经在根除北美和中美和利比亚的丽蝇(screw-worm fly)中取得成功(Krafsur等人,Parasitology Today,1987;3:131-137)。然而,利用该方法控制蚊群的努力至今仍未成功,主要是由于蚊子的生殖策略,包括多产、短的世代时间和在现有种群破坏后使一个地区快速种群恢复的能力。将蚊群替换为不能传播寄生虫的蚊群是抑制蚊子种的一种有效的备选方法。
此外,向昆虫中导入表达能干扰寄生虫生命周期的抗寄生虫剂的外源基因可能也是希望的。
例如,已经提出了用修饰的Wolbachia共生生物向按蚊中导入外源基因(Curtis和Sinkins,Parasitology,1998;116 suppl:111-115)。Wolbachia是一种潜在有用的基因,因为它是母系遗传的,并且在感染的雄性与未感染的雌***配后导致不育。然而,至今没有报道关于蚊子转化的数据,这是由于向蚊子基因组中导入外源DNA的困难。
已经应用P转座因子成功地进行了果蝇的遗传操作。转座因子能用来向果蝇中导入异源基因,来改变昆虫的表型。其它转座因子也曾经被成功导入果蝇基因组中,包括来自黑尾果蝇(D.Melanogaster)的Hobo、来自D.maurifiana的mariner和来自D.hydei的Minos(Blackman等人,EMBO J,1989;8:211-217;Garza等人,Genetics,1991;128:303-310;Loukeris等人,Proc.Natl.Acad.Sci.USA,1995;92:9485-9489)。
在果蝇中用Hertnes、mariner和Minos获得的令人鼓舞的结果支持了利用转座因子作为DNA输送载体在蚊子中实现种系转化的可能性。然而,没有转座因子显示能在按蚊中转座。
外源DNA向按蚊胚中的导入是转化方法中另一个重要的限制步骤。昆虫胚周围是刚性结构——绒毛膜,它在产卵后快速硬化,使得向按蚊胚中注射DNA成为极为困难且耗费时间的方法。产卵几分钟之后,卵已经极硬,如不杀死胚,难以用常用的针头穿透。注射的胚的存活率通常极低,因此需要注射大量胚以获得显著数量的存活者。而且,当果蝇卵的绒毛膜可通过漂白去除时,按蚊胚对卵壳的去除极为敏感,卵壳提供结构支持和保护,并且允许气体交换而使水分损失减为最少。
因此,向按蚊基因组中导入外源基因的可靠技术的建立面临两个主要问题:1)在按蚊中能成功转座的DNA输送载体的发展;和2)克服向蚊子胚中注射DNA的技术困难的新技术的建立。
发明概述
本发明至少部分基于首先操作绒毛膜防止或延迟硬化过程能利于向昆虫胚中注射异源DNA的事实。然后能向昆虫基因组中注射合适的转座因子。
根据本发明的一个方面,一种遗传修饰昆虫胚的方法包括下列步骤:
(i)在防止或延迟昆虫卵绒毛膜硬化的条件下处理昆虫卵;和
(ii)向卵中注射一种转座因子,使该因子能整合到胚基因组中。
该昆虫优选地是蚊子,更优选地是一种按蚊。
根据本发明的另一方面,通过抑制参与硬化过程的酶防止或延迟绒毛膜的硬化。在本发明的方法中可以使用化合物对硝基苯-p’-胍基苯甲酸(p-nitrophenyl-p’-guanidinobenzoate)延迟绒毛膜的硬化。
根据本发明的另一方面,遗传修饰的按蚊可如下获得:
i.在防止或延迟蚊子卵绒毛膜硬化的条件下处理按蚊胚的卵;和
ii.向卵中注射一种转座因子,该转座因子能整合到蚊子胚的基因组中。
根据本发明的另一方面,使用对硝基苯-p’-胍基苯甲酸延迟昆虫卵绒毛膜的硬化。
根据另一方面,用Minos转座因子将异源DNA转移到按蚊胚的基因组中。
本发明提供了一种用于转化昆虫(特别是按蚊)基因组的有效的基因转移技术。
这使得昆虫(特别是按蚊)能被遗传修饰而显示特别的性状,或修饰昆虫以防止致病寄生虫的扩散。该技术的广泛适用性对于技术人员是显然的,他们可以改变现有的遗传操作(如对果蝇实行的),用于其它昆虫,如按蚊。
附图描述
图1显示用于向蚊子胚中转座的载体(MinHyg)。在该图中,ActinP代表来自黑尾果蝇的Actin5C启动子;hspP代表来自黑尾果蝇的热休克启动子hsp70;hspT代表热休克终止序列;AmpR代表氨苄青霉素抗性基因;HygR代表潮霉素抗性基因;ML和MR代表minos转座因子的左臂和右臂,反向重复用黑三角代表;H、E和N分别代表限制酶HindII、EcoRI和NotI。
发明描述
如上所述,本发明的一个重要方面是在防止或延迟昆虫卵绒毛膜硬化的条件下对昆虫卵的处理。绒毛膜的硬化由一系列酶反应介导,第一种酶是酚氧化酶。其它酶包括多巴脱羧酶、多巴胺N-乙酰转移酶和N-乙酰多巴胺去饱和酶。用抑制剂靶向这些酶是延迟或防止绒毛膜硬化过程的一种有用的方法。抑制剂可以是竞争性或非竞争性抑制剂。在本发明中有用的酚氧化酶抑制剂的例子包括谷胱甘肽、二乙基二硫氨基甲酸、1-苯基-3-(2-噻唑基)-2-硫脲和对硝基苯-p’-胍基苯甲酸。其中,优选对硝基苯-p’-胍基苯甲酸。其它抑制剂对于技术人员是显然的,或者可以用标准酶抑制试验鉴定。
这些抑制剂一般溶解于等渗溶液中,以防止胚的膨胀。
能够容易地确定适用于本发明的抑制剂的量。对于对硝基苯-p’-胍基苯甲酸,发现0.1mM的浓度是合适的。
延迟(减缓)而不是防止硬化过程可能是优选的。因此,可以优选使用竞争性抑制剂,它能通过加入过量的酶底物来代替。此外,也可以随时间变化应用抑制剂,从而使酶作用于其天然底物。延迟硬化应当延迟足以向卵中导入核酸物质的一段时间。这可能只需要几个小时的延迟。
向卵中***核酸可以通过显微注射进行。施行方法对于技术人员是显然的,使用常规装置。
核酸分子可以是含有将在昆虫胚中表达的异源基因的载体或质粒形式。也可存在调节序列,包括转录启动子、增强子和起始信号。导入核酸分子的目的可以是产生具有特定遗传性状的转基因昆虫。产生转基因动物和昆虫的技术周知,可能适用于本发明。
利用转座因子将核酸整合到昆虫基因组中。转座酶通常促进整合(转座),转座因子通常包含用于将转座酶引导到正确位置起始切割的反向重复。含有与异源基因结合(在基因融合中)的转座因子的遗传构件可以用常规技术制备,并***昆虫卵中产生转基因昆虫。
除了异源基因之外,转座因子还可含有确保能发生成功表达的调节因子。
可以根据对其它生物(如果蝇)进行的实验鉴定在本发明中有用的转座因子。例如,来自家蝇(Musca domestica)的Hermes(Atkinson等人,Proc.Natl.Acad.Sci.USA,1993;90:9693-9697)能在黑尾果蝇中转座。来自D.mauritania的Mariner(Haymer和Marsh,Dev.Genet.,1986;6:281-291)能在Bactrocera tryoni中转座。
一种优选的转座因子是在Drosophila hydei中发现的Minos(Franz和Savakis,Nucleic Acids Res.,1991;19:6646)。现在发现,minos转座酶能介导向按蚊基因组中的精确***,并使得发生质粒间转座。因此,在一个优选实施方案中,本发明可以应用Minos转座因子进行,以便优选地在minos转座酶的存在下,将异源核酸分子整合到昆虫胚的基因组中。转座因子可以是质粒载体与外源基因一起的形式,并且还含有调节序列,如启动子。在一个优选实施方案中,该启动子是来自黑尾果蝇的actin5C启动子。在另一个优选实施方案中,minos转座酶基因位于分开的辅助质粒上,用于向胚中分开导入。
可以利用转座因子向昆虫胚中整合一种能在体内表达的异源基因。此外,可能需要转座因子的整合来整合一种能用来破坏特定基因表达的异源多核苷酸。例如,一种RNA分子可用于基因沉默。
可以用异源基因控制寄生虫如疟原虫的传播。例如,该基因可以编码一种保护昆虫免受感染的产物,或者编码一种能干扰寄生虫生命周期的抗寄生虫剂。一些抗细菌肽众所周知,包括可以应用的防卫素。此外,也可以用该基因产生不育雄性,它们可以作为遗传控制的工具。已经提出在果蝇中使用性别特异性启动子(Thomas等人,Science,2000;287(5462):2474-2476),并且可以在本发明中使用。也可以应用Wolbachia基因。也可以导入***基因,它们能通过暴露于某些化学物质激活。其它合适的基因对于技术人员是显然的。
转座因子也可以在鉴定具有杀昆虫活性的化合物或产物的试验中使用,或者用于对负责如蚊子对特定寄生虫抗性的基因作图。能利用向基因组中***外源或异源基因鉴定位于基因组中的增强子元件。显著水平的基因产物不可检测,除非转座因子***到邻近含有增强子元件的区域。也可以利用转座因子进行体内定点诱变,如Banga和Boyd,Proc.Natl.Acad.Sci.USA,1992;89:1735-1739所述。
下列实施例说明本发明。
实施例
在下列实验中,利用称为MinHyg的质粒载体(示于图1)完成异源基因向按蚊基因组中的整合。如图1所示,选择绿色荧光蛋白基因GFPS65T(GFP)(Heim等人,Nature,1995;373:663-664)作为报道基因,用来显示完成DNA的成功整合。
选择来自黑尾果蝇actin5C基因的肌动蛋白启动子驱动GFPS65T标记的表达(Fyrberg等人,Cell,1983;33:115-123)。
在诱导型热休克蛋白70(hsp70)启动子控制下的潮霉素基因也被掺入载体中,作为不能完成GFP选择的事件中的选择性标记。
该实验如下进行。使吸血斯氏按蚊在吸血48-72小时后产卵。将卵置于培养皿中,其中含有在溶于等渗缓冲液(150mM NaCl,5mMKCl,10mM HEPES,2.5mM CaCl2,pH7.2)的0.1mM对硝基苯-p’-胍基苯甲酸(NPGB)溶液(Sigma cat.N8010)中浸泡的3mm纸。NPGB不溶于水;它首先在DMSO中溶解,然后加入等渗缓冲液,形成0.1mM终溶液。等渗缓冲液的使用是必需的,因为它防止胚膨胀。在发生第一次产卵30分钟后,从蚊子笼中取出培养皿。然后将卵置于NPGB中直至注射,注射在产卵90-120分钟后进行。将总共约30个胚置于用等渗缓冲液湿润的纸覆盖的载玻片上,其后极对准,并朝向载玻片的内部。胚一开始干燥,就通过施加轻微的压力将它们转移到一端粘贴有一条双面胶带的另一个载玻片上。然后用水饱和卤化碳油覆盖胚,防止进一步干燥。
利用微量加样器吸头(Eppendorf)将玻璃针头(EppendorfFemtotips)中装入2μl DNA溶液。向胚中显微注射100μg/ml无内含子辅助质粒pHSS6hsILMi20(Klinakis等人,Insect Mol.Biol.2000;9(3):269-275)和质粒MinHyg(400μg/ml)的混合物。该辅助质粒提供Minos转座必需的转座酶活性,而质粒MinHyg含有克隆在Minos的反向末端重复中的GFP。显微注射用Eppendorf transjector 5246显微操作器在10×放大倍数下进行。针头以15°角***胚的后极。注射体积通过调节注射压力和时间来控制。注射后,借助于刷子从卤化碳油中轻轻取出胚,转移到新的培养皿中,其中含有用等渗缓冲液浸泡的堆积滤纸层,以防止卵漂浮。然后使它们孵化。在紫外线下分析孵化的幼虫来检测GFP表达。
经荧光监测,平均29%的注射胚存活,约50%的孵化幼虫显示强烈的GFP瞬时表达。到成虫期(G0)的存活率平均为10%,是成功转化的良好预测标志。在得到16%成虫存活率的两次实验中,69只G0蚊子的后代在所分析的10539只G1幼虫中产生92只荧光个体。随后确定92只荧光G1个体来自于最少5只独立的G0起始个体,代表7%的转化频率(5/69存活成虫)。该频率高于在使用以white基因标记物标记的Minos的黑尾果蝇和C.capitata转化实验中所报道的(Loukeris等人,Science,1995;270:2002-2005,和Proc.Natl.Acad.Sci.USA,1995;92:9485-9489)。
这些成功的实验第一次提供了引人注目的证据,证明按蚊的种系转化是可行的,Minos是完成这种转化的出色候选者。
Claims (22)
1.一种遗传修饰昆虫胚的方法,其包括下列步骤:
i.在防止或延迟昆虫卵绒毛膜硬化的条件下处理昆虫卵;和
ii.向卵中注射一种转座因子,使该因子能整合到胚基因组中。
2.根据权利要求1的方法,其中该昆虫是一种按蚊。
3.根据权利要求2的方法,其中蚊子是冈比亚按蚊、***按蚊或斯氏按蚊。
4.根据上述任一权利要求的方法,其中转座因子是minos。
5.根据上述任一权利要求的方法,其进一步包括向卵中注射含有一种能在体内表达的转座酶基因的载体。
6.根据上述任一权利要求的方法,其中转座因子含有一种在整合到胚后能够表达的异源基因。
7.根据权利要求6的方法,其中该异源基因编码一种可防止宿主寄生虫传播的产物。
8.根据权利要求7的方法,其中该产物是一种抗细菌剂。
9.根据权利要求7或权利要求8的方法,其中宿主寄生虫是恶性疟原虫。
10.根据权利要求6的方法,其中该基因是一种***基因。
11.根据权利要求6的方法,其中该基因产物导致雄性不育。
12.根据上述任一权利要求的方法,其中通过抑制参与绒毛膜硬化的酶防止或延迟绒毛膜的硬化。
13.根据权利要求12的方法,其中该酶是酚氧化酶。
14.根据权利要求12或权利要求13的方法,其中该抑制剂是对硝基苯-p’-胍基苯甲酸。
15.一种遗传修饰的按蚊,可如下获得:
i.在防止或延迟蚊子卵绒毛膜硬化的条件下处理按蚊胚的卵;和
ii.向卵中注射一种转座因子,该转座因子能整合到蚊子胚的基因组中。
16.根据权利要求15的蚊子,其中该转座因子是Minos。
17.根据权利要求15或权利要求16的蚊子,其中该转座因子含有如权利要求6-11中任一项所述的异源基因。
18.参与绒毛膜硬化过程的酶的抑制剂的用途,用来防止或延迟绒毛膜的硬化。
19.根据权利要求18的用途,其中该酶是酚氧化酶。
20.根据权利要求18或权利要求19的用途,其中该抑制剂是对硝基苯-p’-胍基苯甲酸。
21.Minos转座因子的用途,用于向按蚊胚的基因组中转移异源基因。
22.根据权利要求21的用途,其中该异源基因如权利要求6-11中任一项所述。
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN107466974A (zh) * | 2017-09-22 | 2017-12-15 | 广州威佰昆生物科技有限公司 | 一种用于显微注射的稻飞虱卵处理方法 |
CN110024749A (zh) * | 2019-04-11 | 2019-07-19 | 遵义市林业科学研究所 | 一种金小蜂林间保育装置及保育方法 |
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EP2059588A4 (en) | 2006-08-29 | 2010-07-28 | Commw Scient Ind Res Org | FATTY ACID SYNTHESIS |
KR102197208B1 (ko) | 2012-06-15 | 2021-01-04 | 커먼웰쓰 사이언티픽 앤 인더스트리알 리서치 오거니제이션 | 식물 세포에서 장쇄 다중불포화 지방산의 생성 |
PE20170253A1 (es) | 2013-12-18 | 2017-04-14 | Nuseed Pty Ltd | Lipido que comprende acidos grasos poliinsaturados de cadena larga |
CA2953008C (en) | 2014-06-27 | 2024-03-19 | Nuseed Pty Ltd | Lipid comprising docosapentaenoic acid |
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US5348874A (en) * | 1992-09-14 | 1994-09-20 | Institute For Molecular Biology And Biotechnology/Forth | Eukaryotic transposable element |
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- 2000-12-13 US US10/148,772 patent/US20030033622A1/en not_active Abandoned
- 2000-12-13 CN CN00817133A patent/CN1409766A/zh active Pending
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107466974A (zh) * | 2017-09-22 | 2017-12-15 | 广州威佰昆生物科技有限公司 | 一种用于显微注射的稻飞虱卵处理方法 |
CN107466974B (zh) * | 2017-09-22 | 2019-12-20 | 广州威佰昆生物科技有限公司 | 一种用于显微注射的稻飞虱卵处理方法 |
CN110024749A (zh) * | 2019-04-11 | 2019-07-19 | 遵义市林业科学研究所 | 一种金小蜂林间保育装置及保育方法 |
Also Published As
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US20030033622A1 (en) | 2003-02-13 |
AP2002002514A0 (en) | 2002-06-30 |
MXPA02005960A (es) | 2003-10-14 |
OA12120A (en) | 2006-05-05 |
JP2004500064A (ja) | 2004-01-08 |
BR0016398A (pt) | 2002-12-03 |
GB9929681D0 (en) | 2000-02-09 |
EP1242607A1 (en) | 2002-09-25 |
WO2001044483A1 (en) | 2001-06-21 |
AU1871901A (en) | 2001-06-25 |
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