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随着基因测序技术的发展,基因编辑系统逐渐成为生物科学领域研究的有力工具,也是昆虫基因功能研究的重要手段。2013年第三代基因编辑技术Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9(CRISPR/Cas9)被首次应用在真核细胞中成功进行了基因编辑[1],随后该技术以其操作简便、材料成本低和效率高等优势,被迅速应用于人类细胞[2-4]、小鼠(Mus musculus)[5]、大鼠(Rattus norvegicus)[6]、斑马鱼(Danio rerio)[7]、酵母(Saccharomyces cerevisiae)[8]、果蝇(Drosophila melanogaster)[9]和家蚕(Bombyx mori)[10]等的研究中。本研究系统综述了CRISPR/Cas9技术在非模式昆虫色素表型、杀虫剂抗性和神经行为学研究中的应用情况及优化改良方案,旨在为昆虫基因学的深入研究和农业害虫的绿色防控提供参考。
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在昆虫基因功能研究中,遗传学手段主要依靠基因可遗传的操纵。1982年,P element在果蝇中实现敲除而使基因功能丧失。然而在非模式昆虫中,直到21世纪90年代才利用Mariner,piggyBac等转座子技术实现,但繁琐、成功率低。虽然基于双链RNA的基因沉默技术在昆虫中广泛使用,但其存在转染效率低、遗传不稳定等缺点。自从2013年张锋和Church两个团队相继报道CRISPR/Cas9系统在人和小鼠细胞中成功实现基因的定点编辑以来[1,4],此技术已广泛应用于昆虫基因功能研究当中,其中主要是农业昆虫(表1),这极大地推进了昆虫科学研究的发展。
表 1 CRISPR/Cas9技术在非模式生物昆虫中的部分应用
目 物种 基因 功能 敲除/敲入 导入形式 参考文献 鞘翅目
Coleoptera马铃薯甲虫
Leptinotarsa decemlineatavest 翅膀发育 敲除 Cas9 RNP [28] 赤拟谷盗
Tribolium castaneumEGFP 荧光基因 敲除/敲入 U6-sgRNA质粒, Cas9 mRNA/sgRNA [29] cardinar 色素沉着 敲除 Cas9 protein/sgRNA [30] vermillion 色素沉着 敲除 Cas9 protein/sgRNA [31] 异色瓢虫
Harmonia axyridisnAChRα6 杀虫剂抗性 敲除 Cas9 protein/sgRNA [32] 鳞翅目
Lepidoptera家蚕
Bombyx moriPHYHD1 卵子大小 敲除 Cas9 protein/sgRNA [33] tim 昼夜行为节律 敲除 U6-sgRNA质粒 [34] BLOS2 色素沉着 敲除 a. Cas9/sgRNA质粒
b. Cas9 mRNA/sgRNA[35] a. NHEJ-related genes,
b. TUDOR-SNa. 细胞生长速率
b. 参与应激颗粒形成a. 敲除
b. 敲入Cas9/gRNA质粒 [36] ggnbp2 配子发生 敲除 U6-sgRNA质粒 [37] 棉铃虫
Helicoverpa armigeraSNMP2 性信息素识别 敲除 Cas9 mRNA/sgRNA [38] ABCC2, ABCC3 杀虫剂抗性 敲除 Cas9 protein/sgRNA [39] 柑橘凤蝶
Papilio xuthusyellow, spineless 色素沉着 敲除 Cas9 protein/sgRNA [40] 小红蛱蝶
Vanessa carduiyellow, spineless 色素沉着 敲除 Cas9 protein/sgRNA [40] 斜纹夜蛾
Spodoptera lituraEbony 色素沉着 敲除 Cas9 mRNA/sgRNA [41] 欧洲玉米螟
Bacillus thuringiensisABCC2 杀虫剂抗性 敲除 Cas9 protein/sgRNA [42] 二化螟
Chilo suppressalisEbony 色素沉着 敲除 Cas9 protein/sgRNA [43] 小菜蛾
Plutella xylostellaABCC2, ABCC3 杀虫剂抗性 敲除 Cas9 protein/sgRNA [44] 番茄夜蛾
Tuta Absolutacinnabar 色素沉着 敲除 Cas9 protein/sgRNA [45] 膜翅目
Hymenoptera印度跳蚁
Harpegnathos saltatorOrco 信息素感知 敲除 Cas9 protein/sgRNA [46] 西方蜜蜂
Apis melliferaMrjp1 雄性发育 敲除 Cas9 mRNA/sgRNA [47] a. Mrjp1,
b. Pax6a. 雄性发育
b. 胚胎器官的形态发育敲除 Cas9 protein/sgRNA [48] yellow-y 色素沉着 敲除 Cas9 protein/sgRNA [49] 丽蝇蛹集金小蜂
Nasonia vitripenniscinnabar 色素沉着 敲除 Cas9 protein/sgRNA [50] cinnabar 色素沉着 敲除 Cas9 RNP [51] 半翅目
Hemiptera褐稻虱
Nilaparvata
lugensCSAD 色素沉着 敲除 Cas9 RNP [52] InR1, InR2 调节发育和寿命 敲除 Cas9 RNP [53] 烟粉虱
Bemisia tabaciwhite 色素沉着 敲除 Cas9 RNP [54] TRP 温度感知 敲除 Cas9 mRNA/sgRNA [55] 翅叶蝉
Homalodisca vitripenniscinnabar, white 色素沉着 敲除 Cas9 protein/sgRNA [56] 绿盲蝽
Apolygus lucorumwhite 色素沉着 敲除 Cas9 protein/sgRNA [57] 直翅目
Orthoptera东亚飞蝗
Locusta migratoriaOr35 气味受体 敲除 Cas9 protein/sgRNA [58] dsRNase2 / 敲除 Cas9 RNP [59] 双斑蟀
Gryllus bimaculatusUbx, abd-A 调节组织器官形态发生 敲除/敲入 Cas9 mRNA/sgRNA [60] 双翅目
Diptera埃及伊蚊
Aedes aegyptiKu70, lig4 DNA连接与转录等功能 敲除/敲入 Cas9 mRNA/sgRNA [61] dsx, sxl 性别发生 敲除 Cas9 protein/sgRNA [62] Op1, Op2 视觉感知 敲除 U6-gRNA质粒 [63] 冈比亚按蚊
Anopheles gambiaeFREP1 疟原虫易感性及自身生长发育 敲除 U6-gRNA质粒 [64] -
早期基因敲除技术集中研究昆虫体色、眼色和翅色等色素基因,对于探索昆虫的遗传发育具有重要意义。在褐稻虱(Nilaparvata lugens)中,Chen等[52]敲除黑色素代谢基因CSAD(cysteine sulfinic acid decarboxylase)诱导G0代的突变,证明CSAD是成虫正常体色的必需基因;Xue等[65]成功验证cinnabar和white基因的沉默会引起褐稻虱红色复眼和白色复眼可遗传突变。Khan等[66]利用CRISPR/Cas9技术敲除棉铃虫(Helicoverpa armigera)体细胞的色素沉着基因white、brown、scarlet和ok,结果发现:white的敲除对卵、一龄幼虫和成虫眼色有影响;scarlet敲除后产生了一龄无色幼虫和黄色成虫;brown敲除不影响其色素沉着表型;ok的敲除使幼虫角质层变透明及眼色变黑。昆虫的翅色通常也是由多基因决定的。研究人员对偏瞳蔽眼蝶(bicyclus anynana)的眼色进行研究,发现通过CRISPR/Cas9系统敲除TH、DDC、aaNAT、yellow和ebony基因会影响其翅膀的结构组成和翅色[67]。
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研究昆虫对杀虫剂的抗性机制对防治害虫有重要作用。2016年,Wang等[68]通过基因组敲除技术证明棉铃虫CAD基因是Bt(Bactospeine)杀虫剂CrylAc毒素受体;2017年,该团队又通过基因敲除分别建立了1个ABCA2基因第二外显子上2 bp缺失的突变品系和1个类似r2突变的5 bp缺失品系,证明了棉铃虫ABCA2对Bt杀虫剂Cry2Aa和Cry2Ab毒素的高抗性[69]。在对亚洲玉米螟(Ostrinia furnacalis)的研究中发现,ABCC2基因对Cry1Fa具有高水平抗性,而对Cry1Ab和Cry1Ac抗性较低[42]。草地贪夜蛾(Spodoptera frugiperda)ABCC2基因的敲除使其对Cry1F的抗性增加118倍[70]。双酰胺类杀虫剂具有胃毒作用,对鳞翅目昆虫防治效果好,但小菜蛾(Plutella xylostella)由于其繁殖能力强和周期短的特点很快产生了抗药性[71],Troczka等[72]发现其抗药性与鱼尼丁受体G4946E的突变有关。随后Zuo等[73]利用CRISPR/Cas9技术在甜菜夜蛾(Spodoptera exigua)中证明鱼尼丁受体G4946E突变,对氯虫苯甲酰胺、氯虫酰胺和氯虫双酰胺的抗性分别提高223、336和1000倍以上。
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化学信息素信号通过昆虫神经外周组织接受,触角神经组织处理,进而在脑部中枢神经系统嗅觉和其他感受器官共同指导昆虫觅食、交配、生殖等[74-75]。Garczynski等[76]通过该技术靶向敲除苹果蠹蛾(Cydia pomonella)的OR1基因,发现对突变雌虫繁殖力影响严重。烟草天蛾(Manduca sexta)的嗅觉受体ORCO被敲除后,其觅食行为发生变化,而产卵行为不变[77]。Ye等[78]通过CRISPR/Cas9敲除棉铃虫编码信息素结合蛋白(Pheromone binding proteins)基因PBP1,证实PBP1在雌虫性信息素的感知中具有重要作用,性信息素的感知与交配有关。敲除斜纹夜蛾信息素结合蛋白基因PBP3引起突变体对性信息素感知能力显著下降[79]。
研究昆虫性别分化有利于农业昆虫的种群控制和害虫防治。家蚕和小菜蛾ser2基因敲除引起雄性个体不育,但并不影响突变体的交配行为,最终将雄性不育表型传递给后代[80]。在家蚕和斜纹夜蛾中,osp基因的敲除使突变雌性的交配行为不变,但所产的卵不发育[81]。Markert等[82]利用TALENS和CRISPR/Cas9系统使君主斑蝶(Danaus plexippus)时钟相关基因cryptochrome2(cry2)和clock(cl)突变,发现clk是位于Z性染色体的性别连锁基因;敲除clk会破坏其羽化的昼夜节律。
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基因敲入难于基因敲除,该技术利用细胞自身的HDR修复机制通过靶向编辑系统将外源基因定点整合到基因组中。果蝇中基因敲入的应用较多。如,Xue等[83]将sgRNA与环状DNA供体注射到果蝇胚胎,成功实现了同源重组介导的荧光基因敲入;Zimmer等[84]使用CRISPR/Cas9系统引入了果蝇nAChRα6受体中的同源突变,发现该受体G275E突变显著增加了果蝇对多杀菌素的抗性;随后Lamb等[85]利用该技术在果蝇的目标位点实现了精确替换。基因敲入在其他农业昆虫中也有一些相关报道。如,2015年,Gillies等[29]将非同源末端连接产生的突变引入赤拟谷盗(Tribolium castaneum),证实这种突变能传递给下一代,并在14%的注射个体中观察到标记的定向敲入;2021年,Matsuoka等[60]通过将供体序列敲入双斑蟀(Gryllus bimaculatus)基因Ubx、abd-A的外显子来破坏目标基因的功能,将成功应用于Hox相关基因的功能分析。
An application and optimization of CRISPR/Cas9 system in functional analysis of insect genes: A review and prospect
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摘要: 主要概述了CRISPR/Cas9技术应用于涉及鞘翅目、鳞翅目、膜翅目、半翅目、直翅目和双翅目的昆虫在色素沉着、抗性、嗅觉、交配和性别发生等方面的研究进展,包括基因敲除和基因敲入技术的应用,同时总结了目前CRISPR/Cas9系统的优化方案,最后对改良CRISPR/Cas9系统在昆虫基因组学研究和农业害虫防控的应用前景进行了展望。利用基因编辑技术对昆虫基因功能进行探究是除基因过表达、RNA干扰等方法外的一种有效策略。CRISPR/Cas9技术是近年来出现并迅速发展的第三代基因编辑工具,以操作简便、成本低和编辑效率高等优点而被广泛应用于生物科学中的各个分支。
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关键词:
- 基因编辑技术 /
- CRISPR/Cas9 /
- 基因敲除 /
- 基因敲入
Abstract: In addition to gene overexpression and RNA interference, it is also an effective strategy to use gene editing technology to explore the function of insect genes. CRISPR/Cas9 technology is a third-generation gene editing tool that has developed rapidly in recent years. It is widely used in diverse areas of life science research due to its simple operation, low material cost, and high editing efficiency. A review was made of the application of CRISPR/Cas9 in the pigmentation, insecticide resistance, olfactory transduction, mating, and sex determination of Coleoptera, Lepidoptera, Hymenoptera, Hemiptera, Orthoptera, and Diptera insects, including gene knockout and gene knockin. And recent optimization strategies for the CRISPR/Cas9 system were summarized, based on which a prospect for application of the optimized CRISPR/Cas9 system in insect functional genomics study and agricultural pest management was made.-
Key words:
- gene editing technology /
- CRISPR/Cas9 /
- gene knockout /
- gene knockin
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表 1 CRISPR/Cas9技术在非模式生物昆虫中的部分应用
目 物种 基因 功能 敲除/敲入 导入形式 参考文献 鞘翅目
Coleoptera马铃薯甲虫
Leptinotarsa decemlineatavest 翅膀发育 敲除 Cas9 RNP [28] 赤拟谷盗
Tribolium castaneumEGFP 荧光基因 敲除/敲入 U6-sgRNA质粒, Cas9 mRNA/sgRNA [29] cardinar 色素沉着 敲除 Cas9 protein/sgRNA [30] vermillion 色素沉着 敲除 Cas9 protein/sgRNA [31] 异色瓢虫
Harmonia axyridisnAChRα6 杀虫剂抗性 敲除 Cas9 protein/sgRNA [32] 鳞翅目
Lepidoptera家蚕
Bombyx moriPHYHD1 卵子大小 敲除 Cas9 protein/sgRNA [33] tim 昼夜行为节律 敲除 U6-sgRNA质粒 [34] BLOS2 色素沉着 敲除 a. Cas9/sgRNA质粒
b. Cas9 mRNA/sgRNA[35] a. NHEJ-related genes,
b. TUDOR-SNa. 细胞生长速率
b. 参与应激颗粒形成a. 敲除
b. 敲入Cas9/gRNA质粒 [36] ggnbp2 配子发生 敲除 U6-sgRNA质粒 [37] 棉铃虫
Helicoverpa armigeraSNMP2 性信息素识别 敲除 Cas9 mRNA/sgRNA [38] ABCC2, ABCC3 杀虫剂抗性 敲除 Cas9 protein/sgRNA [39] 柑橘凤蝶
Papilio xuthusyellow, spineless 色素沉着 敲除 Cas9 protein/sgRNA [40] 小红蛱蝶
Vanessa carduiyellow, spineless 色素沉着 敲除 Cas9 protein/sgRNA [40] 斜纹夜蛾
Spodoptera lituraEbony 色素沉着 敲除 Cas9 mRNA/sgRNA [41] 欧洲玉米螟
Bacillus thuringiensisABCC2 杀虫剂抗性 敲除 Cas9 protein/sgRNA [42] 二化螟
Chilo suppressalisEbony 色素沉着 敲除 Cas9 protein/sgRNA [43] 小菜蛾
Plutella xylostellaABCC2, ABCC3 杀虫剂抗性 敲除 Cas9 protein/sgRNA [44] 番茄夜蛾
Tuta Absolutacinnabar 色素沉着 敲除 Cas9 protein/sgRNA [45] 膜翅目
Hymenoptera印度跳蚁
Harpegnathos saltatorOrco 信息素感知 敲除 Cas9 protein/sgRNA [46] 西方蜜蜂
Apis melliferaMrjp1 雄性发育 敲除 Cas9 mRNA/sgRNA [47] a. Mrjp1,
b. Pax6a. 雄性发育
b. 胚胎器官的形态发育敲除 Cas9 protein/sgRNA [48] yellow-y 色素沉着 敲除 Cas9 protein/sgRNA [49] 丽蝇蛹集金小蜂
Nasonia vitripenniscinnabar 色素沉着 敲除 Cas9 protein/sgRNA [50] cinnabar 色素沉着 敲除 Cas9 RNP [51] 半翅目
Hemiptera褐稻虱
Nilaparvata
lugensCSAD 色素沉着 敲除 Cas9 RNP [52] InR1, InR2 调节发育和寿命 敲除 Cas9 RNP [53] 烟粉虱
Bemisia tabaciwhite 色素沉着 敲除 Cas9 RNP [54] TRP 温度感知 敲除 Cas9 mRNA/sgRNA [55] 翅叶蝉
Homalodisca vitripenniscinnabar, white 色素沉着 敲除 Cas9 protein/sgRNA [56] 绿盲蝽
Apolygus lucorumwhite 色素沉着 敲除 Cas9 protein/sgRNA [57] 直翅目
Orthoptera东亚飞蝗
Locusta migratoriaOr35 气味受体 敲除 Cas9 protein/sgRNA [58] dsRNase2 / 敲除 Cas9 RNP [59] 双斑蟀
Gryllus bimaculatusUbx, abd-A 调节组织器官形态发生 敲除/敲入 Cas9 mRNA/sgRNA [60] 双翅目
Diptera埃及伊蚊
Aedes aegyptiKu70, lig4 DNA连接与转录等功能 敲除/敲入 Cas9 mRNA/sgRNA [61] dsx, sxl 性别发生 敲除 Cas9 protein/sgRNA [62] Op1, Op2 视觉感知 敲除 U6-gRNA质粒 [63] 冈比亚按蚊
Anopheles gambiaeFREP1 疟原虫易感性及自身生长发育 敲除 U6-gRNA质粒 [64] -
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