CN112852806B - 一种靶向草地贪夜蛾Doublesex基因雌性特异E3外显子的sgRNA及其应用 - Google Patents
一种靶向草地贪夜蛾Doublesex基因雌性特异E3外显子的sgRNA及其应用 Download PDFInfo
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Abstract
本发明涉及害虫遗传防控领域,具体地,涉及一种靶向草地贪夜蛾Doublesex基因雌性特异E3外显子的sgRNA及其应用。本发明的靶向草地贪夜蛾Doublesex基因E3外显子的sgRNA在草地贪夜蛾Doublesex基因雌性特异E3外显子上的两个靶点的核苷酸序列如SEQ ID NO.1和SEQ ID NO.2所示。本发明利用CRISPR/Cas9***遗传防控草地贪夜蛾的方法,包括以下步骤:1)获得草地贪夜蛾雌性特异E3外显子序列;2)设计草地贪夜蛾Doublesex基因的E3外显子的sgRNA;3)将Cas9的mRNA或蛋白和步骤2)设计的sgRNA混合,将混合物导入草地贪夜蛾的受精卵,获得雌性特异性不育的草地贪夜蛾。本发明利用CRISPR/Cas9技术实现草地贪夜蛾dsx雌性特异性外显子高效准确的敲除,确定dsx基因作为靶标在草地贪夜蛾遗传防控的重要应用价值。
Description
技术领域
本发明涉及害虫遗传防控领域,具体的,涉及一种靶向草地贪夜蛾Doublesex基因雌性特异E3外显子的sgRNA及其应用。
背景技术
目前我国农作物病虫害防治主要依赖化学防治措施,在控制病虫害的同时,也带来了一系列问题:农药残留、环境污染、害虫抗药性增加、有益物种多样性减少。害虫遗传防控既能显著降低化学农药的使用量、避免农产品中的农药残留,同时也能极大地提升农产品质量安全水平,保护生态环境。基于遗传修饰的昆虫遗传不育技术(SIT),包括雌性不育技术和雄性不育技术,其作为一类物种特异、环境友好、科学高效的害虫遗传防控技术,在害虫防治方面具有极大的应用前景。
草地贪夜蛾Spodoptera frugiperda(J.E.Smith)是2018年***粮农组织向全球发布预警的重要农业害虫,也是我国重要外来入侵物种,其寄主极为广泛、适生范围极广、繁殖能力极强、迁飞扩散速度极快、突发危害重、防控难度较大,传统的害虫防治手段不能实现草地贪夜蛾的精准防控,研究开发草地贪夜蛾的遗传防控技术迫在眉睫。
发明内容
本发明的目的是针对现有技术存在的不足,提供一种靶向草地贪夜蛾Doublesex(dsx)基因雌性特异的E3外显子的sgRNA及其应用,进而利用草地贪夜蛾Doublesex基因在生殖发育中的的重要调控功能进行草地贪夜蛾遗传防控。
本发明的具体技术方案如下:
本发明的靶向草地贪夜蛾Doublesex基因E3外显子的sgRNA,其在草地贪夜蛾Doublesex基因雌性特异E3外显子上的两个靶点的核苷酸序列如SEQ ID NO.1和SEQ IDNO.2所示。
本发明还提供了上述的sgRNA在遗传防控草地贪夜蛾中的应用。
本发明还提供了一种利用CRISPR/Cas9***遗传防控草地贪夜蛾的方法,包括以下步骤:
1)获得草地贪夜蛾雌性特异E3外显子序列;
2)设计草地贪夜蛾Doublesex基因雌性特异E3外显子的sgRNA;
3)将Cas9的mRNA或蛋白和步骤2)设计的sgRNA混合,将混合物导入草地贪夜蛾的受精卵,实现Doublesex基因的E3外显子敲除,获得雌性特异性不育的草地贪夜蛾。
根据本发明所述的方法,其中优选的,上述方法中所述sgRNA在草地贪夜蛾Doublesex基因雌性特异E3外显子上的两个靶点的核苷酸序列,如SEQ ID NO.1和SEQ IDNO.2所示。
进一步的,根据本发明所述的方法,其中优选地,步骤3)将混合物通过显微注射方式导入草地贪夜蛾的受精卵。
本发明还提供了一种用于遗传防控草地贪夜蛾的试剂盒,所述试剂盒包括地贪夜蛾Doublesex基因雌性特异E3外显子的sgRNA和Cas9的mRNA或蛋白。
进一步优选的,试剂盒中所述sgRNA在草地贪夜蛾Doublesex基因雌性特异E3外显子上的两个靶点的核苷酸序列,如SEQ ID NO.1和SEQ ID NO.2所示。
本发明通过草地贪夜蛾全基因组测序及鳞翅目昆虫Doublesex基因(dsx)同源比对获得草地贪夜蛾dsx基因cds序列。分别扩增雌雄不同性别dsx基因转录本,对比不同性别dsx转录本序列差异,获得雌性性别特异的外显子E3。针对草地贪夜蛾E3序列设计特异性靶向的sgRNA,利用CRISPR/Cas9技术实现雌性特异的外显子E3敲除。通过对E3外显子敲除品系进行表型及遗传性能测定,发现dsx基因E3外显子敲除后雌性外生殖器官发育畸形,解剖雌性生殖***发现E3外显子敲除纯合个体受精囊缺失,卵巢发育畸形;杂合个体交配后不产卵,卵巢管靠近侧输卵管处卵粒呈多列排布。初步确定dsx基因作为草地贪夜蛾遗传防控重要靶标的应用价值。
附图说明
图1为本发明实施例中Doublesex基因E3外显子的Cas9蛋白切割位点示意图;
图2为本发明实施例中Doublesex基因E3外显子敲除的基因型检测,其中野生型扩增片段(WT)为511bp,敲除片段(KO)为472bp;
图3为本发明实施例中Doublesex基因E3外显子敲除示意图,其中sgRNA靶位点以下划线标示;
图4为本发明实施例中dsx-E3外显子敲除后蛹生殖节畸形;
图5为本发明实施例中dsx-E3外显子敲除后雌性成虫外生殖器发育畸形;
图6为本发明实施例中dsx-E3外显子敲除后雌性成虫卵巢发育畸形。
具体实施方式
下面结合实施例对本发明的技术方案进行详细说明。以下采用的试剂和生物材料如未特别说明,均为商业化产品。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。实施例中未作具体说明的分子生物学实验方法,均参照《分子克隆实验指南》(第四版)J.萨姆布鲁克一书中所列的具体方法进行,或者按照试剂盒和产品说明书进行。
实施例1Doublesex基因不同转录本PCR扩增
通过草地贪夜蛾全基因组Blast获得dsx基因候选序列,设计引物序列如下:
F:ATGGTGTCCGTGGGCTCGTG(SEQ ID NO.3)
R:CGCCGCCTGCGCACTTGTAG(SEQ ID NO.4)
取草地贪夜蛾成虫雌雄各3头,分雌雄两组分别混合,分别提取雌雄成虫的RNA反转录获得雌雄成虫cDNA,以此cDNA为模板,利用上述引物扩增,扩增体系及程序如下:
2×Phanta Master Mix:25uL
引物F:1uL
引物R:1uL
cDNA:1uL
水:22uL
程序:95℃/3min;95℃/10sec,64℃/10sec,72℃/90sec;72℃/5min,扩增34个循环。
回收上述条带TA克隆并测序,获得dsx基因雌雄不同转录本编码区序列如下:
sfDSX-M(雄)(SEQ ID NO.5)
sfDSX-F1(雌)(SEQ ID NO.6)
sfDSX-F2(雌)(SEQ ID NO.7)
sfDSX-F3(雌)(SEQ ID NO.8)
sfDSX-F4(雌)(SEQ ID NO.9)
实施例2Doublesex基因雌性特异性外显子E3敲除及基因型鉴定
如图1所示,采用sgRNAcas9设计软件对dsx基因E3外显子进行sgRNA靶位点扫描,以全基因组为参考序列进行脱靶风险评估,选取得分最高且无脱靶风险的N18NGG序列(图1下划线所示,其中Cas9蛋白切割位点以^表示),位点1序列为:GTTCGACGGGTTAGAGCTGAGG(PAM序列以粗体表示)(SEQ ID NO.1),位点2序列为:GATAATCAACGAATACGCGCGG(PAM序列以粗体表示)(SEQ ID NO.2);根据上述序列设计2对特异性引物:
E3-1F:TAATACGACTCACTATAGGATAATCAACGAATACGCG(SEQ ID NO.10)
E3-1R:TTCTAGCTCTAAAACCGCGTATTCGTTGATTATCC(SEQ ID NO.11)
E3-4F:TAATACGACTCACTATAGGTTCGACGGGTTAGAGCTG(SEQ ID NO.12)
E3-4R:TTCTAGCTCTAAAACCAGCTCTAACCCGTCGAACC(SEQ ID NO.13)
利用Precision gRNA Synthesis Kit(Invitrogen,A29377)试剂盒体外转录成sgRNA。将sgRNA与Cas9蛋白通过显微注射的方式导入草地贪夜蛾受精卵中,正常饲养,取5龄末期蜕皮进行基因型检测,检测引物如下:
E3F511:CGTAAACGACGATGTGCTGT(SEQ ID NO.14)
E3R511:GAGAAATGTCTCTCCCGCCTT(SEQ ID NO.15)
扩增体系及程序如下:
DreamTaq Green PCR Master Mix(2X):12.5uL
引物F:1uL
引物R:1uL
DNA:4uL
水:6.5uL
程序:95℃/3min;95℃/30sec,58℃/30sec,72℃/30sec;72℃/5min,扩增34个循环。
检测结果如图2所示。其中,野生型扩增片段(WT)为511bp,敲除片段(KO)为472bp,显示敲除成功。对上述片段进行测序,结果如图3所示,在Cas9蛋白切割位点附近有预期的目的片段缺失,测序峰图中下划线所示为sgRNA靶位点Cas9蛋白切割残余序列。
实施例3Doublesex基因雌性特异性外显子E3敲除品系表型分析
观察dsx基因敲除雌性个体内、外生殖器官发育情况,拍照记录,结果如图4-6所示。图4中可见dsx-E3外显子敲除后蛹生殖节畸形。图5中可见dsx-E3外显子敲除后雌性成虫外生殖器发育畸形。图6中可见dsx-E3外显子敲除后雌性成虫卵巢发育畸形。
实施例4Doublesex基因雌性特异性外显子E3敲除个体遗传性能测定
将雌性不育系杂合个体分别与野生型个体(1:2)雄性进行杂交配对,通过观测交配行为及解剖检测交配囊变化,确定雌性个体交配情况,交配后成虫连续饲养7天,统计产卵情况。结果显示,dsx-E3 KO雌虫不论基因型为杂合还是纯合,其交配行为没有明显异常,能与雄虫成功交配,但交配后的雌虫不产卵。
最后所应说明的是,以上实施例仅用以说明本发明的技术方案而非限制。尽管参照实施例对本发明进行了详细说明,本领域的普通技术人员应该理解,对本发明的技术方案进行修改或者等同替换,都不脱离本发明技术方案的精神和范围,其均应涵盖在本发明的权利要求范围当中。
序列表
<110> 中国农业科学院深圳农业基因组研究所
<120> 一种靶向草地贪夜蛾Doublesex基因雌性特异E3外显子的sgRNA及其应用
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> Artificial
<400> 1
gttcgacggg ttagagctga gg 22
<210> 2
<211> 22
<212> DNA
<213> Artificial
<400> 2
gataatcaac gaatacgcgc gg 22
<210> 3
<211> 20
<212> DNA
<213> Artificial
<400> 3
atggtgtccg tgggctcgtg 20
<210> 4
<211> 20
<212> DNA
<213> Artificial
<400> 4
cgccgcctgc gcacttgtag 20
<210> 5
<211> 804
<212> DNA
<213> 草地贪夜蛾(Spodoptera frugiperda)
<400> 5
atggtgtccg tgggctcgtg gaggcgccgc acgcccgacg actgcgagga gcgctccgag 60
cccggcgcct ccagctccgc cgtgccgcgc gcgccgccga actgcgcgcg ctgtcggaac 120
caccggctca agatcgagct gaagggccac aagcggtact gcaagtaccg caactgtact 180
tgcgagaagt gtatactgac ggctgaccgg cagcgagtga tggcacaaca gactgctatg 240
aggcgtgccc aggcgcagga cgaggcgcgc gcgcgggccg ggatgcaggc gctgggcgtg 300
gagctggagc agccggagcc gccggtggtg aaggcgccgc gcagccccgc ggtgccgccg 360
ccgcgctcgc tgggctcggc cagctgcgac tcggtcccgg gctcgcccgg agtgtccccg 420
tacgcgccgc tgccgccttc cataccgctg ccgcaagcga tgccgcctct gctgccgcca 480
caacagccgg cggtttcctt agaaaactta gtggataact gtaacaagct gctggagaag 540
ttccactact cctgggagat gatgcctctg gtcctggtca tcctgaatta cgcaggcagt 600
gacgtggacg aggcatcacg gaaaatagac gaagcccact ggatagtcca ccaatggcgg 660
atgtacgagc ggtccatatg ttccctgctg gagctgcagg cgcgcaaggg atcctgctcc 720
atgtgcttca cggagtacgc tccacccttg ctgccgctgc ccctcaccac gcagcgaccc 780
tcgccgccgc ctgcgcactt gtag 804
<210> 6
<211> 727
<212> DNA
<213> 草地贪夜蛾(Spodoptera frugiperda)
<400> 6
atggtgtccg tgggctcgtg gaggcgccgc acgcccgacg actgcgagga gcgctccgag 60
cccggcgcct ccagctccgc cgtgccgcgc gcgccgccga actgcgcgcg ctgtcggaac 120
caccggctca agatcgagct gaagggccac aagcggtact gcaagtaccg caactgtact 180
tgcgagaagt gtatactgac ggctgaccgg cagcgagtga tggcacaaca gactgctatg 240
cggcgtgccc aggcgcagga cgaggcgcgc gcgcgggccg ggatgcaggc gctgggcgtg 300
gagctggagc agccggagcc gccggtggtg aaggcgccgc gcagccccgc ggtgccgccg 360
ccgcgctcgc tgggctcggc cagctgcgac tcggtcccgg gctcgcccgg agtgtccccg 420
tacgcgccgc tgccgccttc cataccgctg ccgcaagcga tgccgcctct gctgccgcca 480
caacagccgg cggtttcctt agaaaactta gtggataact gtaacaaact gctggagaag 540
ttccactact cctgggagat gatgcctctg gtcctggtca tcctgaatta cgcaggcagt 600
gacgtggacg aggcatcacg gaaaatagac gaaggaaaga tgataatcaa cgaatacgcg 660
cggaagaaca atctgaacgt gttcgacggg ttagagctga ggaactcgac acgccagtac 720
ggacatt 727
<210> 7
<211> 783
<212> DNA
<213> 草地贪夜蛾(Spodoptera frugiperda)
<400> 7
atggtgtccg tgggctcgtg gaggcgccgc acgcccgacg actgcgagga gcgctccgag 60
cccggcgcct ccagctccgc cgtgccgcgc gcgccgccga actgcgcgcg ctgtcggaac 120
caccggctca agatcgagct gaagggccac aagcggtact gcaagtaccg caactgtact 180
tgcgagaagt gtatactgac ggctgaccgg cagcgagtga tggcacaaca gactgctatg 240
cggcgtgccc aggcgcagga cgaggcgcgc gcgcgggccg ggatgcaggc gctgggcgtg 300
gagctggagc agccggagcc gccggtggtg aaggcgccgc gcagccccgc ggtgccgccg 360
ccgcgctcgc tgggctcggc cagctgcgac tcggtcccgg gctcgcccgg agtgtccccg 420
tacgcgccgc tgccgccctc cataccgctg ccgcaagcga tgccgcctct gctgccgcca 480
caacagccgg cggtttcctt agaaaactta gtggataact gtaacaagct gctggagaag 540
ttccactact cctgggagat gatgcctctg gtcctggtca tcctgaatta cgcaggcagt 600
gacgtggacg aggcatcacg gaaaatagac gaaggaaaga tgataatcaa cgaatacgcg 660
cggaagaaca atctgaacgt gttcgacggg ttagagctga ggaactcgac acgccagaaa 720
atggcggaaa ttaataatat aagtggtgta ctatcgtcgt cgatgaagtt attttgcgaa 780
tga 783
<210> 8
<211> 747
<212> DNA
<213> 草地贪夜蛾(Spodoptera frugiperda)
<400> 8
atggtgtccg tgggctcgtg gaggcgccgc acgcccgacg actgcgagga gcgctccgag 60
cccggcgcct ccagctccgc cgtgccgcgc gcgccgccga actgcgcgcg ctgtcggaac 120
caccggctca agatcgagct gaagggccac aagcggtact gcaagtaccg caactgtact 180
tgcgagaagt gtatactgac ggctgaccgg cagcgagtga tggcacaaca gactgctatg 240
cggcgtgccc aggcgcagga cgaggcgcgc gcgcgggccg ggatgcaggc gctgggcgtg 300
gagctggagc agccggagcc gccggtggtg aaggcgccgc gcagccccgc ggtgccgccg 360
ccgcgctcgc tgggctcggc cagctgcgac tcggtcccgg gctcgcccgg agtgtccccg 420
tacgcgccgc tgccgccctc cataccgctg ccgcaagcga tgccgcctct gctgccgcca 480
caacagccgg cggtttcctt agaaaactta gtggataact gtaacaagct gctggagaag 540
ttccactact cctgggagat gatgcctctg gtcctggtca tcctgaatta cgcaggcagt 600
gacgtggacg aggcatcacg gaaaatagac gaaggaaaga tgataatcaa cgaatacgcg 660
cggaagaaca atctgaacgt gttcgacggg ttagagctga ggaactcgac acgccatgac 720
cggacgaagg aaaatggcgg aaattaa 747
<210> 9
<211> 759
<212> DNA
<213> 草地贪夜蛾(Spodoptera frugiperda)
<400> 9
atggtgtccg tgggctcgtg gaggcgccgc acgcccgacg actgcgagga gcgctccgag 60
cccggcgcct ccagctccgc cgtgccgcgc gcgccgccga actgcgcgcg ctgtcggaac 120
caccggctca agatcgagct gaagggccac aagcggtact gcaagtaccg caactgtact 180
tgcgagaagt gtatactgac ggctgaccgg cagcgagtga tggcacaaca gactgctatg 240
cggcgtgccc aggcgcagga cgaggcgcgc gcgcgggccg ggatgcaggc gctgggcgtg 300
gagctggagc agccggagcc gccggtggtg aaggcgccgc gcagccccgc ggtgccgccg 360
ccgcgctcgc tgggctcgac cagctgcgac tcggtcccgg gctcgcccgg agtgtccccg 420
tacgcgccgc tgccgccttc cataccgctg ccgcaagcga tgccgcctct gctgccgcca 480
caacagccgg cggtttcctt agaaaactta gtggataact gtaacaaact gctggagaag 540
ttccactact cctgggagat gatgcctctg gtcctggtca tcctgaatta cgcaggcagt 600
gacgtggacg aggcatcacg gaaaatagac gaaggaaaga tgataatcaa cgaatacgcg 660
cggaagaaca atctgaacgt gttcgacggg ttagagctga ggaactcgac acgccatgac 720
cggacgaagg tggtgaaatt cgaaatgcaa cctcagtga 759
<210> 10
<211> 37
<212> DNA
<213> Artificial
<400> 10
taatacgact cactatagga taatcaacga atacgcg 37
<210> 11
<211> 35
<212> DNA
<213> Artificial
<400> 11
ttctagctct aaaaccgcgt attcgttgat tatcc 35
<210> 12
<211> 37
<212> DNA
<213> Artificial
<400> 12
taatacgact cactataggt tcgacgggtt agagctg 37
<210> 13
<211> 35
<212> DNA
<213> Artificial
<400> 13
ttctagctct aaaaccagct ctaacccgtc gaacc 35
<210> 14
<211> 20
<212> DNA
<213> Artificial
<400> 14
cgtaaacgac gatgtgctgt 20
<210> 15
<211> 21
<212> DNA
<213> Artificial
<400> 15
gagaaatgtc tctcccgcct t 21
Claims (5)
1.一种靶向草地贪夜蛾Doublesex基因雌性特异E3外显子的sgRNA,其特征在于,所述sgRNA在草地贪夜蛾Doublesex基因雌性特异E3外显子上的两个靶点的核苷酸序列,如SEQID NO.1和SEQ ID NO.2所示。
2.权利要求1所述的sgRNA在遗传防控草地贪夜蛾中的应用。
3.一种利用CRISPR/Cas9***遗传防控草地贪夜蛾的方法,包括以下步骤:
1)获得草地贪夜蛾雌性特异E3外显子序列;
2)设计靶向草地贪夜蛾Doublesex基因雌性特异E3外显子的sgRNA,所述sgRNA在草地贪夜蛾Doublesex基因雌性特异E3外显子上的两个靶点的核苷酸序列,如SEQ ID NO.1和SEQ ID NO.2所示;
3)将Cas9的mRNA或蛋白和步骤2)设计的sgRNA混合,将混合物导入草地贪夜蛾的受精卵,实现Doublesex基因的E3外显子敲除,获得雌性特异性不育的草地贪夜蛾。
4.根据权利要求3所述的方法,其特征在于,步骤3)将混合物通过显微注射方式导入草地贪夜蛾的受精卵。
5.一种用于遗传防控草地贪夜蛾的试剂盒,其特征在于,所述试剂盒包括草地贪夜蛾Doublesex基因雌性特异E3外显子的sgRNA和Cas9的mRNA或蛋白,所述sgRNA在草地贪夜蛾Doublesex基因雌性特异E3外显子上的两个靶点的核苷酸序列,如SEQ ID NO.1和SEQ IDNO.2所示。
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