CN108998467A - 一种抗虫基因t-dna载体及其应用 - Google Patents

一种抗虫基因t-dna载体及其应用 Download PDF

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CN108998467A
CN108998467A CN201810671300.0A CN201810671300A CN108998467A CN 108998467 A CN108998467 A CN 108998467A CN 201810671300 A CN201810671300 A CN 201810671300A CN 108998467 A CN108998467 A CN 108998467A
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insect gene
anti insect
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dna carrier
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于小星
许超
沈志成
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Zhejiang University ZJU
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis

Abstract

本发明公开了一种抗虫基因T‑DNA载体的构建及其应用。本发明将一个复合启动子用于启动一个抗虫基因的表达，所述的复合启动子包含有花椰菜花叶病毒CaMV 35S启动子以及水稻肌动蛋白Actin1的内含子序列。所述的抗虫基因由Cry1Ab和Vip3A通过连接肽连接构成。本发明所述的重组载体构建完成后转入玉米，转基因玉米中抗虫蛋白的表达量显著提高。本发明的重组载体可用于制备抗虫植物细胞，所述植物包括玉米、水稻等。利用本发明的重组载体转化得到的转基因植物，可以高效杀灭粘虫、斜纹夜蛾、玉米螟、棉铃虫、甜菜夜蛾等主要鳞翅目害虫。

Description

一种抗虫基因T-DNA载体及其应用
(一)技术领域
本发明涉及一种抗虫基因T-DNA载体的构建及其应用。
(二)背景技术
害虫给全球农业生产带来每年约80亿美元的损失，害虫的防治目前主要依靠使用化学农药，但是农药的残留会对人体健康和环境带来不良影响。利用生物技术方法培育含有抗虫基因的转基因抗虫农作物，可以大幅度降低化学杀虫剂的使用，有效保护农作物免受害虫为害，目前转基因抗虫玉米、棉花等已经大量推广种植。
转基因作物抗虫的关键技术是获得性能优良的杀虫蛋白质，杀虫蛋白质有多种。比较常用的是BT杀虫晶体蛋白，如Cry1Ab、Cry1C等，它们已被大量运用于转基因抗虫作物；近年来在BT中发现的另一种营养期杀虫蛋白也被证明具有较好的抗虫活性，如Vip3A等。单个杀虫毒素往往杀虫谱比较窄；同时，长期大量地使用单一的杀虫蛋白质，还可能引起害虫抗性的产生和发展。因此，获得高杀虫活性的新型蛋白质杀虫毒素，对提高杀虫能力和减缓害虫抗性的发生具有重要的应用价值。
(三)发明内容
本发明目的是提供一种高表达抗虫蛋白的重组载体的DNA序列及其构建方法，以及在获得转基因抗虫农作物中的应用。
本发明采用的技术方案是：
本发明提供一种抗虫基因T-DNA载体，所述T-DNA载体包含了一个具有花椰菜花叶病毒CaMV 35S启动子以及水稻肌动蛋白Actin1内含子序列来启动抗虫基因表达的完整表达框，具体所述T-DNA载体包含抗虫基因表达框，所述抗虫基因表达框是由抗虫基因、复合启动子和终止子拼接构成；复合启动子由CaMV 35s和Actin-intron拼接而成。
进一步，所述的抗虫基因由Cry1Ab和Vip3A连接而成。
进一步，所述抗虫基因是将编码BT杀虫晶体蛋白Cry1Ab的核苷酸序列(SEQ IDNO:1所示)与编码BT营养期杀虫蛋白Vip3A的核苷酸序列(SEQ ID NO:2所示)通过连接肽基因序列连接构成，完整的抗虫基因的核苷酸序列为SEQ ID NO:3所示。
进一步，所述CaMV 35s核苷酸序列为SEQ ID NO:4所示，所述Actin-intron核苷酸序列为SEQ ID NO:5所示。
进一步，所述终止子为终止子Pepc-ter，核苷酸序列为SEQ ID NO:6所示。
本发明所述T-DNA核苷酸序列为SEQ ID NO:7所示。
本发明还涉及一种所述抗虫基因T-DNA载体在制备抗虫植物细胞中的应用，所述植物为单子叶植物，优选为玉米或水稻。
本行业的技术人员均知道以下理论：
1)一种用于制备抗虫植物细胞的转化载体中必须包含整个抗虫基因的表达框，由启动子、基因、终止子三个组件构成；
2)并非任意的启动子都能高效启动抗虫基因的表达，不同的启动子对于抗虫蛋白的表达效果相差很大。
与现有技术相比，本发明有益效果主要体现在：
1)由CaMV 35s与水稻肌动蛋白Actin1内含子序列拼接而成的复合启动子，可以高效启动抗虫基因的表达；
2)将上述抗虫基因的表达框的T-DNA转入植物后，获得的转基因株系中抗虫蛋白的表达量能够显著提高。
(四)具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
本发明以下实施例中所使用的分子生物学和生物化学方法均为已知的技术。在Ausubel编写的John Wiley and Sons公司出版的Current Protocols in MolecularBiology，和J.Sambrook等编写的Cold Spring Harbor Laboratory Press(2001)出版的Molecular Cloning:A Laboratory Manual,3rd ED.等文献均有详细的说明。
实施例1、抗虫基因表达框的构建
由上海生工人工合成抗虫基因Cry1Ab核苷酸序列为SEQ ID NO:1，Vip3A核苷酸序列为SEQ ID NO:2，Cry1Ab基因与Vip3A基因通过连接肽基因序列连接为一个完整的抗虫基因，该抗虫基因的序列为SEQ ID NO:3。由上海生工人工合成启动子CaMV 35S的核苷酸序列为SEQ ID NO:4，编码水稻肌动蛋白的内含子Actin-intron核苷酸序列为SEQ ID NO:5。
抗虫基因的构建过程：Cry1Ab片段由BamHI和XmaI酶切获得，Vip3A片段由XmaI和SacI酶切获得。将上述两个片段以T4DNA聚合酶连接到中间载体中，得到一个两端的酶切位点为BamHI和SacI的完整的抗虫基因。
启动子35S/Actin-intron的构建过程：CaMV 35S片段由HindIII和XhoI酶切获得，Actin-intron片段由XhoI和BamHI酶切获得。将上述两个片段以T4DNA聚合酶连接到中间载体中，得到一个两端的酶切位点为HindIII和BamHI的完整的复合启动子。将上述完整的抗虫基因和完整的复合启动子分别酶切，连同终止子(玉米磷酸烯醇式丙酮酸羧化酶基因的终止子Pepc-ter，序列为SEQ ID NO:6)连接到转化载体pCambia1300-G10(Zhao,Q.,et al.(2014)"Expression of Cry1Ab and Cry2Ab by a polycistronic transgene with aself-cleavage peptide in rice."Plos One 9(10):e110006.)中，得到用于制备植物抗虫细胞的转化载体pCam-1Ab-v3A，载体中T-DNA部分的序列为SEQ ID NO:7。
相同条件下，以水稻肌动蛋白启动子pOsActin1替换上述p35S/Actin-intron，其余均保持一致，作为对照组CK。所得的对照T-DNA载体用于转化制备植物抗虫细胞。
实施例2、转基因玉米的获得
玉米转化事件所用的方法是农杆菌介导方法，根据Frame等报道的方法和培养基配方(Plant Physiol,2002,129:13-22)进行转化，具体步骤如下：取授粉后8～10天的Hi-2玉米穗。收集所有的未成熟胚(大小为1.0～1.5mm)，将含有实施例1构建的T-DNA载体pCam-1Ab-v3A的农杆菌与未成熟胚共培育2～3天(22℃)。转移未成熟胚到愈伤诱导培养基上挂(含200mg/L的timentin杀农杆菌)，28℃暗培养10～14天。将所有的愈伤组织转移到带有2.0mM草甘膦的筛选培养基上，28℃暗培养2～3周。
转移所有的组织到新鲜的筛选培养基上，28℃暗培养2～3周。随后转移所有筛选后成活的胚性组织到再生培养基上，28℃暗培养10～14天，每皿一个株系。转移胚性组织到新鲜的再生培养基上，26℃光照培养10～14天。转移所有发育完全的植株到生根培养基上，26℃光照培养直到根发育完全，获得转基因玉米植株。
同样条件下，用含对照组T-DNA载体的农杆菌转化玉米，获得对照转基因玉米植株。
实施例3、转基因农作物抗虫能力的测定
利用玉米螟和棉铃虫测定通过实施例2获得的10个转基因玉米系的抗虫活性。取实施例2获得的转基因玉米嫩叶饲喂一龄幼虫，每叶接虫10头，28℃暗培养5天后记录数据。新生玉米螟一龄幼虫取食10个转基因玉米系后，全部100％死亡；新生棉铃虫一龄幼虫取食10个转基因玉米系后，有8个系的棉铃虫100％死亡，其他两个系的棉铃虫死亡率在60％～90％之间。
同时，通过实施例2的方法，在玉米中转入对照组CK的T-DNA，获得pOsActin1启动抗虫基因表达的对照转基因玉米株系。利用玉米螟和棉铃虫进行上述抗虫活性鉴定。结果显示，新生玉米螟一龄幼虫取食10个转基因玉米系后，全部100％死亡；新生棉铃虫一龄幼虫取食10个转基因玉米系后，有2个系的棉铃虫100％死亡，其余6个系的棉铃虫死亡率在30％～60％之间。
进一步，对转基因玉米株系进行抗虫蛋白的ELISA鉴定。结果显示，在转基因株系中，p35S/Actin-intron启动抗虫蛋白的表达量比对照组中高10-30倍(如下表1所示)。
表1转基因玉米株系中的抗虫蛋白表达量测定
最后，还需要注意的是，以上列举的仅是本发明的若干实施例。显然，本发明不限于以上实施例，还可以有许多延伸和拓展。本领域的普通技术人员能从本发明公开的内容直接到导出或联想到的所有延伸，均应认为是本发明的保护范围。
序列表
<110> 浙江大学
<120> 一种抗虫基因T-DNA载体及其应用
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gacctgagca acaaggagac caagctgatc gtgccgccga tcagcttcat cagcaacatc 1620
gtggagaacg gcaacctgga gggcgagaac ctggagccgt ggatcgccaa caacaagaac 1680
gcctacgtgg accacaccgg cggcatcaac ggcaccaagg tgctgtacgt gcacaaggac 1740
ggcgagttca gccagttcgt gggcggcaag ctgaagagca agaccgagta cgtgatccag 1800
tacatcgtga agggcaaggc cagcatctac ctgaaggacg agaacaccgg ctacatccac 1860
tacgaggaca ccaacaacaa cctggaggac taccagacca tcaacaagag gttcaccacc 1920
ggcaccgacc tgaagggcgt gtacctgatc ctgaagagcc agaacggcga cgaggcctgg 1980
ggcgacaact tcatcatcct ggagatcagc ccgagcgaga agctgctgag cccggagctg 2040
atcaacacca acaactggac cagcaccggc agcaccaaca tcagcggcaa caccctgacc 2100
ctgtaccagg gcggcagggg catcctgaag cagaacctgc agctggacag cttcagcacc 2160
tacagggtgt acttcagcgt gagcggcgac gccaacgtga ggatcaggaa cagcagggag 2220
gtgctgttcg agaagaggta catgagcggc gccaaggacg tgagcgagat gttcaccacc 2280
aagttcgaga aggacaactt ctacatcgag ctgagccagg gcaacaacct gtacggcggc 2340
ccgatcgtgc acttctacga cgtgagcatc aagtaagagc tc 2382
<210> 3
<211> 4366
<212> DNA
<213> 未知(Unknown)
<400> 3
ggatccaaca atggacaaca accccaacat caacgagtgc atcccctaca actgcctgag 60
caaccccgag gtggaggtgc tgggcggcga gcgcatcgag accggctaca cccccatcga 120
catcagcctg agcctgaccc agttcctgct gagcgagttc gtgcccggcg ccggcttcgt 180
gctgggcctg gtggacatca tctggggcat cttcggcccc agccagtggg acgccttcct 240
ggtgcagatc gagcagctga tcaaccagcg catcgaggag ttcgcccgca accaggccat 300
cagccgcctg gagggcctga gcaacctgta ccaaatctac gccgagagct tccgcgagtg 360
ggaggccgac cccaccaacc ccgccctgcg cgaggagatg cgcatccagt tcaacgacat 420
gaacagcgcc ctgaccaccg ccatccccct gttcgccgtg cagaactacc aggtgcccct 480
gctgagcgtg tacgtgcagg ccgccaacct gcacctgagc gtgctgcgcg acgtcagcgt 540
gttcggccag cgctggggct tcgacgccgc caccatcaac agccgctaca acgacctgac 600
ccgcctgatc ggcaactaca ccgaccacgc cgtgcgctgg tacaacaccg gcctggagcg 660
cgtgtggggt cccgacagcc gcgactggat caggtacaac cagttccgcc gcgagctgac 720
cctgaccgtg ctggacatcg tgagcctgtt ccccaactac gacagccgca cctaccccat 780
ccgcaccgtg agccagctga cccgcgagat ttacaccaac cccgtgctgg agaacttcga 840
cggcagcttc cgcggcagcg cccagggcat cgagggcagc atccgcagcc cccacctgat 900
ggacatcctg aacagcatca ccatctacac cgacgcccac cgcggcgagt actactggag 960
cggccaccag atcatggcca gccccgtcgg cttcagcggc cccgagttca ccttccccct 1020
gtacggcacc atgggcaacg ctgcacctca gcagcgcata gtggcacagc tgggccaggg 1080
agtgtaccgc accctgagca gcaccctgta ccgtcgacct ttcaacatcg gcatcaacaa 1140
ccagcagctg agcgtgctgg acggcaccga gttcgcctac ggcaccagca gcaacctgcc 1200
cagcgccgtg taccgcaaga gcggcaccgt ggacagcctg gacgagatcc cccctcagaa 1260
caacaacgtg ccacctcgac agggcttcag ccaccgtctg agccacgtga gcatgttccg 1320
cagtggcttc agcaacagca gcgtgagcat catccgtgca cctatgttca gctggattca 1380
ccgcagtgcc gagttcaaca acatcatccc cagcagccag atcacccaga tccccctgac 1440
caagagcacc aacctgggca gcggcaccag cgtggtgaag ggccccggct tcaccggcgg 1500
cgacatcctg cgccgcacca gccccggcca gatcagcacc ctgcgcgtga acatcaccgc 1560
ccccctgagc cagcgctacc gcgtccgcat ccgctacgcc agcaccacca acctgcagtt 1620
ccacaccagc atcgacggcc gccccatcaa ccagggcaac ttcagcgcca ccatgagcag 1680
cggcagcaac ctgcagagcg gcagcttccg caccgtgggc ttcaccaccc ccttcaactt 1740
cagcaacggc agcagcgtgt tcaccctgag cgcccacgtg ttcaacagcg gcaacgaggt 1800
gtacatcgac cgcatcgagt tcgtgcccgc cgaggtgacc ttcgaggccg agtacgacct 1860
ggagagggct cagaaggccg tgaacgagct gttcaccagc agcaaccaga tcggcctgaa 1920
gaccgacgtg accgactacc acatcgatca ggtgcgaggc cccggtggag caggtggagc 1980
aggtggaggg ggtaaaggaa acaacaccaa gctgaacgcc agggccctgc cgagcttcat 2040
cgactacttc aacggcatct acggcttcgc caccggcatc aaggacatca tgaacatgat 2100
cttcaagacc gacaccggcg gcaacctgac cctggacgag atcctgaaga accagcagct 2160
gctgaacgag atcagcggca agctggacgg cgtgaacggc agcctgaacg acctgatcgc 2220
ccagggcaac ctgaacaccg agctgagcaa ggagatcctg aagatcgcca acgagcagaa 2280
ccaggtgctg aacgacgtga acaacaagct ggacgccatc aacaccatgc tgcacatcta 2340
cctgccgaag atcaccagca tgctgagcga cgtgatgaag cagaactacg ccctgagcct 2400
gcaggtggag tacctgagca agcagctgaa ggagatcagc gacaagctgg acgtgatcaa 2460
cgtgaacgtg ctgatcaaca gcaccctgac cgagatcacc ccggcctacc agaggatcaa 2520
gtacgtgaac gagaagttcg aggagctgac cttcgccacc gagaccaccc tgaaggtgaa 2580
gaaggacagc agcccggccg acatcctgga cgagctgacc gagctgaccg agctggccaa 2640
gagcgtgacc aagaacgacg tggacggctt cgagttctac ctgaacacct tccacgacgt 2700
gatggtgggc aacaacctgt tcggcaggag cgccctgaag accgccagcg agctgatcgc 2760
caaggagaac gtgaagacca gcggcagcga ggtgggcaac gtgtacaact tcctgatcgt 2820
gctgaccgcc ctgcaggcca aggccttcct gaccctgacc acctgcagga agctgctggg 2880
cctggccgac atcgactaca ccagcatcat gaacgagcac ctgaacaagg agaaggagga 2940
gttcagggtg aacatcctgc cgaccctgag caacaccttc agcaacccga actacgccaa 3000
ggtgaagggc agcgacgagg acgccaagat gatcgtggag gccaagccgg gccacgccct 3060
ggtgggcttc gagatcagca acgacagcat gaccgtgctg aaggtgtacg aggccaagct 3120
gaagcagaac taccaggtgg acaaggacag cctgagcgag gtgatctaca gcgacatgga 3180
caagctgctg tgcccggacc agagcgagca gatctactac accaacaaca tcgtgttccc 3240
gaacgagtac gtgatcacca agatcgactt caccaagaag atgaagaccc tgaggtacga 3300
ggtgaccgcc aacagctacg acagcagcac cggcgagatc gacctgaaca agaagaaggt 3360
ggagagcagc gaggccgagt acaggaccct gagcgccaac aacgacggcg tgtacatgcc 3420
gctgggcgtg atcagcgaga ccttcctgac cccgatcaac ggcttcggcc tgcaggccga 3480
cgagaacagc aggctgatca ccctgacctg caagagctac ctgagggagc tgctgctggc 3540
caccgacctg agcaacaagg agaccaagct gatcgtgccg ccgatcagct tcatcagcaa 3600
catcgtggag aacggcaacc tggagggcga gaacctggag ccgtggatcg ccaacaacaa 3660
gaacgcctac gtggaccaca ccggcggcat caacggcacc aaggtgctgt acgtgcacaa 3720
ggacggcgag ttcagccagt tcgtgggcgg caagctgaag agcaagaccg agtacgtgat 3780
ccagtacatc gtgaagggca aggccagcat ctacctgaag gacgagaaca ccggctacat 3840
ccactacgag gacaccaaca acaacctgga ggactaccag accatcaaca agaggttcac 3900
caccggcacc gacctgaagg gcgtgtacct gatcctgaag agccagaacg gcgacgaggc 3960
ctggggcgac aacttcatca tcctggagat cagcccgagc gagaagctgc tgagcccgga 4020
gctgatcaac accaacaact ggaccagcac cggcagcacc aacatcagcg gcaacaccct 4080
gaccctgtac cagggcggca ggggcatcct gaagcagaac ctgcagctgg acagcttcag 4140
cacctacagg gtgtacttca gcgtgagcgg cgacgccaac gtgaggatca ggaacagcag 4200
ggaggtgctg ttcgagaaga ggtacatgag cggcgccaag gacgtgagcg agatgttcac 4260
caccaagttc gagaaggaca acttctacat cgagctgagc cagggcaaca acctgtacgg 4320
cggcccgatc gtgcacttct acgacgtgag catcaagtaa gagctc 4366
<210> 4
<211> 799
<212> DNA
<213> 未知(Unknown)
<400> 4
aagcttaggt acctggtgga gcacgacact ctcgtctact ccaagaatat caaagataca 60
gtctcagaag accaaagggc tattgagact tttcaacaaa gggtaatatc gggaaacctc 120
ctcggattcc attgcccagc tatctgtcac ttcatcaaaa ggacagtaga aaaggaaggt 180
ggcacctaca aatgccatca ttgcgataaa ggaaaggcta tcgttcaaga tgcctctgcc 240
gacagtggtc ccaaagatgg acccccaccc acgaggagca tcgtggaaaa agaagacgtt 300
ccaaccacgt cttcaaagca agtggattga tgtgataaca tggtggagca cgacactctc 360
gtctactcca agaatatcaa agatacagtc tcagaagacc aaagggctat tgagactttt 420
caacaaaggg taatatcggg aaacctcctc ggattccatt gcccagctat ctgtcacttc 480
atcaaaagga cagtagaaaa ggaaggtggc acctacaaat gccatcattg cgataaagga 540
aaggctatcg ttcaagatgc ctctgccgac agtggtccca aagatggacc cccacccacg 600
aggagcatcg tggaaaaaga agacgttcca accacgtctt caaagcaagt ggattgatgt 660
gatatctcca ctgacgtaag ggatgacgca caatcccact atccttcgca agaccttcct 720
ctatataagg aagttcattt catttggaga ggacacgctg aaatcaccag tctctctcta 780
caaatctatc tctctcgag 799
<210> 5
<211> 549
<212> DNA
<213> 未知(Unknown)
<400> 5
ctcgagacca ccaccaccac caccacctcc tcccccctcg ctgccggacg acgagctcct 60
cccccctccc cctccgccgc cgccggtaac caccccgccc ctctcctctt tctttctccg 120
tttttttttt cgtctcggtc tcgatctttg gccttggtag tttgggtggg cgagagcggc 180
ttcgtcgccc agatcggtgc gcgggagggg cgggatctcg cggctggcgt ctccgggcgt 240
gagtcggccc ggatcctcgc ggggaatggg gctctcggat gtagatctgc gatccgccgt 300
tgttggggga gatgatgggg ggtttaaaat ttccgccatg ctaaacaaga tcaggaagag 360
gggaaaaggg cactatggtt tatattttta tatatttctg ctgcttcgtc aggcttagat 420
gtgctagatc tttctttctt ctttttgtgg tagaatttga atccctcagc attgttcatc 480
ggtagttttt cttttcatga tttgtgacaa atgcagcctc gtgcggagct tttttgtagg 540
tagggatcc 549
<210> 6
<211> 203
<212> DNA
<213> 未知(Unknown)
<400> 6
gagctctaga tctgttctgc acaaagtgga gtagtcagtc atcgatcagg aaccagacac 60
cagactttta ttcatacagt gaagtgaagt gaagtgcagt gcagtgagtt gctggttttt 120
gtacaactta gtatgtattt gtatttgtaa aatacttcta tcaataaaat ttctaattcc 180
taaaaccaaa atccaggggt acc 203
<210> 7
<211> 10046
<212> DNA
<213> 未知(Unknown)
<400> 7
taaacgctct tttctcttag gtttacccgc caatatatcc tgtcaaacac tgatagttta 60
aactgaaggc gggaaacgac aatctgatcc aagctcaagc tgctctagca ttcgccattc 120
aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg 180
gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca 240
cgacgttgta aaacgacggc cagtgccaag ctttggtgga gcacgacact ctcgtctact 300
ccaagaatat caaagataca gtctcagaag accaaagggc tattgagact tttcaacaaa 360
gggtaatatc gggaaacctc ctcggattcc attgcccagc tatctgtcac ttcatcaaaa 420
ggacagtaga aaaggaaggt ggcacctaca aatgccatca ttgcgataaa ggaaaggcta 480
tcgttcaaga tgcctctgcc gacagtggtc ccaaagatgg acccccaccc acgaggagca 540
tcgtggaaaa agaagacgtt ccaaccacgt cttcaaagca agtggattga tgtgataaca 600
tggtggagca cgacactctc gtctactcca agaatatcaa agatacagtc tcagaagacc 660
aaagggctat tgagactttt caacaaaggg taatatcggg aaacctcctc ggattccatt 720
gcccagctat ctgtcacttc atcaaaagga cagtagaaaa ggaaggtggc acctacaaat 780
gccatcattg cgataaagga aaggctatcg ttcaagatgc ctctgccgac agtggtccca 840
aagatggacc cccacccacg aggagcatcg tggaaaaaga agacgttcca accacgtctt 900
caaagcaagt ggattgatgt gatatctcca ctgacgtaag ggatgacgca caatcccact 960
atccttcgca agaccttcct ctatataagg aagttcattt catttggaga ggacacgctg 1020
aaatcaccag tctctctcta caaatctatc tctctcgaga ccaccaccac caccaccacc 1080
tcctcccccc tcgctgccgg acgacgagct cctcccccct ccccctccgc cgccgccggt 1140
aaccaccccg cccctctcct ctttctttct ccgttttttt tttcgtctcg gtctcgatct 1200
ttggccttgg tagtttgggt gggcgagagc ggcttcgtcg cccagatcgg tgcgcgggag 1260
gggcgggatc tcgcggctgg cgtctccggg cgtgagtcgg cccggatcct cgcggggaat 1320
ggggctctcg gatgtagatc tgcgatccgc cgttgttggg ggagatgatg gggggtttaa 1380
aatttccgcc atgctaaaca agatcaggaa gaggggaaaa gggcactatg gtttatattt 1440
ttatatattt ctgctgcttc gtcaggctta gatgtgctag atctttcttt cttctttttg 1500
tggtagaatt tgaatccctc agcattgttc atcggtagtt tttcttttca tgatttgtga 1560
caaatgcagc ctcgtgcgga gcttttttgt aggtagggat ccaacaatgg acaacaaccc 1620
caacatcaac gagtgcatcc cctacaactg cctgagcaac cccgaggtgg aggtgctggg 1680
cggcgagcgc atcgagaccg gctacacccc catcgacatc agcctgagcc tgacccagtt 1740
cctgctgagc gagttcgtgc ccggcgccgg cttcgtgctg ggcctggtgg acatcatctg 1800
gggcatcttc ggccccagcc agtgggacgc cttcctggtg cagatcgagc agctgatcaa 1860
ccagcgcatc gaggagttcg cccgcaacca ggccatcagc cgcctggagg gcctgagcaa 1920
cctgtaccaa atctacgccg agagcttccg cgagtgggag gccgacccca ccaaccccgc 1980
cctgcgcgag gagatgcgca tccagttcaa cgacatgaac agcgccctga ccaccgccat 2040
ccccctgttc gccgtgcaga actaccaggt gcccctgctg agcgtgtacg tgcaggccgc 2100
caacctgcac ctgagcgtgc tgcgcgacgt cagcgtgttc ggccagcgct ggggcttcga 2160
cgccgccacc atcaacagcc gctacaacga cctgacccgc ctgatcggca actacaccga 2220
ccacgccgtg cgctggtaca acaccggcct ggagcgcgtg tggggtcccg acagccgcga 2280
ctggatcagg tacaaccagt tccgccgcga gctgaccctg accgtgctgg acatcgtgag 2340
cctgttcccc aactacgaca gccgcaccta ccccatccgc accgtgagcc agctgacccg 2400
cgagatttac accaaccccg tgctggagaa cttcgacggc agcttccgcg gcagcgccca 2460
gggcatcgag ggcagcatcc gcagccccca cctgatggac atcctgaaca gcatcaccat 2520
ctacaccgac gcccaccgcg gcgagtacta ctggagcggc caccagatca tggccagccc 2580
cgtcggcttc agcggccccg agttcacctt ccccctgtac ggcaccatgg gcaacgctgc 2640
acctcagcag cgcatagtgg cacagctggg ccagggagtg taccgcaccc tgagcagcac 2700
cctgtaccgt cgacctttca acatcggcat caacaaccag cagctgagcg tgctggacgg 2760
caccgagttc gcctacggca ccagcagcaa cctgcccagc gccgtgtacc gcaagagcgg 2820
caccgtggac agcctggacg agatcccccc tcagaacaac aacgtgccac ctcgacaggg 2880
cttcagccac cgtctgagcc acgtgagcat gttccgcagt ggcttcagca acagcagcgt 2940
gagcatcatc cgtgcaccta tgttcagctg gattcaccgc agtgccgagt tcaacaacat 3000
catccccagc agccagatca cccagatccc cctgaccaag agcaccaacc tgggcagcgg 3060
caccagcgtg gtgaagggcc ccggcttcac cggcggcgac atcctgcgcc gcaccagccc 3120
cggccagatc agcaccctgc gcgtgaacat caccgccccc ctgagccagc gctaccgcgt 3180
ccgcatccgc tacgccagca ccaccaacct gcagttccac accagcatcg acggccgccc 3240
catcaaccag ggcaacttca gcgccaccat gagcagcggc agcaacctgc agagcggcag 3300
cttccgcacc gtgggcttca ccaccccctt caacttcagc aacggcagca gcgtgttcac 3360
cctgagcgcc cacgtgttca acagcggcaa cgaggtgtac atcgaccgca tcgagttcgt 3420
gcccgccgag gtgaccttcg aggccgagta cgacctggag agggctcaga aggccgtgaa 3480
cgagctgttc accagcagca accagatcgg cctgaagacc gacgtgaccg actaccacat 3540
cgatcaggtg cgaggccccg gtggagcagg tggagcaggt ggagggggta aaggaaacaa 3600
caccaagctg aacgccaggg ccctgccgag cttcatcgac tacttcaacg gcatctacgg 3660
cttcgccacc ggcatcaagg acatcatgaa catgatcttc aagaccgaca ccggcggcaa 3720
cctgaccctg gacgagatcc tgaagaacca gcagctgctg aacgagatca gcggcaagct 3780
ggacggcgtg aacggcagcc tgaacgacct gatcgcccag ggcaacctga acaccgagct 3840
gagcaaggag atcctgaaga tcgccaacga gcagaaccag gtgctgaacg acgtgaacaa 3900
caagctggac gccatcaaca ccatgctgca catctacctg ccgaagatca ccagcatgct 3960
gagcgacgtg atgaagcaga actacgccct gagcctgcag gtggagtacc tgagcaagca 4020
gctgaaggag atcagcgaca agctggacgt gatcaacgtg aacgtgctga tcaacagcac 4080
cctgaccgag atcaccccgg cctaccagag gatcaagtac gtgaacgaga agttcgagga 4140
gctgaccttc gccaccgaga ccaccctgaa ggtgaagaag gacagcagcc cggccgacat 4200
cctggacgag ctgaccgagc tgaccgagct ggccaagagc gtgaccaaga acgacgtgga 4260
cggcttcgag ttctacctga acaccttcca cgacgtgatg gtgggcaaca acctgttcgg 4320
caggagcgcc ctgaagaccg ccagcgagct gatcgccaag gagaacgtga agaccagcgg 4380
cagcgaggtg ggcaacgtgt acaacttcct gatcgtgctg accgccctgc aggccaaggc 4440
cttcctgacc ctgaccacct gcaggaagct gctgggcctg gccgacatcg actacaccag 4500
catcatgaac gagcacctga acaaggagaa ggaggagttc agggtgaaca tcctgccgac 4560
cctgagcaac accttcagca acccgaacta cgccaaggtg aagggcagcg acgaggacgc 4620
caagatgatc gtggaggcca agccgggcca cgccctggtg ggcttcgaga tcagcaacga 4680
cagcatgacc gtgctgaagg tgtacgaggc caagctgaag cagaactacc aggtggacaa 4740
ggacagcctg agcgaggtga tctacagcga catggacaag ctgctgtgcc cggaccagag 4800
cgagcagatc tactacacca acaacatcgt gttcccgaac gagtacgtga tcaccaagat 4860
cgacttcacc aagaagatga agaccctgag gtacgaggtg accgccaaca gctacgacag 4920
cagcaccggc gagatcgacc tgaacaagaa gaaggtggag agcagcgagg ccgagtacag 4980
gaccctgagc gccaacaacg acggcgtgta catgccgctg ggcgtgatca gcgagacctt 5040
cctgaccccg atcaacggct tcggcctgca ggccgacgag aacagcaggc tgatcaccct 5100
gacctgcaag agctacctga gggagctgct gctggccacc gacctgagca acaaggagac 5160
caagctgatc gtgccgccga tcagcttcat cagcaacatc gtggagaacg gcaacctgga 5220
gggcgagaac ctggagccgt ggatcgccaa caacaagaac gcctacgtgg accacaccgg 5280
cggcatcaac ggcaccaagg tgctgtacgt gcacaaggac ggcgagttca gccagttcgt 5340
gggcggcaag ctgaagagca agaccgagta cgtgatccag tacatcgtga agggcaaggc 5400
cagcatctac ctgaaggacg agaacaccgg ctacatccac tacgaggaca ccaacaacaa 5460
cctggaggac taccagacca tcaacaagag gttcaccacc ggcaccgacc tgaagggcgt 5520
gtacctgatc ctgaagagcc agaacggcga cgaggcctgg ggcgacaact tcatcatcct 5580
ggagatcagc ccgagcgaga agctgctgag cccggagctg atcaacacca acaactggac 5640
cagcaccggc agcaccaaca tcagcggcaa caccctgacc ctgtaccagg gcggcagggg 5700
catcctgaag cagaacctgc agctggacag cttcagcacc tacagggtgt acttcagcgt 5760
gagcggcgac gccaacgtga ggatcaggaa cagcagggag gtgctgttcg agaagaggta 5820
catgagcggc gccaaggacg tgagcgagat gttcaccacc aagttcgaga aggacaactt 5880
ctacatcgag ctgagccagg gcaacaacct gtacggcggc ccgatcgtgc acttctacga 5940
cgtgagcatc aagtaagagc tctagatctg ttctgcacaa agtggagtag tcagtcatcg 6000
atcaggaacc agacaccaga cttttattca tacagtgaag tgaagtgaag tgcagtgcag 6060
tgagttgctg gtttttgtac aacttagtat gtatttgtat ttgtaaaata cttctatcaa 6120
taaaatttct aattcctaaa accaaaatcc aggggtaccg catgcctaca gtgcagcgtg 6180
acccggtcgt gcccctctct agagataatg agcattgcat gtctaagtta taaaaaatta 6240
ccacatattt tttttgtcac acttgtttga agtgcagttt atctatcttt atacatatat 6300
ttaaacttta ctctacgaat aatataatct atagtactac aataatatca gtgttttaga 6360
gaatcatata aatgaacagt tagacatggt ctaaaggaca attgagtatt ttgacaacag 6420
gactctacag ttttatcttt ttagtgtgca tgtgttctcc tttttttttg caaatagctt 6480
cacctatata atacttcatc cattttatta gtacatccat ttagggttta gggttaatgg 6540
tttttataga ctaatttttt tagtacatct attttattct attttagcct ctaaattaag 6600
aaaactaaaa ctctatttta gtttttttat ttaataattt agatataaaa tagaataaaa 6660
taaagtgact aaaaattaaa caaataccct ttaagaaatt aaaaaaacta aggaaacatt 6720
tttcttgttt cgagtagata atgccagcct gttaaacgcc gtcgacgagt ctaacggaca 6780
ccaaccagcg aaccagcagc gtcgcgtcgg gccaagcgaa gcagacggca cggcatctct 6840
gtcgctgcct ctggacccct ctcgagagtt ccgctccacc gttggacttg ctccgctgtc 6900
ggcatccaga aattgcgtgg cggagcggca gacgtgagcc ggcacggcag gcggcctcct 6960
cctcctctca cggcacggca gctacggggg attcctttcc caccgctcct tcgctttccc 7020
ttcctcgccc gccgtaataa atagacaccc cctccacacc ctctttcccc aacctcgtgt 7080
tgttcggagc gcacacacac acaaccagat ctcccccaaa tccacccgtc ggcacctccg 7140
cttcaaggta cgccgctcgt cctccccccc cccccctctc taccttctct agatcggcgt 7200
tccggtccat ggttagggcc cggtagttct acttctgttc atgtttgtgt tagatccgtg 7260
tttgtgttag atccgtgctg ctagcgttcg tacacggatg cgacctgtac gtcagacacg 7320
ttctgattgc taacttgcca gtgtttctct ttggggaatc ctgggatggc tctagccgtt 7380
ccgcagacgg gatcgatttc atgatttttt ttgtttcgtt gcatagggtt tggtttgccc 7440
ttttccttta tttcaatata tgccgtgcac ttgtttgtcg ggtcatcttt tcatgctttt 7500
ttttgtcttg gttgtgatga tgtggtctgg ttgggcggtc gttctagatc ggagtagaat 7560
tctgtttcaa actacctggt ggatttatta attttggatc tgtatgtgtg tgccatacat 7620
attcatagtt acgaattgaa gatgatggat ggaaatatcg atctaggata ggtatacatg 7680
ttgatgcggg ttttactgat gcatatacag agatgctttt tgttcgcttg gttgtgatga 7740
tgtggtgtgg ttgggcggtc gttcattcgt tctagatcgg agtagaatac tgtttcaaac 7800
tacctggtgt atttattaat tttggaactg tatgtgtgtg tcatacatct tcatagttac 7860
gagtttaaga tggatggaaa tatcgatcta ggataggtat acatgttgat gtgggtttta 7920
ctgatgcata tacatgatgg catatgcagc atctattcat atgctctaac cttgagtacc 7980
tatctattat aataaacaag tatgttttat aattattttg atcttgatat acttggatga 8040
tggcatatgc agcagctata tgtggatttt tttagccctg ccttcatacg ctatttattt 8100
gcttggtact gtttcttttg tcgatgctca ccctgttgtt tggtgttact tctgcaggtc 8160
gactctagaa acaatggcgg cgaccatggc gtccaacgct gcggctgcgg ctgcggtgtc 8220
cctggaccag gccgtggctg cgtcggcagc gttctcgtcg cggaagcagc tgcggctgcc 8280
tgccgcagcg cgcggaggga tgcgggtgcg ggtgcgggcg cggggtcggc gggaggcggt 8340
ggtggtggcg tccgcgtcgt cgtcgtcggt ggcagcgccg gcggcgaagg ctgagatgct 8400
acacggtgca agcagccggc cggcaaccgc tcgcaaatct tccggccttt cgggaacggt 8460
caggattccg ggcgataagt ccatatccca ccggtcgttc atgttcggcg gtcttgccag 8520
cggtgagacg cgcatcacgg gcctgcttga aggtgaggac gtgatcaata ccgggaaggc 8580
catgcaggct atgggagcgc gtatccgcaa ggaaggtgac acatggatca ttgacggcgt 8640
tgggaatggc ggtctgctcg cccctgaggc ccctctcgac ttcggcaatg cggcgacggg 8700
ctgcaggctc actatgggac tggtcggggt gtacgacttc gatagcacgt tcatcggaga 8760
cgcctcgctc acaaagcgcc caatgggccg cgttctgaac ccgttgcgcg agatgggcgt 8820
acaggtcaaa tccgaggatg gtgaccgttt gcccgttacg ctgcgcgggc cgaagacgcc 8880
taccccgatt acctaccgcg tgccaatggc atccgcccag gtcaagtcag ccgtgctcct 8940
cgccggactg aacactccgg gcatcaccac ggtgatcgag cccatcatga ccagggatca 9000
taccgaaaag atgcttcagg ggtttggcgc caacctgacg gtcgagacgg acgctgacgg 9060
cgtcaggacc atccgccttg agggcagggg taaactgact ggccaagtca tcgatgttcc 9120
gggagacccg tcgtccacgg ccttcccgtt ggttgcggcg ctgctcgtgc cggggagtga 9180
cgtgaccatc ctgaacgtcc tcatgaaccc gaccaggacc ggcctgatcc tcacgcttca 9240
ggagatggga gccgacatcg aggtgatcaa cccgcgcctg gcaggcggtg aagacgttgc 9300
ggatctgcgc gtgcgctcct ctaccctgaa gggcgtgacg gtcccggaag atcgcgcgcc 9360
gtccatgata gacgagtatc ctattctggc cgtcgccgct gcgttcgccg aaggggccac 9420
ggtcatgaac ggtcttgagg aactccgcgt gaaggaatcg gatcgcctgt cggcggtggc 9480
caatggcctg aagctcaacg gtgttgactg cgacgagggt gagacctcac tcgtggtccg 9540
tggccggcct gatggcaagg gcctcggcaa cgccagtgga gcggccgtcg ccacgcacct 9600
cgatcatcgc atcgcgatgt ccttcttggt gatgggtctc gtctcagaga acccggtgac 9660
cgtcgatgac gccacgatga tagcgacgag cttcccagag ttcatggatc tgatggcggg 9720
cctcggggcc aagatcgaac tgtctgacac gaaggccgct tgactcgagt ttctccataa 9780
taatgtgtga gtagttccca gataagggaa ttagggttcc tatagggttt cgctcatgtg 9840
ttgagcatat aagaaaccct tagtatgtat ttgtatttgt aaaatacttc tatcaataaa 9900
atttctaatt cctaaaacca aaatccagta ctaaaatcca gatcccccga attaattcgg 9960
cgttaattca gtacattaaa aacgtccgca atgtgttatt aagttgtcta agcgtcaatt 10020
tgtttacacc acaatatatc ctgcca 10046

Claims (8)

1.一种抗虫基因T-DNA载体，其特征在于所述T-DNA载体包含抗虫基因表达框，所述抗虫基因表达框由抗虫基因、复合启动子和终止子拼接构成；所述复合启动子由CaMV 35s和Actin-intron拼接而成。
2.如权利要求1所述抗虫基因T-DNA载体，其特征在于所述的抗虫基因由Cry1Ab和Vip3A连接而成。
3.如权利要求2所述抗虫基因T-DNA载体，其特征在于所述Cry1Ab基因的核苷酸序列为SEQ ID NO:1所示，Vip3A基因的核苷酸序列是SEQ ID NO:2所示。
4.如权利要求1所述抗虫基因T-DNA载体，其特征在于所述CaMV 35s核苷酸序列为SEQID NO:4所示，所述Actin-intron核苷酸序列为SEQ ID NO:5所示。
5.如权利要求1抗虫基因T-DNA载体，其特征在于所述终止子为终止子Pepc-ter，核苷酸序列为SEQ ID NO:6所示。
6.如权利要求1所述抗虫基因T-DNA载体，其特征在于所述T-DNA核苷酸序列为SEQ IDNO:7所示。
7.一种权利要求1所述抗虫基因T-DNA载体在制备抗虫植物细胞中的应用。
8.如权利要求7所述的应用，其特征在于所述植物为玉米或水稻。
CN201810671300.0A 2018-06-26 2018-06-26 一种抗虫基因t-dna载体及其应用 Pending CN108998467A (zh)

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