CN106459988A - 可选择标志物基因在甜菜原生质体转化方法和系统中的用途 - Google Patents

可选择标志物基因在甜菜原生质体转化方法和系统中的用途 Download PDF

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CN106459988A
CN106459988A CN201580031418.1A CN201580031418A CN106459988A CN 106459988 A CN106459988 A CN 106459988A CN 201580031418 A CN201580031418 A CN 201580031418A CN 106459988 A CN106459988 A CN 106459988A
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盖伊·魏恩斯
马克·勒菲弗
夏夫高达·塔姆曼纳高达
米里耶勒·隆梅尔
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SESVanderHave NV
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Abstract

本发明涉及用于甜菜原生质体转化的方法，包括以下步骤：‑从分离自甜菜植物的气孔保卫细胞获得原生质体，‑用包含感兴趣的核苷酸序列和选择标志物序列的核酸构建体转化所述原生质体，‑以对所述原生质体体外培养致死的浓度向所述原生质体的体外培养应用一种或多种ALS抑制剂，和‑从整合了包含感兴趣的序列和选择标志物序列的核酸构建体的存活的原生质体再生甜菜植物，其中所述选择标志物序列是在其序列中携带在氨基酸113位从丙氨酸至酪氨酸的突变的突变BvALS113序列。

Description

可选择标志物基因在甜菜原生质体转化方法和系统中的用途
技术领域
本发明处于植物生物技术领域，更具体地，涉及植物转化方法和方式(means)，具体地，涉及通过使用新型选择标志物遗传序列，优选地突变BvALS可选择标志物遗传序列用于转化甜菜原生质体、细胞、组织(愈伤组织)和/或植物的方法和方式。
本发明还涉及从所述转化的甜菜原生质体、细胞或组织再生转基因甜菜植物的方法，并且涉及通过该方法获得的转基因植物和种子。
背景技术
甜菜(Beta vulgaris L.)是重要的农作物，其参与世界约20％的糖生产。即使对基因修饰的甜菜植物的需求日益提高，甜菜植物仍是难以转化的植物和难以植物再生的材料。仍需要改善和创新以获得这种植物可靠、有效的转化和再生方法。
通常并且更具体地，对于难以转化的植物，如甜菜，用于选择转化的原生质体、细胞或组织的适当、有效的方式或方法的可用性是至关重要的。
此外，由于选择标志物通常存在于转化植物中，因此核苷酸序列，如编码对抗生素耐受性的基因的用途是不可接受的，这是由于将使用后续的并且通常复杂、昂贵的方法来从转化植物中除去选择标志物。
由于甜菜是温带和亚热带重要的农作物，因此在其生长期间，广泛使用除草剂来控制杂草增殖。这些新开发的除草剂包括乙酰乳酸合酶(Acetolactate synthase)(ALS)，也称为乙酰羟酸合酶(Acetohydroxyacid synthase)(AHAS)抑制剂。根据美国专利5 013659、5 141 870和5 378 824，编码乙酰乳酸合酶(ALS)的基因是已知的。
发明目标
本发明的目标在于提供不具有现有技术缺点的转化甜菜原生质体、细胞、组织(愈伤组织)和植物的新方法和新方式，具体地，涉及基于新选择标志物的使用用于改善包含和表达一种或多种感兴趣的基因的基因修饰的甜菜原生质体、细胞、组织和植物的选择的方法和方式。
发明内容
本发明涉及通过使用BvALS113突变的遗传序列作为选择(可选择)标志物基因转化甜菜原生质体、细胞、组织和植物的新方法和新方式。
优选地，本发明涉及使用核酸构建体(或者包含该核酸构建体的载体)的甜菜原生质体、细胞、组织和/或植物的转化方法，并且涉及包含感兴趣的核苷酸序列(优选地，基因)和选择标志物序列(或者由其组成)的该核酸构建体，当与野生型ALS序列相比时，所述选择标志物序列是在其序列中携带在氨基酸113位丙氨酸至酪氨酸突变的突变BvALS113序列。
优选地，在根据本发明所述的核酸构建体、载体或方法中，感兴趣的核苷酸序列选自由以下各项组成的组：编码赋予对昆虫耐受性、对线虫耐受性或对植物疾病耐受性的肽的序列，或者编码抗盐或氢应激的肽的核苷酸序列，或者编码一种或多种酶、抗真菌肽或抗细菌肽的序列，或它们的混合物。
有利地，根据本发明并且在本发明所述的方法中使用的核酸构建体包含在载体中，所述载体还包含用于感兴趣的核苷酸序列以及可能的选择标志物序列在甜菜原生质体、细胞、组织或植物中的表达的(适当的)调控序列。这些调控序列优选地选自由以下各项组成的组：在植物中有活性的启动子、转录终止和/或聚腺苷酰化信号序列，更优选地，(来自根癌农杆菌(Agrobacterium tumefaciens))的(CAMV)35S启动子序列和Nos终止子序列。
优选地，根据本发明的选择(可选择)标志物序列是当与其相应野生型序列(优选地，序列SEQ.ID.NO：1)相比时，在其(野生型)ALS序列中在氨基酸113位包含突变(L-丙氨酸(Ala)突变为L-酪氨酸(Tyr))的序列SEQ.ID.NO：3。
有利地，根据本发明所述的方法包括以下步骤：
-从分离自甜菜植物的气孔保卫细胞获得原生质体，
-用上述包含感兴趣的核苷酸序列和选择标志物序列的核酸构建体(或载体)转化这些原生质体，
-以对这些体外培养的原生质体致死的浓度(大于(约)99.9％)，将一种或多种ALS抑制剂应用于这些原生质体的体外培养，并从这些体外培养的原生质体的存活的原生质体再生一种或多种甜菜植物，并且其中存活的原生质体是整合了包含该感兴趣的核苷酸序列和该选择标志物序列(提供对一种或多种ALS抑制剂的耐受性)的核酸构建体的原生质体。
在根据本发明所述的方法中，术语“大于(约)99.9％的”表示大于99％；99.1％；99.2％；99.3％；99.4％；99.5％；99.6％；99.7％或者99.8％。
更有利地，在根据本发明所述的方法中，对于甲酰胺磺隆(foramsulfuron)，以(约)5×10-9M至(约)1×10-8M的浓度，对于乙氧嘧磺隆(ethoxysulfuron)，以(约)5×10-11M至(约)5×10-10M的浓度应用ALS抑制剂。
适合的ALS抑制剂优选地选自由以下各项组成的组：磺酰脲除草剂、磺酰氨基羰基三唑啉酮除草剂、咪唑啉酮除草剂、三唑并嘧啶除草剂和嘧啶基(硫代)苯甲酸酯除草剂。更优选地，根据本发明所述的方法包括以下步骤：应用存在于包含至少一种磺酰脲除草剂和至少一种三唑并嘧啶除草剂的组合物中的一些ALS抑制剂。
优选的ALS抑制剂是磺酰脲除草剂，其选自由以下各项组成的组：甲酰胺磺隆(ALF)、碘甲磺隆(iodosulfuron)、酰嘧磺隆(amidosulfuron)、乙氧嘧磺隆(ALE)、氯嘧磺隆(chloramsulfuron)或它们的混合物。
其它适合的ALS抑制剂为噻酮磺隆(thiencarbazone-methyl)和三唑并嘧啶除草剂。
本领域技术人员还可以选择与上述ALS序列相互作用的其它合适的除草剂，并且其中SEQ.ID.NO：3中上述突变(其中与野生型序列SEQ.ID.NO：1相比，在氨基酸113位存在L-丙氨酸(Ala)向L-酪氨酸(Tyr)的突变)可以赋予原生质体、细胞和植物对该ALS抑制剂或ALS抑制剂的混合物的抗性。
在根据本发明所述的方法中，愈伤组织是由(良好再生的)气孔保卫细胞原生质体的生长所造成的。
有利地，当在适合的培养基中生长时，如含有聚合物的培养基(即，如含有海藻酸盐或琼脂糖的培养基)，通过这些(良好再生的)原生质体获得的愈伤组织具有出芽和再生为活的甜菜植物的能力。
在根据本发明所述的方法中，ALS抑制剂是(或者包含)甲酰胺磺隆，如应用于含有海藻酸盐的培养基上的1周大(或者应用于3周大)的原生质体体外培养(更具体地，包含从这些培养的原生质体再生的愈伤组织的体外培养)的甲酰胺磺隆。
在根据本发明所述的方法中，优选地通过农杆菌(Agrobacterium)介导的过程(或方法)转化原生质体，并且所述方法优选地还包含其中通过将转化的植物与相同植物未转化的品种杂交(从植物基因组除去)消除选择标志物序列的步骤。
本发明还涉及包含感兴趣的核苷酸序列(优选地，基因)和选择标志物序列(或者由其组成)的核酸构建体，所述选择标志物序列是在其序列中携带在氨基酸113位从丙氨酸至酪氨酸突变的突变BvALS113序列，并且涉及包含该核酸构建体和用于该核酸构建体的感兴趣的核苷酸序列和可能的选择标志物序列在甜菜原生质体、细胞、组织和/或植物中表达的一种或多种调控序列的载体。
本发明最后的方面涉及通过转化方法获得或者包含根据本发明所述的核酸构建体或载体的原生质体、细胞、组织(愈伤组织)或植物，更具体地，涉及在其基因组中整合了感兴趣的核苷酸序列的甜菜植物。在以下实施例中，将参考作为本发明非限制性实施方式提供的附图详细描述本发明。
附图说明
图1表示根据本发明所述的pS189载体。
图2表示BvALS基因的序列，包括所使用的序列SEQ ID NO：3
图3表示通过本发明所述的pS189载体转化的甜菜原生质体的存活性。
具体实施方式
出于它们从气孔保卫细胞原生质体再生的能力，选择了一些甜菜植物基因型。通过它们体外生长和分化的能力，选择原生质体。还根据生长的愈伤组织出芽的能力以及生长的愈伤组织再生植物的比例进行选择。优选地，所选基因型具有大于0.25％的能够体外生长的气孔保卫细胞原生质体。本领域技术人员可以(例如)将作为NCIMB 42050或NCIMB42051提交保存的植物作为包含高比例生长气孔保卫细胞原生质体的适合基因型。
在根据本发明所述的方法中，当在适合的培养基中生长时，气孔保卫细胞原生质体具有分化(生长)和再生(优选地，通过甜菜愈伤组织)的能力。愈伤组织是指未分化细胞块(mass)。在本领域中，愈伤组织可以得自外植体(explant)，如来自叶或子叶的胚胎或薄壁组织(parenchyma)来源的外植体。
在附图1中描述了根据本发明所使用的载体，并且所述载体可以包含由均受组成型(CAMV)35S启动子和Nos终止子序列控制的由本发明的感兴趣的核苷酸序列和选择标志物序列组成(包含它们或由它们组成)的核酸构建体。在根据本发明的该核酸构建体或载体中，通过IDT(整合DNA技术)合成了在位置337含有核苷酸TAT而不是GCA(将位置113处相应的氨基酸从丙氨酸氨基酸(Ala)改变为酪氨酸氨基酸(Tyr))的甜菜ALS核苷酸序列，并且将所述甜菜ALS核苷酸序列插入到载体pIDT blue中。然后，将核酸构建体作为Kpnl-Bglll片段插入到pMJB3质粒中在用于组成型表达的2x35S启动子和来自根癌农杆菌(Agrobacterium tumefaciens)的Nos终止子之间。将所得质粒(根据本发明的载体)称为pS189并且如图1所示。
根据两种优选的ALS抑制剂(甲酰胺磺隆和乙氧嘧磺隆)的杀灭曲线浓度确定在根据本发明所述的方法中使用的最佳除草剂(ALS抑制剂)浓度并且如下所示：
为了使用用于转化甜菜原生质体选择的除草剂的最佳浓度，根据以下浓度(表1和表2)对野生型甜菜原生质体建立杀灭曲线(killing curve)。根据杀灭曲线实验所获得的结果，为了与具有携带突变的BvALS核苷酸序列的pS189质粒DNA的转化的甜菜原生质体一起使用，对甲酰胺磺隆选择5×10-9M的浓度，对于乙氧嘧磺隆选择5×10-11M的浓度。
表1：用于评价甜菜原生质体的最佳致死剂量浓度的甲酰胺磺隆的浓度
表2：用于评价甜菜原生质体的最佳致死剂量浓度的乙氧嘧磺隆的浓度
在含有5×10-9M甲酰胺磺隆(ALF)和5×10-11M乙氧嘧磺隆(ALE)浓度的两种选择培养基条件下，根据标准PEG转化系统，使用质粒DNA(pS189)进行转化实验。通过从每个转化实验回收的愈伤组织的数目，观察到仅选择了转化的原生质体。作为对照实验，将未转化的原生质体置于不导致任何愈伤组织或细胞存活的甲酰胺磺隆和乙氧嘧磺隆选择培养基上。到目前为止，对ALF进行了7个转化实验，对ALE进行了6个实验。由ALF实验，获得了约1799个芽和89株再生小植株(plantlet)(表3)。
对这89个原代转化事件(primary transformation event)筛选了倍体水平(ploidy level)，并且导致产生了27个二倍体事件(diploid event)。将对这些事件进行用于通过PCR确认转基因存在的其它分子分析、通过taqman分析的拷贝数分析和Southern印迹分析。
表3：以甲酰胺磺隆作为选择培养基的pS189质粒转化实验概述
可以使用多种转化方法，例如，PEG添加或农杆菌(Agrobacterium)介导的转化载体(农杆菌(Agrobacterium)载体)来将异源核苷酸序列插入到对农杆菌(Agrobacterium)感染敏感的原生质体或细胞中。
根据本发明所述的核酸构建体或载体优选地包含启动子、包含感兴趣的核苷酸序列，优选地感兴趣的基因产物以及选择标志物序列的编码序列以及调控序列，如聚腺苷酰化信号和转录激活序列(增强子，如烟草花叶病毒(TMV)或烟草蚀刻病毒(TEV)的翻译激活子序列)及其它转录终止子(增强子)序列。本领域技术人员可以选择用于获得选择标志物序列和感兴趣的核苷酸序列在所选细胞、组织和植物中表达的其它适合的序列。
(组成型)启动子序列可以得自植物或病毒并且包含(但不限于)花椰菜花叶病毒(CAMV)或来自圆环病(circovirus))的35S或19S启动子或者分离自植物基因，或种子特异性的启动子，如Napin启动子、云扁豆蛋白(phaseolin)启动子、麦谷蛋白(glutenin)启动子、向日葵蛋白(helianthinin)启动子、白蛋白启动子、oesosin启动子、SAT1启动子、SAT3启动子和诱导型启动子，如Pal启动子、HMG启动子、RuBisCO启动子和得自根癌农杆菌(agrobacterium tumefaciens)的T-DNA基因的启动子，如胭脂氨酸(nopaline)启动子和甘露碱合酶(mannopine synthase)启动子。
因此，本发明还涉及适合于转化甜菜植物细胞(可以使用农杆菌(Agrobacterium)介导的方法)并且包含至少(异源)核酸构建体的载体，所述核酸构建体包含根据本发明的感兴趣的序列和选择标志物序列或者由它们组成。
可以使用多种农杆菌(Agrobacterium)株，包括(但不限于)根癌农杆菌(Agrobacterium tumefaciens)和发根植物单胞菌(Agrobacterium rhizogenes)。适合的根癌农杆菌(Agrobacterium tumefaciens)株包括A208EHA101和LBA4404株。适合的发根植物单胞菌(Agrobacterium rhizogenes)株包括K599株。
可以将选择标志物序列与感兴趣的核苷酸序列同时引入原生质体或细胞，优选地，在相同载体上并且受相同调控序列(相同启动子)控制，但是也可以以汇聚(convergent)/发散(divergent)或同线(collinear)方式或者通过同时用于转化植物原生质体或细胞的两种载体的施用结合。
在根据本发明所述的核酸构建体或载体中，感兴趣的核苷酸序列是受在植物细胞中具有活性的调控序列(如启动子序列，其在所选植物细胞或感兴趣的植物中是功能性的)控制的编码感兴趣的蛋白质的基因以赋予转化植物新型的农艺学性质或者改善转化植物的农艺学品质。优选地，感兴趣的这些序列选自由以下各项组成的组：编码赋予对某些昆虫的耐受性、赋予对线虫的耐受性、赋予对某些疾病的耐受性的蛋白质的序列、编码特异性酶的序列和/或编码抗细菌或抗真菌肽或蛋白的序列。
序列表
<110> 西斯凡德尔哈维公众公司
<120> 可选择标志物基因在甜菜原生质体转化方法和系统中的用途
<130> SESS0012
<150> PCT/EP2014/062288
<151> 2014-06-12
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 1998
<212> DNA
<213> 甜菜
<400> 1
atggcggcta ccttcacaaa cccaacattt tccccttcct caactccatt aaccaaaacc 60
ctaaaatccc aatcttccat ctcttcaacc ctcccctttt ccacccctcc caaaacccca 120
actccactct ttcaccgtcc cctccaaatc tcatcctccc aatcccacaa atcatccgcc 180
attaaaacac aaactcaagc accttcttct ccagctattg aagattcatc tttcgtttct 240
cgatttggcc ctgatgaacc cagaaaaggg tccgatgtcc tcgttgaagc tcttgagcgt 300
gaaggtgtta ccaatgtgtt tgcttaccct ggtggtgcat ctatggaaat ccaccaagct 360
ctcacacgct ctaaaaccat ccgcaatgtc ctccctcgcc atgaacaagg cggggttttc 420
gccgccgagg gatatgctag agctactgga aaggttggtg tctgcattgc gacttctggt 480
cctggtgcta ccaacctcgt atcaggtctt gctgacgctc tccttgattc tgtccctctt 540
gttgccatca ctggccaagt tccacgccgt atgattggca ctgatgcttt tcaggagact 600
ccaattgttg aggtgacaag gtctattact aagcataatt atttagtttt ggatgtagag 660
gatattccta gaattgttaa ggaagccttt tttttagcta attctggtag gcctggacct 720
gttttgattg atcttcctaa agatattcag cagcaattgg ttgttcctga ttgggatagg 780
ccttttaagt tgggtgggta tatgtctagg ctgccaaagt ccaagttttc gacgaatgag 840
gttggacttc ttgagcagat tgtgaggttg atgagtgagt cgaagaagcc tgtcttgtat 900
gtgggaggtg ggtgtttgaa ttctagtgag gagttgagga gatttgttga gttgacaggg 960
attccggtgg ctagtacttt gatggggttg gggtcttacc cttgtaatga tgaactgtct 1020
cttcatatgt tggggatgca cgggactgtt tatgccaatt atgcggtgga taaggcggat 1080
ttgttgcttg ctttcggggt taggtttgat gatcgtgtga ccgggaagct cgaggcgttt 1140
gctagccgtg ctaagattgt gcatattgat attgactctg ctgagattgg gaagaacaag 1200
cagccccatg tgtccatttg tgctgatgtt aaattggcat tgcggggtat gaataagatt 1260
ctggagtcta gaatagggaa gctgaatttg gatttctcca agtggagaga agaattaggt 1320
gagcagaaga aggaattccc actgagtttt aagacatttg gggatgcaat tcctccacaa 1380
tatgccattc aggtgcttga tgagttgacc aatggtaatg ctattataag tactggtgtt 1440
gggcagcacc aaatgtgggc tgcgcagcat tacaagtaca gaaaccctcg ccaatggctg 1500
acctctggtg ggttgggggc tatggggttt gggctaccag ccgccattgg agctgcagtt 1560
gctcgaccag atgcagtggt tgtcgatatt gatggggatg gcagttttat tatgaatgtt 1620
caagagttgg ctacaattag ggtggaaaat ctcccagtta agataatgct gctaaacaat 1680
caacatttag gtatggttgt ccaatgggaa gataggttct ataaagctaa ccgggcacat 1740
acataccttg gaaacccttc caaatctgct gatatcttcc ctgatatgct caaattcgct 1800
gaggcatgtg atattccttc tgcccgtgtt agcaacgtgg ctgatttgag ggccgccatt 1860
caaacaatgt tggatactcc agggccgtac ctgctcgatg tgattgtacc gcatcaagag 1920
catgtgttgc ctatgattcc aagtggtgcc ggtttcaagg ataccattac agagggtgat 1980
ggaagaacct cttattga 1998
<210> 2
<211> 665
<212> PRT
<213> 甜菜
<400> 2
Met Ala Ala Thr Phe Thr Asn Pro Thr Phe Ser Pro Ser Ser Thr Gln
1 5 10 15
Leu Thr Lys Thr Leu Lys Ser Gln Ser Ser Ile Ser Ser Thr Leu Pro
20 25 30
Phe Ser Thr Pro Pro Lys Thr Pro Thr Pro Leu Phe His Arg Pro Leu
35 40 45
Gln Ile Ser Ser Ser Gln Ser His Lys Ser Ser Ala Ile Lys Thr Gln
50 55 60
Thr Gln Ala Pro Ser Ser Pro Ala Ile Glu Asp Ser Ser Phe Val Ser
65 70 75 80
Arg Phe Gly Pro Asp Glu Pro Arg Lys Gly Ser Asp Val Leu Val Glu
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Ala Leu Glu Arg Glu Gly Val Thr Asn Val Phe Ala Tyr Pro Gly Gly
100 105 110
Ala Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Lys Thr Ile Arg
115 120 125
Asn Val Leu Pro Arg His Glu Gln Gly Gly Val Phe Ala Ala Glu Gly
130 135 140
Tyr Ala Arg Ala Thr Gly Lys Val Gly Val Cys Ile Ala Thr Ser Gly
145 150 155 160
Pro Gly Ala Thr Asn Leu Val Ser Gly Leu Ala Asp Ala Leu Leu Asp
165 170 175
Ser Val Pro Leu Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile
180 185 190
Gly Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser
195 200 205
Ile Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg
210 215 220
Ile Val Lys Glu Ala Phe Phe Leu Ala Asn Ser Gly Arg Pro Gly Pro
225 230 235 240
Val Leu Ile Asp Leu Pro Lys Asp Ile Gln Gln Gln Leu Val Val Pro
245 250 255
Asp Trp Asp Arg Pro Phe Lys Leu Gly Gly Tyr Met Ser Arg Leu Pro
260 265 270
Lys Ser Lys Phe Ser Thr Asn Glu Val Gly Leu Leu Glu Gln Ile Val
275 280 285
Arg Leu Met Ser Glu Ser Lys Lys Pro Val Leu Tyr Val Gly Gly Gly
290 295 300
Cys Leu Asn Ser Ser Glu Glu Leu Arg Arg Phe Val Glu Leu Thr Gly
305 310 315 320
Ile Pro Val Ala Ser Thr Leu Met Gly Leu Gly Ser Tyr Pro Cys Asn
325 330 335
Asp Glu Leu Ser Leu His Met Leu Gly Met His Gly Thr Val Tyr Ala
340 345 350
Asn Tyr Ala Val Asp Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg
355 360 365
Phe Asp Asp Arg Val Thr Gly Lys Leu Glu Ala Phe Ala Ser Arg Ala
370 375 380
Lys Ile Val His Ile Asp Ile Asp Ser Ala Glu Ile Gly Lys Asn Lys
385 390 395 400
Gln Pro His Val Ser Ile Cys Ala Asp Val Lys Leu Ala Leu Arg Gly
405 410 415
Met Asn Lys Ile Leu Glu Ser Arg Ile Gly Lys Leu Asn Leu Asp Phe
420 425 430
Ser Arg Trp Arg Glu Glu Leu Gly Glu Gln Lys Lys Glu Phe Pro Leu
435 440 445
Ser Phe Lys Thr Phe Gly Asp Ala Ile Pro Pro Gln Tyr Ala Ile Gln
450 455 460
Val Leu Asp Glu Leu Thr Asn Gly Asn Ala Ile Ile Ser Thr Gly Val
465 470 475 480
Gly Gln His Gln Met Trp Ala Ala Gln His Tyr Lys Tyr Arg Asn Pro
485 490 495
Arg Gln Trp Leu Thr Ser Gly Gly Leu Gly Ala Met Gly Phe Gly Leu
500 505 510
Pro Ala Ala Ile Gly Ala Ala Val Ala Arg Pro Asp Ala Val Val Val
515 520 525
Asp Ile Asp Gly Asp Gly Ser Phe Ile Met Asn Val Gln Glu Leu Ala
530 535 540
Thr Ile Arg Val Glu Asn Leu Pro Val Lys Ile Met Leu Leu Asn Asn
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Gln His Leu Gly Met Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala
565 570 575
Asn Arg Ala His Thr Tyr Leu Gly Asn Pro Ser Lys Ser Ala Asp Ile
580 585 590
Phe Pro Asp Met Leu Lys Phe Ala Glu Ala Cys Asp Ile Pro Ser Ala
595 600 605
Arg Val Ser Asn Val Ala Asp Leu Arg Ala Ala Ile Gln Thr Met Leu
610 615 620
Asp Thr Pro Gly Pro Tyr Leu Leu Asp Val Ile Val Pro His Gln Glu
625 630 635 640
His Val Leu Pro Met Ile Pro Ser Gly Ala Gly Phe Lys Asp Thr Ile
645 650 655
Thr Glu Gly Asp Gly Arg Thr Ser Tyr
660 665
<210> 3
<211> 1998
<212> DNA
<213> 甜菜
<400> 3
atggcggcta ccttcacaaa cccaacattt tccccttcct caactccatt aaccaaaacc 60
ctaaaatccc aatcttccat ctcttcaacc ctcccctttt ccacccctcc caaaacccca 120
actccactct ttcaccgtcc cctccaaatc tcatcctccc aatcccacaa atcatccgcc 180
attaaaacac aaactcaagc accttcttct ccagctattg aagattcatc tttcgtttct 240
cgatttggcc ctgatgaacc cagaaaaggg tccgatgtcc tcgttgaagc tcttgagcgt 300
gaaggtgtta ccaatgtgtt tgcttaccct ggtggttatt ctatggaaat ccaccaagct 360
ctcacacgct ctaaaaccat ccgcaatgtc ctccctcgcc atgaacaagg cggggttttc 420
gccgccgagg gatatgctag agctactgga aaggttggtg tctgcattgc gacttctggt 480
cctggtgcta ccaacctcgt atcaggtctt gctgacgctc tccttgattc tgtccctctt 540
gttgccatca ctggccaagt tccacgccgt atgattggca ctgatgcttt tcaggagact 600
ccaattgttg aggtgacaag gtctattact aagcataatt atttagtttt ggatgtagag 660
gatattccta gaattgttaa ggaagccttt tttttagcta attctggtag gcctggacct 720
gttttgattg atcttcctaa agatattcag cagcaattgg ttgttcctga ttgggatagg 780
ccttttaagt tgggtgggta tatgtctagg ctgccaaagt ccaagttttc gacgaatgag 840
gttggacttc ttgagcagat tgtgaggttg atgagtgagt cgaagaagcc tgtcttgtat 900
gtgggaggtg ggtgtttgaa ttctagtgag gagttgagga gatttgttga gttgacaggg 960
attccggtgg ctagtacttt gatggggttg gggtcttacc cttgtaatga tgaactgtct 1020
cttcatatgt tggggatgca cgggactgtt tatgccaatt atgcggtgga taaggcggat 1080
ttgttgcttg ctttcggggt taggtttgat gatcgtgtga ccgggaagct cgaggcgttt 1140
gctagccgtg ctaagattgt gcatattgat attgactctg ctgagattgg gaagaacaag 1200
cagccccatg tgtccatttg tgctgatgtt aaattggcat tgcggggtat gaataagatt 1260
ctggagtcta gaatagggaa gctgaatttg gatttctcca agtggagaga agaattaggt 1320
gagcagaaga aggaattccc actgagtttt aagacatttg gggatgcaat tcctccacaa 1380
tatgccattc aggtgcttga tgagttgacc aatggtaatg ctattataag tactggtgtt 1440
gggcagcacc aaatgtgggc tgcgcagcat tacaagtaca gaaaccctcg ccaatggctg 1500
acctctggtg ggttgggggc tatggggttt gggctaccag ccgccattgg agctgcagtt 1560
gctcgaccag atgcagtggt tgtcgatatt gatggggatg gcagttttat tatgaatgtt 1620
caagagttgg ctacaattag ggtggaaaat ctcccagtta agataatgct gctaaacaat 1680
caacatttag gtatggttgt ccaatgggaa gataggttct ataaagctaa ccgggcacat 1740
acataccttg gaaacccttc caaatctgct gatatcttcc ctgatatgct caaattcgct 1800
gaggcatgtg atattccttc tgcccgtgtt agcaacgtgg ctgatttgag ggccgccatt 1860
caaacaatgt tggatactcc agggccgtac ctgctcgatg tgattgtacc gcatcaagag 1920
catgtgttgc ctatgattcc aagtggtgcc ggtttcaagg ataccattac agagggtgat 1980
ggaagaacct cttattga 1998
<210> 4
<211> 665
<212> PRT
<213> 甜菜
<400> 4
Met Ala Ala Thr Phe Thr Asn Pro Thr Phe Ser Pro Ser Ser Thr Gln
1 5 10 15
Leu Thr Lys Thr Leu Lys Ser Gln Ser Ser Ile Ser Ser Thr Leu Pro
20 25 30
Phe Ser Thr Pro Pro Lys Thr Pro Thr Pro Leu Phe His Arg Pro Leu
35 40 45
Gln Ile Ser Ser Ser Gln Ser His Lys Ser Ser Ala Ile Lys Thr Gln
50 55 60
Thr Gln Ala Pro Ser Ser Pro Ala Ile Glu Asp Ser Ser Phe Val Ser
65 70 75 80
Arg Phe Gly Pro Asp Glu Pro Arg Lys Gly Ser Asp Val Leu Val Glu
85 90 95
Ala Leu Glu Arg Glu Gly Val Thr Asn Val Phe Ala Tyr Pro Gly Gly
100 105 110
Tyr Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Lys Thr Ile Arg
115 120 125
Asn Val Leu Pro Arg His Glu Gln Gly Gly Val Phe Ala Ala Glu Gly
130 135 140
Tyr Ala Arg Ala Thr Gly Lys Val Gly Val Cys Ile Ala Thr Ser Gly
145 150 155 160
Pro Gly Ala Thr Asn Leu Val Ser Gly Leu Ala Asp Ala Leu Leu Asp
165 170 175
Ser Val Pro Leu Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile
180 185 190
Gly Thr Asp Ala Phe Gln Glu Thr Pro Ile Val Glu Val Thr Arg Ser
195 200 205
Ile Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg
210 215 220
Ile Val Lys Glu Ala Phe Phe Leu Ala Asn Ser Gly Arg Pro Gly Pro
225 230 235 240
Val Leu Ile Asp Leu Pro Lys Asp Ile Gln Gln Gln Leu Val Val Pro
245 250 255
Asp Trp Asp Arg Pro Phe Lys Leu Gly Gly Tyr Met Ser Arg Leu Pro
260 265 270
Lys Ser Lys Phe Ser Thr Asn Glu Val Gly Leu Leu Glu Gln Ile Val
275 280 285
Arg Leu Met Ser Glu Ser Lys Lys Pro Val Leu Tyr Val Gly Gly Gly
290 295 300
Cys Leu Asn Ser Ser Glu Glu Leu Arg Arg Phe Val Glu Leu Thr Gly
305 310 315 320
Ile Pro Val Ala Ser Thr Leu Met Gly Leu Gly Ser Tyr Pro Cys Asn
325 330 335
Asp Glu Leu Ser Leu His Met Leu Gly Met His Gly Thr Val Tyr Ala
340 345 350
Asn Tyr Ala Val Asp Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg
355 360 365
Phe Asp Asp Arg Val Thr Gly Lys Leu Glu Ala Phe Ala Ser Arg Ala
370 375 380
Lys Ile Val His Ile Asp Ile Asp Ser Ala Glu Ile Gly Lys Asn Lys
385 390 395 400
Gln Pro His Val Ser Ile Cys Ala Asp Val Lys Leu Ala Leu Arg Gly
405 410 415
Met Asn Lys Ile Leu Glu Ser Arg Ile Gly Lys Leu Asn Leu Asp Phe
420 425 430
Ser Arg Trp Arg Glu Glu Leu Gly Glu Gln Lys Lys Glu Phe Pro Leu
435 440 445
Ser Phe Lys Thr Phe Gly Asp Ala Ile Pro Pro Gln Tyr Ala Ile Gln
450 455 460
Val Leu Asp Glu Leu Thr Asn Gly Asn Ala Ile Ile Ser Thr Gly Val
465 470 475 480
Gly Gln His Gln Met Trp Ala Ala Gln His Tyr Lys Tyr Arg Asn Pro
485 490 495
Arg Gln Trp Leu Thr Ser Gly Gly Leu Gly Ala Met Gly Phe Gly Leu
500 505 510
Pro Ala Ala Ile Gly Ala Ala Val Ala Arg Pro Asp Ala Val Val Val
515 520 525
Asp Ile Asp Gly Asp Gly Ser Phe Ile Met Asn Val Gln Glu Leu Ala
530 535 540
Thr Ile Arg Val Glu Asn Leu Pro Val Lys Ile Met Leu Leu Asn Asn
545 550 555 560
Gln His Leu Gly Met Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala
565 570 575
Asn Arg Ala His Thr Tyr Leu Gly Asn Pro Ser Lys Ser Ala Asp Ile
580 585 590
Phe Pro Asp Met Leu Lys Phe Ala Glu Ala Cys Asp Ile Pro Ser Ala
595 600 605
Arg Val Ser Asn Val Ala Asp Leu Arg Ala Ala Ile Gln Thr Met Leu
610 615 620
Asp Thr Pro Gly Pro Tyr Leu Leu Asp Val Ile Val Pro His Gln Glu
625 630 635 640
His Val Leu Pro Met Ile Pro Ser Gly Ala Gly Phe Lys Asp Thr Ile
645 650 655
Thr Glu Gly Asp Gly Arg Thr Ser Tyr
660 665

Claims (13)

1.一种用于转化甜菜原生质体的方法，包括以下步骤：
-从分离自甜菜植物的气孔保卫细胞获得原生质体，
-用包含感兴趣的核苷酸序列和选择标志物序列的核酸构建体转化所述原生质体，
-以对所述原生质体体外培养致死的浓度，向所述原生质体的体外培养应用一种或多种ALS抑制剂，和
-从整合了包含感兴趣的所述序列和所述选择标志物序列的核酸构建体的存活的原生质体再生甜菜植物，其中所述选择标志物序列是在其序列中携带在氨基酸113位从丙氨酸至酪氨酸的突变的突变BvALS113序列。
2.根据权利要求1所述的方法，其中所述突变BvALS113序列包含SEQ.ID.NO：3或由其组成。
3.根据权利要求1或2所述的方法，其中所述核酸序列进一步包括在甜菜原生质体、细胞、组织和/或植物中用于表达感兴趣的所述核苷酸序列和所述选择标志物序列的一种或多种调控序列。
4.根据以上权利要求中任一项所述的方法，其中所述ALS抑制剂选自由以下各项组成的组：磺酰脲除草剂、磺酰氨基羰基三唑啉酮除草剂、咪唑啉酮除草剂、三唑并嘧啶除草剂、嘧啶基(硫代)苯甲酸酯除草剂、噻酮磺隆除草剂或它们的混合物。
5.根据权利要求4所述的方法，其中所述磺酰脲除草剂选自由以下各项组成的组：甲酰胺磺隆、碘甲磺隆、酰嘧磺隆、乙氧嘧磺隆、氯嘧磺隆或它们的混合物。
6.根据以上权利要求中任一项所述的方法，其中对于甲酰胺磺隆，以5×10-9M至1×10- 8M之间的浓度，对于乙氧嘧磺隆，以5×10-11M至5×10-10M之间的浓度应用所述ALS抑制剂。
7.根据以上权利要求中任一项所述的方法，其中通过农杆菌介导的方法转化所述原生质体。
8.根据以上权利要求中任一项所述的方法，包含以下步骤，其中通过将转化植物与相同植物的未转化品种杂交来消除所述选择标志物序列。
9.根据以上权利要求中任一项所述的方法，其中感兴趣的所述核苷酸序列编码选自由以下各项组成的组的肽：赋予对昆虫的耐受性、对线虫的耐受性或对植物疾病的耐受性的肽、保护植物抵抗盐或氢应激的肽、编码一种或多种酶或者编码抗真菌肽或抗细菌肽的肽或它们的混合物。
10.一种核酸构建体，包含感兴趣的核苷酸序列和选择标志物序列或者由感兴趣的核苷酸序列和选择标志物序列组成，所述选择标志物序列是在其序列中携带在氨基酸113位从丙氨酸至酪氨酸的突变的突变BvALS113序列。
11.根据权利要求10所述的核酸构建体，其中所述突变BvALS113序列包含SEQ.ID.NO：3或由其组成。
12.一种载体，包含权利要求10或11所述的核酸构建体以及用于将所述核酸构建体的感兴趣的核苷酸序列和选择标志物序列在甜菜原生质体、细胞、组织和/或植物中表达的一种或多种调控序列。
13.通过根据以上权利要求1-9中任一项所述的方法获得的或者包含根据以上权利要求10或12中任一项所述的核酸构建体或载体的转基因甜菜原生质体、细胞组织或植物。
CN201580031418.1A 2014-06-12 2015-06-12 可选择标志物基因在甜菜原生质体转化方法和系统中的用途 Pending CN106459988A (zh)

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