CN101252831A - 大豆事件mon89788和检测它的方法 - Google Patents

大豆事件mon89788和检测它的方法 Download PDF

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CN101252831A
CN101252831A CNA2006800275135A CN200680027513A CN101252831A CN 101252831 A CN101252831 A CN 101252831A CN A2006800275135 A CNA2006800275135 A CN A2006800275135A CN 200680027513 A CN200680027513 A CN 200680027513A CN 101252831 A CN101252831 A CN 101252831A
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M·马尔文
J·赖恩哈特
N·泰勒
E·迪金森
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Abstract

本发明提供了包含转化事件MON89788的大豆植物和种子,以及这些事件独特的DNA分子。本发明还提供了检测样品中这些DNA分子的存在的方法。

Description

大豆事件MON89788和检测它的方法
发明背景
本申请要求2005年5月27日提交的美国临时申请NO.60/685,584的优先权,通过引用将其完整公开内容合并在此。
1.发明领域
本发明涉及新的和独特的转基因大豆转化事件,称为MON89788,由此衍生的大豆品种,以及其植物部分、种子和产物。本发明还涉及用于检测植物部分提取物或种子提取物中特异于MON89788的DNA分子存在的分析。
2.相关领域的说明
大豆(Glycine max)是世界的许多区域中的重要作物。已经将生物技术方法应用于大豆用于改善产品的农业性状和品质。大豆生产中的一种这样的重要农学性状是除草剂耐受性,特别是对草甘膦除草剂的耐受性。除草剂耐受性大豆事件将是对于管理杂草有用的性状。
N-磷酰甲基甘氨酸,也称为草甘膦,是一种在广谱的植物品种上具有活性的公知的除草剂。草甘膦是
Figure A20068002751300051
(Monsanto Co.,St.Louis,MO)的活性成分,是在环境中具有理想的短半衰期的安全的除草剂。当施用到植物表面时,草甘膦在植物中全身性地移动。由于其对莽草酸途径的抑制,草甘膦是植物毒性的,莽草酸途径提供了用于合成芳香族氨基酸的前体。草甘膦抑制在植物中发现的5-烯醇丙酮-3-磷酸莽草酸合酶(EPSPS)。
草甘膦耐受性可以通过EPSPS变体的表达来实现,所述EPSPS变体具有对草甘膦的更低亲和性,并因而在存在草甘膦的情况下保持了它们的催化活性(美国专利NO.5,633,435;5,094,945;4,535,060和6,040,497)。在植物组织中降解草甘膦的酶(美国专利No.5,463,175)也能够赋予对草甘膦的细胞耐受性。这些基因被用于耐受草甘膦的转基因作物的生产,因而容许以作物损害的最小牵涉将草甘膦用于有效的杂草控制。例如,草甘膦耐受性已经被遗传工程化到玉米(美国专利No.5,554,798)、小麦(美国专利6,689,880)、棉花(美国专利6,740,488)、大豆(WO 9200377)和canola(美国专利申请20040018518)中。关于草甘膦耐受性的转基因以及关于对其他除草剂耐受性的转基因,例如bar基因(Toki et al.,1992;Thompson et al.,1987;phosphinothricin转乙酰酶(DeBlock et al.,1987),关于对glufosinate除草剂的耐受性)作为可选择标记物或可计分标记物也是有用的,并且可以提供有用的表型,用于选择与其他农业上有用性状联系的植物。
已知在植物中外源基因表达受到它们的染色体位置的影响,也许是由于染色质结构(例如,异染色质)或转录调节元件(例如,增强子)靠近整合位点的邻近(Weising et al.,1988)。为此,常常必须筛选大量的事件来鉴定以导入的目标基因的最优表达为特征的事件。例如,已经在植物和其他有机体中观察到在事件之中可能存在着在导入的基因的表达水平上的广泛变动。在表达的空间或时间模式上也可能存在差异,例如,在各种植物组织中转基因的相对表达的差异,其可能不相应于根据导入的基因构建体中存在的转录调节元件所预期的模式。为此,普遍的是生产数百到数千个不同的事件并从这些事件中筛选出具有用于商业目的的期望的转基因表达水平和模式的单一事件。具有期望的转基因表达水平或模式的事件对于利用常规育种方法通过有性异型杂交将转基因渗入到其他的遗传背景中是有用的。这种杂交的子代维持了原始转化体的转基因表达特征。这种策略被用于在许多品种中确保可靠的基因表达,这些品种很好的适应了当地的生长条件。
能够检测特定事件的存在以确定有性杂交的子代是否包含目标转基因将是有益的。此外,检测特定事件的方法对于遵守需要上市前批准和对来源于重组农作物的食物的标记,比方说,是很有帮助的。通过任何公知的多核酸检测方法例如聚合酶链式反应(PCR)或利用多核酸探针的DNA杂交,有可能检测转基因的存在。这些检测方法通常集中于经常使用的遗传元件,例如启动子、终止子、标记基因等等。结果,除非邻近于插入的转基因DNA的染色体DNA(“侧翼DNA”)的序列是已知的,这种方法对于辨别不同的事件,特别是那些利用相同的DNA构建体产生的事件可能是没有用的。已经讨论了事件特异性的PCR分析,例如,Windels等(1999),他们利用跨越了插入转基因和侧翼DNA之间的交界的引物集,特别是一个包括来自插入物的序列的引物以及包括来自侧翼DNA的序列的第二引物,通过PCR鉴别了草甘膦耐受的大豆事件40-3-2。转基因植物事件特异性DNA检测方法也已经在美国专利NO.6,893,826;6,825,400;6,740,488;6,733,974和6,689,880;6,900,014和6,818,807中描述,通过完全引用将它们合并在此。
本发明涉及草甘膦耐受性大豆事件MON89788(也称为MON19788或GM_A19788),并涉及这些大豆植物中含有的DNA分子,其在来自MON89788的植物和其子代以及植物组织的检测方法中是有用的。
发明概述
本发明提供了称为MON89788(也称为MON19788)的大豆转基因事件和其子代,具有以登记号No.PTA-6708在美国典型培养物保藏所(ATCC)保藏的代表性的种子。本发明的另一个方面是大豆事件MON89788的植物和种子的植物细胞或可再生部分。本发明并且包括大豆事件MON89788的植物部分,其包括但不限于细胞、花粉、胚珠、花、芽、根、叶和来自MON89788的产物,例如大豆饼、粉和油。
本发明的一个方面提供了组合物和方法,用于检测来自大豆事件MON89788植物或种子,或来自植物部分或种子的产物的DNA转基因/基因组连接区的存在。提供了DNA分子,其包含选自由SEQ ID NO:1和SEQ ID NO:2、以及其互补物构成的组的至少一个转基因/基因组连接DNA分子,其中连接分子跨越插入位点和基因组DNA,所述插入位点包含插入到大豆细胞基因组中的异源DNA,所述基因组DNA来自大豆细胞、侧翼于插入位点大豆事件MON89788。在本发明的一个方面,这种连接序列可以定义为分别包含SEQ ID NO:9的核苷酸1093-1113或5396-5416。在本发明的其他方面,接点可以定义为包括例如侧翼基因组和转基因的其他部分,并可以定义为包含由SEQ ID NO:9的1073-1113、1043-1113、1093-1133、1093-1163、1043-1163、5376-5416、5346-5416、5396-5436、5396-5416、5396-5466或5346-5466所给出的一个或多个序列。这些序列以及包含这些序列的植物和种子因而形成了本发明的一个方面。
提供了新的DNA分子,其是来自大豆事件MON89788的DNA转基因/基因组区域SEQ ID NO:3或其互补物。在其基因组中包含SEQ IDNO:3的大豆植物和种子是本发明的一个方面。SEQ ID NO:3进一步包含完整的SEQ ID NO:1。
根据本发明的另一个方面,提供了DNA分子,其是DNA转基因/基因组区域SEQID NO:4或其互补物,其中这个DNA分子在大豆事件MON89788中是新的。在其基因组中包含SEQ ID NO:4的大豆植物和种子是本发明的一个方面。SEQ ID NO:4进一步包含完整的SEQ ID NO:2。
根据本发明的另一个方面,提供了两个核酸分子用于DNA检测方法中,其中第一核酸分子包含SEQ ID NO:3的DNA分子的转基因区域的任何部分的至少11个或更多个连续多核苷酸,第二核酸是SEQ ID NO:3的5′侧翼大豆基因组DNA区域的任何部分的类似长度的分子,其中这些核酸分子在一起使用时在产生扩增子的DNA扩增方法中作为引物是有用的。在DNA扩增方法中使用这些引物产生的扩增子对于大豆事件MON89788 DNA是诊断性的。通过所描述的引物产生的扩增子是本发明的一个方面,所述引物同源于或互补于包含SEQ ID NO:1的SEQ ID NO:3的一部分。
根据本发明的另一个方面,提供了两个核酸分子用于DNA检测方法中,其中第一核酸分子包含SEQ ID NO:4的DNA分子的转基因区域的任何部分的至少11个或更多个连续多核苷酸,第二核酸是SEQ ID NO:4的3′侧翼大豆基因组DNA的任何部分的类似长度,其中这些核酸分子在一起使用时在产生扩增子的DNA扩增方法中作为引物是有用的。在DNA扩增方法中使用这些引物产生的扩增子对于大豆事件MON89788 DNA是诊断性的。通过所描述的引物产生的扩增子是本发明的一个方面,所述引物同源于或互补于包含SEQ ID NO:2的SEQ ID NO:4的一部分。
来自SEQ ID NO:3或SEQ ID NO:4,或SEQ ID NO:9或其互补物的任何核酸引物对是本发明的另一个实施方式,所述引物对当用于DNA扩增反应时产生对大豆事件MON89788衍生的组织为诊断性的扩增子,例如分别包含SEQ ID NO:1或SEQ ID NO:2,或SEQ ID NO:9的任何部分的扩增子。在特定的实施方式中,所述引物对可以由引物A(SEQ IDNO:5)和引物D(SEQ ID NO:8)组成。
本发明的另一个方面是大豆植物、或种子、或来自包含事件MON89788的植物或种子的产物,其中当从所述大豆植物、或种子、或产物分离时,所述基因组DNA在包含SEQ ID NO:1或SEQ ID NO:2的DNA扩增方法中产生扩增子。
本发明的另一个方面是大豆植物、或种子、或来自包含事件MON89788的植物或种子的产物,其中当从所述大豆植物、或种子、或产物分离时,所述基因组DNA在DNA扩增方法中扩增子,其中DNA引物分子SEQ ID NO:5和SEQ ID NO:6被用于所述DNA扩增方法中。
本发明的又一个方面是包含MON89788的大豆植物、种子、产物或来自所述植物或种子的商品,其中当基因组DNA从所述大豆植物、或种子、或产物分离时,在DNA扩增方法中产生扩增子,其中DNA引物分子SEQ ID NO:7和SEQ ID NO:8用于所述DNA扩增方法中。所述产物或商品可以包含,无限制地,食物或饲料产品,其包含或来源于包含事件MON89788的大豆植物的一种或多种以下产物:卵磷脂、脂肪酸、甘油、固醇、食用油、脱脂大豆片、包括脱脂的和烘烤的大豆粉的大豆粉、豆浆凝块、豆腐、大豆粉、大豆蛋白浓缩物、分离的大豆蛋白、水解植物蛋白、组织化大豆蛋白和大豆蛋白纤维。
根据本发明的另一个方面,提供了检测样品中特异地相应于大豆事件MON89788 DNA的DNA存在的方法。这种方法包括:(a)使包含DNA的样品与DNA引物对接触;(b)进行核酸扩增反应,从而产生扩增子;和(c)检测所述扩增子,其中所述扩增子包含SEQ ID NO:1或SEQ IDNO:2。包含DNA引物分子的试剂盒是本发明的进一步的方面,所述DNA引物当用于DNA扩增方法时产生包含SEQ ID NO:1或SEQ ID NO:2的扩增子。
根据本发明的另一个方面,提供了检测样品中特异地相应于大豆事件MON89788 DNA的DNA存在的方法。这种方法包括:(a)使包含DNA的样品与探针接触,所述探针在严格杂交条件下与来自大豆事件MON89788的基因组DNA杂交并且在严格杂交条件下不与对照大豆植物DNA杂交;(b)将所述样品和探针置于严紧杂交条件下;和(c)检测探针对大豆事件MON89788 DNA的杂交,其中所述探针包含SEQ IDNO:1或SEQ ID NO:2。所述样品可以包括包含大豆事件MON89788的子代种子、植物或植物部分,或来自包含MON89788的植物的任何以下的产物:卵磷脂、脂肪酸、甘油、固醇、食用油、脱脂大豆片、包括脱脂的和烘烤的大豆粉的大豆粉、豆浆凝块、豆腐、大豆粉、大豆蛋白浓缩物、分离的大豆蛋白、水解植物蛋白、组织化大豆蛋白和大豆蛋白纤维。包含DNA探针的试剂盒是本发明的进一步的方面,所述探针包含同源于或互补于SEQ ID NO:1或SEQ ID NO:2的DNA分子。包含DNA分子的试剂盒也是本发明的一方面,所述DNA分子包含SEQ ID NO:18、SEQ ID NO:19或SEQ ID NO:20,或它们的互补物。
根据本发明的另一个方面,提供了产生耐受草甘膦施用的大豆植物的方法,包括步骤:使包含事件MON89788的第一亲本草甘膦耐受性大豆植物与缺乏草甘膦耐受性的第二亲本大豆植物有性杂交,从而产生多个子代植物;和(b)选择耐受草甘膦施用的子代植物。育种方法可以另外包括步骤:使包含大豆事件MON89788的亲本植株与对草甘膦也是耐受性的第二亲本大豆植物杂交,以及通过与每个亲本中发现的草甘膦耐受性表型遗传性联系的分子标记DNA来选择草甘膦耐受性子代。
本发明的另一个方面是在包含MON89788的大豆植物的田地中控制杂草的方法,其中所述方法包括使用包含事件MON89788的大豆种子种植田地,所述代表性的种子作为ATCC登记号NO.PTA-6708保藏,容许所述种子萌芽,以及使用有效剂量的草甘膦处理所述植物来控制所述田地中的杂草生长。
本发明的上述及其他方面将根据以下的详细说明和附随的附图变得更为显而易见。
附图的简要说明
附图1.在包含事件MON89788的大豆植物的基因组中转基因插入物的结构。
附图2A-2B.来自大豆的商品产物的加工。
详细说明
本发明涉及提供了草甘膦耐受性的称为MON89788的新的大豆转化事件,以及所述植物部分和种子和从植物、植物部分、种子产生的产物,以及包含所述事件的产物。本发明提供了在包含MON89788的大豆细胞的基因组中是新的的DNA分子,以及可以用于各种DNA检测方法以鉴定样品中的MON89788 DNA的DNA分子。本发明提供了在含有MON89788的植物的田地中控制杂草的方法,通过使用草甘膦除草剂处理包含植物的所述田地中的杂草,所述植物包含事件MON89788。
提供以下定义和方法来更好地定义本发明和指导本领域的普通技术人员实施本发明。除非另作说明,根据本领域普通技术人员的常规的用法来理解术语。分子生物学中的常见术语的定义也可以在Rieger et al.(1991)和Lewin(1994)中找到。使用如37 CFR§1.822中阐述的DNA碱基命名法。
如在此使用的,术语“大豆”是指Glycine max,并且包括可以与大豆培育的所有植物品种。
如在此使用的,术语“包含”是指“包括但不限于”。“草甘膦”是指N-膦酰甲基甘氨酸和它的盐,是Roundup
Figure A20068002751300111
除草剂(Monsanto Co.)的活性成分。用“草甘膦除草剂”处理是指使用Roundup
Figure A20068002751300112
、Roundup Ultra、Roundup Pro
Figure A20068002751300114
、除草剂或含有草甘膦的任何其他除草剂制剂处理。草甘膦的商业制剂的实例包括,无限制地,由Monsanto Company作为ROUNDUP
Figure A20068002751300115
、ROUNDUPULTRA、ROUNDUP
Figure A20068002751300117
ULTRAMAX、ROUNDUP
Figure A20068002751300118
CT、ROUNDUP
Figure A20068002751300119
EXTRA、ROUNDUP
Figure A200680027513001110
BIACTIVE、ROUNDUPBIOFORCE、RODEO
Figure A200680027513001112
POLARIS
Figure A200680027513001113
、SPARK
Figure A200680027513001114
和ACCORD
Figure A200680027513001115
除草剂出售的那些,所有这些都含有草甘膦作为其异丙胺盐;ROUNDUP
Figure A200680027513001116
WEATHERMAX(草甘膦钾盐),由Monsanto Company作为ROUNDUP
Figure A200680027513001117
DRY和RIVAL除草剂出售的那些,其含有草甘膦作为其铵盐;由Monsanto Company作为ROUNDUPGEOFORCE出售的那些,其含有草甘膦作为其钠盐;以及由Syngenta Crop Protection作为TOUCHDOWN
Figure A200680027513001120
除草剂出售的,其含有草甘膦作为其三甲基磺酸盐。使用任何这些草甘膦除草剂制剂处理包含了包含事件MON89788的草甘膦耐受性大豆植物的田地将控制所述田地中的杂草生长,并且不影响包含MON89788的大豆植物的生长或产量。
通过用异源DNA,即,包括目标转基因的核酸构建体对植物细胞进行转化,由转基因插入到植物基因组中产生的植物群体的再生,和选择以对特定基因组位置的插入为特征的特定的植物,来产生转基因“事件”。术语“事件”指包括异源DNA的原始转化体和该转化体的子代。术语“事件”还指通过在转化体和另一个包括异源转基因DNA和侧翼基因组DNA的品种之间进行有性的异型杂交生产的子代。术语“事件”还指来自原始转化体的、包含插入的DNA和紧密邻近于插入的DNA的侧翼基因组序列的DNA,期待着将其转移到子代中,该子代做为包括插入的DNA的一个亲本系(例如,原始转化体和自交的子代)与不含有插入的DNA的亲本系进行有性杂交的结果,接受了包括目标转基因的插入的DNA。
通过首先使第一亲本大豆植物与第二亲本大豆植物有性杂交,从而产生多个第一子代植物;然后选择对草甘膦除草剂的施用是耐受性的子代植物,可以培育草甘膦耐受性大豆植物,所述第一亲本大豆植物由从包含MON89788的转基因草甘膦耐受性大豆植物生长出的大豆植物、或作为表达草甘膦耐受性表型的这种植物的杂交的子代的大豆植物组成,所述第二亲本植物缺乏对草甘膦的耐受性。这些步骤可以进一步包括使草甘膦耐受性子代植物与第二亲本大豆植物或第三亲本大豆植物回交,然后通过草甘膦施用或通过与性状相关的分子标记物的鉴定来选择子代,从而产生耐受草甘膦除草剂施用的大豆植物。可以使用分子标记物,其包含事件MON89788中转基因的插入的5′和3′位点鉴定出的接点DNA分子。
还应理解的是,两种不同的转基因植物也可以被配对来产生含有两个独立地隔离的、外源转基因的后代。如早先描述的对亲本植物进行回交和用非转基因植物进行异型杂交也是预期的,无性繁殖也是同样的。通常用于不同性状和作物的其他育种方法的描述可以在几份参考文件之一中找到,例如,Fehr,(1987)。
“探针”是分离的核酸,在上面附有常规的可检测的标记或报告分子,例如,放射性同位素、配体、化学发光剂或酶。这种探针与目标核酸的链互补,就本发明来说,与来自包含事件MON89788的大豆植物的基因组DNA链互补,不论该基因组DNA是来自大豆植物或种子还是来自包括来自该事件的DNA的植物或种子的样品或提取物。根据本发明的探针不仅包括脱氧核糖核酸或核糖核酸,还包括特异性地与目标DNA序列结合并可用于检测该目标DNA序列的存在的聚酰胺及其他探针材料。
“引物”是分离的多核酸,其通过核酸杂交与互补的目标多核酸链退火来形成在引物和目标多核酸链之间的杂交体,然后通过聚合酶,例如DNA聚合酶沿目标多核酸链延伸。本发明的引物对涉及它们用于目标多核酸分子的扩增的用途,例如,通过聚合酶链式反应(PCR)或其他常规的核酸扩增方法。
探针和引物的长度一般是11个多核苷酸或更多,优选的是18个多核苷酸或更多、更优选的24个多核苷酸或30个多核苷酸或更多。这种探针和引物在高度严格杂交条件下与目标分子特异性地杂交。优选的,根据本发明的探针和引物具有与目标分子的完全的序列同一性,然而不同于目标序列并且保持了在高度严格条件下与目标序列杂交的能力的探针可以通过常规方法设计。
例如,在Sambrook et al.(1989);Ausubel et al.(1992);和Inniset al.(1990)中描述了制备和使用探针和引物的方法。PCR引物对(引物集)可以来自已知序列,例如,通过使用为此目的设计的计算机程序,例如Primer(Version 0.5,1991,Whitehead Institute for BiomedicalResearch,Cambridge,MA)。
基于在此公开的侧翼基因组DNA和插入物序列的引物和探针(SEQID NO:1-4和9)可以用于通过常规方法确认,如有必要,用于纠正所公开的序列,例如,通过从包含MON89788的种子保藏物分离相应DNA分子,并确定该分子的核酸序列。其他的相关DNA分子可以从包含MON89788的细胞的基因组分离,所述DNA分子包含转基因插入物和基因组侧翼区域,这些分子的片段可以用作引物或探针。
本发明的核酸探针和引物在严格条件下与目标DNA序列杂交。可使用任何常规的核酸杂交或扩增方法可以用于鉴定样品中来自MON89788事件的DNA的存在。核酸分子或其片段能够在某些情况下与其他核酸分子特异性杂交。如此处使用的,如果两个核酸分子能形成反平行的双链核酸结构并且有足够长度以在高严格条件下维持这种结构,则称为两个核酸分子能相互特异性地杂交。如果核酸分子显示出完全的互补性,则称核酸分子是另一个核酸分子的“互补物”。如此处使用的,当一个分子的每一个核苷酸与另一个分子的核苷酸互补时,称为分子显示出“完全的互补性”。如果分子的相互杂交具有足够的稳定性以允许它们在至少常规的“低严格”条件下保持相互的退火,称两个分子是“最低度互补的”。类似地,如果分子的相互杂交具有足够的稳定性以允许它们在常规的“高严格”条件下保持相互的退火,称所述分子是“互补的”。Sambrook etal.,1989,and by Haymes et al.(1985)描述了常规的严格条件。因而从完全互补性的偏离是可允许的,只要这种偏离不完全地排除了分子形成双链结构的能力。为了使核酸分子作为引物或探针,仅需在序列中充分的互补,以使得在所采用的特定溶剂和盐浓度下能形成稳定的双链结构。
如此处使用的,基本上同源的序列是在高度严格条件下同与其相比较的核酸序列的互补物特异性的杂交的核酸序列。促进DNA杂交的适合的严格条件,例如,6.0×氯化钠/柠檬酸钠(SSC)约45℃,之后是在50℃用2.0×SSC洗涤,对本领域的技术人员是公知的,或可在CurrentProtocols in Molecular Biology,John Wiley & Sons,N.Y.(1989),6.3.1-6.3.6中找到。例如,洗涤步骤中的盐浓度可以选自低度严格的约2.0×SSC、50℃到高度严格的约0.2×SSC、50℃。此外,洗涤步骤中的温度可以从低度严格条件的室温下约22℃,升高到高度严格条件的约65℃。温度和盐度可以都变化,或者温度或盐浓度保持不变而另一个变量发生改变。在优选的实施方式中,本发明的核酸将在中度严格条件下,例如在约2.0×SSC和约65℃下特异性地杂交SEQ ID NO:1-4中所列核酸分子、和其9个互补物或两者的片段的一个或多个。在特别优选的实施方式中,本发明的核酸将在高度严格条件下杂交SEQ ID NO:1-4所列核酸分子、和其9个互补物或两者的片段的一个或多个。在本发明的一个方面中,本发明的优选的标记物核酸分子包含如SEQ ID NO:1或SEQ IDNO:2所列的核酸序列,或其互补物或两者的片段。在本发明的另一个方面中,本发明的优选的标记物核酸分子与SEQ ID NO:1或SEQ ID NO:2中所列核酸序列或其互补物或两者的片段享有80%到1 00%或90%到100%的序列同一性。包含SEQ ID NO:1或SEQ ID NO:2,或其互补物或两者的片段的分子标记物DNA分子可以用作植物育种方法中的标记物来鉴定遗传杂交的子代,类似于在Cregan et al.(1997)中对于单一序列重复DNA标记物分析所描述的方法,通过完全引用将它的所有合并在此。探针对目标DNA分子的杂交可以通过许多本领域技术人员已知的方法进行检测,这些可包括但不局域于,荧光标签、放射性标签、基于抗体的标签和化学发光标签。
关于使用特定的扩增引物对进行目标核酸序列的扩增(例如,通过PCR),“严格条件”是允许引物对仅与目标核酸序列杂交的条件,具有相应的野生型序列(或其互补物)的引物将与所述目标核酸序列结合,优选的在DNA热扩增反应中产生独特的扩增产物,扩增子。
术语“特异于(目标序列)”是指探针或引物在严格杂交条件下仅与包含目标序列的样品中的目标序列杂交。
如在此使用的,“扩增的DNA”或“扩增子”是指作为核酸模板的部分的目标核酸序列的核酸扩增的产物。例如,为了确定产自有性杂交的大豆植物是否含有转基因事件MON89788,或采集自田地的大豆样品是否包含MON89788,或大豆提取物,例如粗粉、粉或油是否包含MON89788。从大豆植物组织样品或提取物提取的DNA可以经历使用引物对的核酸扩增方法,所述引物对包括来源于邻近插入的异源转基因DNA的插入位点的基因组区域的引物,和来源于插入的异源转基因DNA的第二引物,来产生对于事件DNA的存在是诊断性的扩增子。扩增子具有一定长度并具有序列,所述序列对所述事件也是诊断性的。扩增子的长度可根据引物对加上一个核苷酸碱基对、或加上约五十个核苷酸碱基对,或加上约两百五十个核苷酸碱基对,或加上约三百五十个核苷酸碱基对或更多的组合长度而变化。
做为选择,引物对可以来源于插入的DNA的两侧的侧翼基因组序列,以产生包括整个插入核苷酸序列的扩增子。来自植物基因组序列的引物对的成员可以被定位在距插入的转基因DNA分子一定距离上,该距离可以从一个核苷酸碱基对到约两万核苷酸碱基对间变化。术语“扩增子”的使用要特别排除可在DNA热扩增反应中形成的引物二聚物。
可以通过本领域已知的各种核酸扩增反应方法的任一种,包括聚合酶链式反应(PCR)来实现核酸扩增。各种扩增方法是本领域已知的,特别地,在美国专利NO.4,683,195和4,683,202中,以及在Innis et al.(1990)中描述了。PCR扩增方法已经发展到可扩增多达22kb的基因组DNA和多达42kb的噬菌体DNA(Cheng等,et al.,1994)。这些方法以及本领域的其他DNA扩增方法可以用于本发明的实践中。来自大豆事件MON89788的异源DNA插入物的序列或侧翼序列可以通过利用来自此处提供的序列的引物从事件基因组中扩增这种分子,然后对PCR扩增子或对分离的克隆转基因/基因组DNA进行标准的DNA测序,来证实,如果有必要,来纠正。
可以提供多种技术来检测这些方法生产的扩增子。一个这种方法是Genetic Bit Ananlysis(Nikiforov,et al.,1994),其中设计一DNA寡核苷酸,其覆盖邻近的侧翼基因组DNA序列和插入的DNA转基因序列。将寡聚核苷酸固定在微孔平板的孔中。在对目标区域进行PCR之后(使用在插入的序列中的一个引物,和在邻近侧翼基因组序列中的一个引物),单链PCR产物可与固定的寡聚核苷酸杂交并充当模板,用于利用DNA聚合酶和特异于期待的下一个碱基的标记的ddNTP进行单碱基延伸反应。读出过程可以是基于荧光的或基于ELISA的。信号指示了由于成功的扩增、杂交和单碱基延伸导致的插入物/侧翼基因组序列的存在。
另一种方法是Winge(2000)描述的Pyrosequencing技术。在这个方法中设计一寡核苷酸,其覆盖邻近的基因组DNA和插入物DNA接点。使寡核苷酸与来自目标区域的单链PCR产物(一个引物在插入的序列中,一个在侧翼基因组序列中)杂交,在存在DNA聚合酶、ATP、硫酸化酶、荧光素酶、三磷酸腺苷双磷酸酶、腺苷酸5’磷酸和萤光素的情况下孵育。分别地添加DNTPs,测量产生光信号的掺入。光信号指示了由于成功的扩增、杂交和单碱基或多碱基延伸导致的转基因插入物/侧翼序列的存在。
Chen等(1999)描述的荧光偏振是可以用于检测本发明的扩增子的一种方法。使用这种方法设计一寡核苷酸,其覆盖基因组侧翼的和插入的DNA接点。使寡核苷酸与来自目标区域的单链PCR产物(一个引物在插入的DNA序列中,一个在侧翼基因组DNA序列中)杂交,在存在DNA聚合酶和荧光标记的ddNTP的情况下孵育。单碱基延伸导致ddNTP的掺入。利用荧光计测量偏振的变化可以测量掺入。偏振的变化指示了由于成功的扩增、杂交和单碱基延伸导致的转基因插入物/侧翼基因组序列的存在。
Taqman
Figure A20068002751300161
(PE Applied Biosystems,Foster City,CA)被描述为一种对DNA序列的存在进行检测和定量的方法,可以根据厂家提供的说明完全理解。简要地,设计一寡核苷酸探针,其覆盖基因组侧翼的和插入的DNA接点。在存在热稳定聚合酶和dNTP的情况下,FRET探针和PCR引物(一个引物在插入的DNA序列中以及一个在侧翼基因组序列中)进行循环。FRET探针的杂交引起FRET探针上荧光部分从淬灭部分裂解和释放。荧光信号指示了由于成功的扩增和杂交产生的侧翼基因组/转基因插入物序列的存在。
在如Tyangi et al.(1996)中描述的,Molecular Beacons已经被描述用于序列检测中。简要地,设计一FRET寡核苷酸探针,其覆盖侧翼基因组和插入物DNA接点。该FRET探针的独特的结构造成其含有二级结构,该二级结构使荧光和淬灭部分保持邻近。在存在热稳定聚合酶和dNTP的情况下,FRET探针和PCR引物(一个引物在插入的DNA序列中以及一个在侧翼基因组序列中)进行循环。在成功的PCR扩增之后,FRET探针对目标序列的杂交引起探针二级结构的消除和荧光部分与淬灭部分的空间分隔。产生荧光信号。荧光信号指示了由于成功的扩增和杂交产生的侧翼基因组/转基因插入物序列的存在。
其他描述的方法,例如microfluidics(美国专利公开2006068398,美国专利NO.6,544,734)提供了分离和扩增DNA样品的方法和设备。光染料用于检测和测定特定的DNA分子(WO/05017181)。包含用于检测DNA分子的电子传感器或结合特定DNA分子的纳珠并因而可被检测的纳试管(nanotube)设备(WO/06024023)对于检测本发明的DNA分子是有用的。
可以使用在此公开的组合物和DNA检测领域描述的或已知的方法来开发DNA检测试剂盒。所述试剂盒对于样品中大豆事件DNA的鉴定是有用的,可以用于培育含DNA的大豆植物的方法。所述试剂盒可以含有DNA引物或探针,其同源于或互补于SEQ ID NO:1-4和9,或含有同源于或互补于DNA的转基因遗传元件中所含DNA的DNA引物或探针,这些DNA序列可以用于DNA扩增反应,或作为DNA杂交方法中的探针。在大豆基因组中含有的以及在附图1中说明的转基因遗传元件的DNA结构包含:侧翼于转基因插入物的大豆A3244基因组的5′基因组区域,包含来自Agrobacterium tumefaciens(根癌土壤杆菌)的右侧边界区域(RB)的一部分的插入物,嵌合的启动子FMV/Tsf1和相关的连接元件(美国专利6,660,911;也称为FMV/E1F1a)可操作连接到Arabidopsis EPSPS叶绿体转运肽编码序列(在此称为CTP2或TS-AtEPSPS CTP2,美国专利5,633,435,可操作连接到草甘膦抗性EPSPS(在此称为CP4 EPSPS或aroA:CP4,分离自Agrobacterium tumefaciens菌株CP4,以及被修改用于增强植物细胞中的表达的编码序列,美国专利5,633,435),可操作连接到来自豌豆核酮糖1,5-二磷酸羧化酶(在此称为E9 3’或T-Ps.RbcS:E9,Coruzzi et al.,(1984),来自Agrobacterium tumefaciens的左侧边界(LB)区域的一部分,以及侧翼于转基因插入物的大豆A3244基因组的3′基因组区域。在DNA扩增方法中作为引物有用的DNA分子可以来源于大豆事件MON89788中含有的转基因插入物的遗传元件的序列。这些引物分子可以用作引物集的部分,所述引物集也包括来源于侧翼于转基因插入物的大豆基因组的DNA引物分子。通过土壤杆菌介导的方法,例如美国专利6,384,301和7,002,058(通过完全引用合并在此)中描述的方法,通过转化大豆系A3244(美国专利5,659,114)产生了大豆事件MON89788。
本发明的发明人发现,在基因组中包含MON89788 T型基因组区域(T型是转基因与植物基因组的相关单体型区域的组合)的大豆株系,相对于包含早先的40-3-2 T型基因组区域的株系具有改善的产量。在包括从美国多个区域采集的产量数据的重复田间试验中展现了这一点(美国专利申请60/685584)。
以下实施例被包括进来以说明本发明的某些优选实施方式的实例。本领域的技术人员应当理解,在根据当前方法的实施例中公开的技术,发明人发现在本发明的实践中运作良好,因此可以认为构成了其实践的优选模式的实施例。然而,本领域的技术人员根据当前公开的内容应当理解,可以在公开的具体方案中进行许多变化,在不背离本发明的精神和范围的情况下仍获得相似的或类似的结果。
实施例
实施例1
对于MON89788基因组DNA为诊断性的扩增子的产生
来自转基因大豆事件MON89788的DNA从包含大豆种子、营养组织或粗粉的组织提取。使用Qiagen’s DNeasy Plant Miniprep试剂盒根据厂家的说明(Qiagen Corp.Valencia,CA)从组织分离DNA。
产生PCR产物,其包含在包含MON89788的植物基因组中侧翼于TDNA(包含转基因的转化DNA)插入物的5′末端的基因组DNA的一部分。这个DNA产物包含SEQ ID NO:3。可以进行PCR,使用被设计以与侧翼于转基因插入物的5′末端的基因组DNA序列杂交的一个引物(DNA引物A,SEQ ID NO:5,参见附图1),和与之配对的位于转基因启动子区域的第二引物(DNA引物B,SEQ ID NO:)(美国专利6,660,911,SEQ ID NO:28,通过引用合并在此,在SEQ ID NO:9中)。
从转基因插入物的3′末端产生PCR产物,其包含侧翼于包含MON89788的植物基因组中T-DNA插入物的3′末端的基因组DNA的一部分。这个DNA产物包含SEQ ID NO:4。可以进行PCR,使用被设计以与侧翼于每个事件的插入物的3′末端的基因组DNA序列杂交的引物(DNA引物D,SEQ ID NO:8),和与之配对的位于插入物的3′末端的T-Ps.RbcS:E9 3′转录终止序列中的第二引物(DNA引物C,SEQ ID NO:7)。
PCR模板包括~50ng基因组DNA。利用来自非转基因大豆品种的~50ng基因组DNA作为阴性对照。每个PCR反应含有用于REDAccuTaqTM LADNA Polymerase Mix(Sigma-Aldrich,St Louis,MO)的5μl 10×缓冲液,200μM每种dNTP(Sigma-Aldrich),0.4μM每种引物,和2.5单位JumpStartTM REDTaqTM DNA聚合酶(Sigma-Aldrich),50μl总体积反应。在以下循环条件下进行PCR反应:94℃3分钟(min)1个循环;94℃30秒(s)、58℃30s、72℃30s或1min 32或35个循环;72℃10min 1个循环。
DNA事件引物对被用于产生对MON89788基因组DNA为诊断性的扩增子。这些事件引物对包括但不限于,引物A和B(SEQ ID NO:5和6),和事件引物对C和D(SEQ ID NO:7和8),用于所描述的DNA扩增方法中。除了这些引物对之外,可以使用来自SEQ ID NO:3或SEQ ID NO:4、或其互补物的任何引物对,当用于DNA扩增反应时分别产生包含对于大豆MON89788事件衍生的组织为诊断性的SEQ ID NO:1或SEQ IDNO:2的扩增子。表1和表2中说明的DNA扩增条件可以用于使用合适的事件引物对产生MON89788的诊断性扩增子。用于产生对MON89788为诊断性的扩增子的这些方法的任何修改处于本领域技术人员的能力之内。当在DNA扩增方法中测试时产生对大豆事件MON89788为诊断性的扩增子的、推定含有包含MON89788的大豆植物或种子DNA的提取物,或来自包含MON89788的植物的产物,可以被用作扩增的模板,来确定是否存在MON89788。
通过使用来自SEQ ID NO:3或SEQ ID NO:4的至少一个引物来产生扩增子,所述引物当用于PCR方法中时产生事件MON89788的诊断性扩增子。例如,使用表2中示出的Stratagene Robocycler、MJ Engine、Perkin-Elmer 9700或Eppendorf Mastercycler Gradient热循环仪,或通过本领域技术人员已知的方法和设备,来进行MON89788扩增子的产生。
表1.用于大豆MON89788 5’转基因插入物/基因组连接区的鉴定的PCR步骤和反应混合物条件。
步骤    试剂                 数量              注释
  1   无核酸酶的水 添加到终体积20μl -
  2   10×反应缓冲液(与MgCl2) 2.0μl 1×缓冲液终浓度,1.5mM MgCl2终浓度
  3   dATP、dCTP、dGTP和dTTP的10mM溶液 0.4μl 每种dNTP 200μM终浓度
  4   事件引物A(SEQ ID NO:5重悬浮在1×TE缓冲液或无核酸酶水中到10μM的浓度) 0.2μl 0.1μM终浓度
  5   事件引物B(SEQ ID NO:6重悬浮在1×TE缓冲液或无核酸酶水中到10μM浓度) 0.2μl 0.1μM终浓度
  6   RNase,无DNase(500μg/ml) 0.1μl 50ng/反应
  7   RED Taq DNA聚合酶(1单位/μl) 1.0μl(建议在下一步之前转换吸管) 1单位/反应
  8   提取的DNA(模板):·要分析的样品:*单独的叶*集中的叶(最大10叶子/集中)·阴性对照·阴性对照·阳性对照 ·10-200ng基因组DNA·200ng基因组DNA·50ng非转基因大豆基因组DNA·无模板DNA(DNA重悬浮在其中的溶液)·50ng包含MON89788的大豆基因组DNA -
轻轻地混合,和如果必要(热循环仪上没有保温帽),在每个反应上添加1-2滴矿物油。使用以下循环参数(表2)在Stratagene Robocycler(Stratagene,La Jolla,CA)、MJ Engine(MJR-Biorad,Hercules,CA)、Perkin-Elmer 9700(Perkin Elmer,Boston,MA)或Eppendorf MastercyclerGradient(Eppendorf,Hamburg,Germany)热循环仪上进行PCR。MJEngine或Eppendorf Mastercycler Gradient热循环仪应当在计算的模式下运行。将斜坡速度设定在最大值运行Perkin-Elmer 9700热循环仪。
表2.热循环仪条件
循环数  设置:Stratagene Robocycler
1       94℃ 3分钟
34      94℃ 1分钟
        64℃ 1分钟
        72℃ 1分钟30秒
1       72℃ 10分钟
 循环数 设置:MJ Engine或Perkin-Elmer 9700
 1 94℃ 3分钟
 34 94℃ 30秒64℃ 30秒72℃ 1分钟
 1 72℃ 10分钟
 循环数 设置:Eppendorf Mastercycler Gradient
 1 94℃ 3分钟
 34 94℃ 15秒64℃ 15秒72℃ 1分钟
 1 72℃ 10分钟
实施例2
转基因/基因组区域的序列测定和Southern分析
PCR产物的DNA测序提供了可以用于设计其他DNA分子的DNA,所述其他DNA分子作为引物和探针用于包含MON89788的大豆植物或种子的鉴定。代表5′和3′转基因/基因组序列的预期大小的PCR产物通过在2.0%琼脂糖凝胶上通过电泳分离PCR产物来分离。分离出包括5′和3′DNA区域的PCR产物,所述5′和3′DNA区域跨越了插入到大豆基因组中的转基因之间的插入物接点。MON89788的5′和3′PCR产物通过琼脂糖凝胶电泳来纯化,随后使用QIAquick Gel提取试剂盒(目录#_28704,QiagenInc.,Valencia,CA)从琼脂糖基质分离。然后对纯化的PCR产物测序(例如,ABI PrismTM 377,PE Biosystems,Foster City,CA)并分析(例如,DNASTAR序列分析软件,DNASTAR Inc.,Madison,WI)。
DNA序列被确定为代表了如附图1中说明的事件MON89788的转基因/基因组区域的核苷酸碱基对片段,并鉴定为SEQ ID NO:9。SEQ IDNO:9中含有的基因组和转基因元件在表3中描述。5′和3′侧翼区域被包括在SEQ ID NO:9中,在SEQ ID NO:21和22中给出。
接点序列是相对短的多核苷酸分子,其是新的DNA序列,当在多核酸检测分析中检测到时对于MON89788 DNA是诊断性的。SEQ ID NO:1和SEQ ID NO:2中的接点序列为MON89788中转基因片段的插入位点和大豆基因组DNA的每一侧的10个多核苷酸。更长或更短的多核苷酸接点序列可以从SEQ ID NO:3或SEQ ID NO:4中选择。接点分子(5′连接区SEQ ID NO:1,和3′连接区SEQ ID NO:2)作为DNA探针或作为DNA引物分子在DNA检测方法中是有用的。
对于事件MON89788开发了在Taqman方法(Roche MolecularSystems,Inc.,Pleasanton,CA)中使用的、用于检测事件特异性DNA分子的引物和探针。引物分子被称为SQ2824(SEQ ID NO:10)、SQ2826(SEQ ID NO:11)、SQ 1141(SEQ ID NO:12)、SQ1142(SEQ IDNO:13)、SQ5543(SEQ ID NO:14),探针分子被称为PB871-6FAM(SEQ ID NO:15)、PB2191-VIC(SEQ ID NO:16)和PB57-VIC(SEQID NO:17)。所述引物和探针根据厂家说明书在Taqman
Figure A20068002751300222
方法中使用,来提供对于包含MON89788的DNA的诊断性扩增子。包括加工的产物例如粗粉的大豆组织作为这种方法的DNA来源是有用的。用于从大豆粗粉产生扩增子的其他引物包括SEQ ID NO:18-20。
表3.在包含MON89788的大豆的基因组中含有的转基因/基因组DNA片段(SEQ ID NO:9)的基因组和遗传元件注释。
遗传元件1     序列中的位置2   功能(参考文件)
侧翼于插入物5′末端的序列     1-1103   大豆基因组DNA
5’连接区     1093-1113   跨越转基因插入物的DNA区域
B3-右边界     1104-1145   来自Agrobacterium tumefaciens、含有用于T-DNA转移的右边界序列的DNA区域(Depickeret al.,1982)
间插序列     1146-1215   DNA克隆中使用的序列
P4-FMV/Tsf1     1216-2255   由来自玄参花叶病毒的35S启动子的增强子序列(Richins et al.,1987)和来自Arabidopsisthaliana的Tsf1基因的启动子(编码延伸因子EF-1alpha(Axelos,et al.,1989)构成的嵌合启动子
L5-Tsf1     2256-2301   来自Arabidopsis thaliana的编码延伸因子EF-1alpha的Tsf1基因的5′非翻译前导区(外显子1)(Axelos et al.,1989)
I6-Tsf1     2302-2923   来自Arabidopsis thaliana的编码延伸因子EF-1alpha的Tsf1基因的内含子(Axelos et al.,1989)
间插序列     2924-2932   DNA克隆中使用的序列
TS 7 -CTP2     2933-3160   编码来自Arabidopsis thaliana的ShkG基因的叶绿体转运肽的序列,所述ShkG基因编码EPSPS(Klee et al.,1987)
CS8-cp4 epsps     3161-4528   来自Agrobacterium sp.菌株CP4的aroA基因的密码子优化的编码序列,所述aroA基因编码CP4EPSPS蛋白(Padgette et al.,1996;Barry et al.,1997)
间插序列     4529-4570   DNA克隆中使用的序列
 遗传元件1   序列中的位置2  功能(参考文件)
 T9-E9   4571-5213  来自Pisum sativum的RbcS2基因的3′非翻译序列,所述RbcS2基因编码核酮糖小亚基(Coruzziet al.,1984)
 间插序列   5214-5256  DNA克隆中使用的序列
 B-左侧边界   5257-5406  来自Agrobacterium tumefaciens、含有用于T-DNA转移的左边界序列的DNA区域(Barkeret al.,1983)
 3’连接区   5396-5416  跨越转基因插入物的DNA区域
 侧翼于插入物3′末端的序列   5407-6466  大豆基因组DNA
Southern印迹分析
来自包含MON89788的植物的基因组DNA和对照大豆基因组DNA(各~15μg)在包括15μl相应厂家的缓冲液(NEB,Beverly,MA)的150μl总体积中用各种限制性内切酶(140U)消化。限制性内切酶,例如Bgl11、BamH1、Nco1、Hind111和Bc11,被用于MON89788的Southern分析。在合适的温度下进行核酸内切酶消化至少6小时。在孵育之后,用3M醋酸钠和2.5倍体积乙醇沉淀DNA。随后,用70%洗涤DNA,干燥,重悬浮在40μl TBE中。加载缓冲液(0.2×)添加到样品中,然后在30伏在琼脂糖凝胶(0.8%)上进行电泳16-18小时。凝胶用溴化乙锭染色,然后用脱嘌呤溶液(0.125N HCL)处理10min,用变性溶液(0.5M氢氧化钠,1.5M氯化钠)处理30分钟,最后用中和溶液(0.5M Trizma碱,1.5M氯化钠)处理30分钟。将DNA转移到Hybond-N膜上(Amersham Pharmacia Biotech,Buckingamshire,England)使用Turboblotter(Schleicher and Schuell,Dassel,Germany)4-6小时,然后使用紫外光固定在膜上。
膜在45℃与20ml DIG Easy Hyb溶液(Roche Molecular Biochemicals,Indianapolis,IN;cat.#_1603558)预杂交2-4小时。使用Radprime DNA标记试剂盒(Invitrogen,Carlsbad,CA;cat.#_18428-011)制造与SEQ IDNO:1、或SEQ ID NO:2、或SEQ ID NO:3、或SEQ ID NO:4、或其部分同源或互补的放射性DNA探针(32P dCTP)。使用SEPHADEX G-50柱(Invitrogen)除去未结合的核苷酸。将预杂交溶液替换为10ml预暖的DIG Easy Hyb溶液,所述溶液含有变性的探针达1百万计数每ml的终浓度。在45℃进行印迹杂交16-18小时。
在45℃用低严格溶液(5×SSC,0.1×SDS)洗涤印迹,然后在65℃用更高严格溶液(0.1×SSC,0.1%SDS)重复洗涤。将印迹暴光于荧光屏(Amersham Biosciences,Piscataway,NJ)>2小时,使用Data Storm 860机(Amersham Biosciences)读取曝光。这些例示的方法和条件可以由检测样品中DNA的本领域技术人员修改。
实施例3
杂草控制
在包含MON89788的大豆田地中控制杂草生长。田地种植上包含MON89788的大豆种子,容许种子萌芽成为植株,用含有草甘膦的除草剂制剂处理植物的田地。约0.25lb ae/A(草甘膦酸数量磅数/英亩)到3或更高lb ae/A处理速率的有效剂量的草甘膦制剂施用到田地中。以控制田地中杂草所需的、生长季节期间的一次或多次处理的频率,施用的速率在约0.75lb ae/A到1.5lb ae/A的范围。从处理的植物收获来自包含MON89788的植物的种子。
Monsanto Company LLC的保藏物、以上公开的和在权利要求中叙述的代表事件MON89788的大豆种子已经根据布达佩斯条约保藏在美国典型培养物保藏所(ATCC),10801University Boulevard,Manassas,Va.20110。包含事件MON89788(也称为MON19788或GM_A19788)的保藏物的ATCC登记号是PTA-6708,2005年5月11日保藏。该保藏物将在保藏所维持30年、或在最后一次请求后5年、或在本专利的有效期内,哪一个时间更长,根据需要就在该时期内进行替换。
已经说明和描述了本发明的原理,对本领域的技术人员显而易见的是,可以在安排和细节上对本发明进行修改而不背离这种原理。我们要求所有的修改都处于附加的权利要求的精神和范围内。
在本说明书中引用的所有出版物和公开的专利文件在此通过引用来合并,其程度与特定的或单独的指示通过引用来合并每个单独的出版物或专利申请相同。
参考文献
以下参考文件通过引用特别地合并在此,达到提供示范性程序或对在此阐述的那些补充的其他细节的程度。
美国专利4,535,060
美国专利4,683,195
美国专利4,683,202
美国专利5,094,945
美国专利5,463,175
美国专利5,554,798
美国专利5,633,435
美国专利5,633,435
美国专利5,659,114
美国专利6,040,497
美国专利6,384,301
美国专利6,544,734
美国专利6,660,911
美国专利6,660,911
美国专利6,689,880
美国专利6,689,880
美国专利6,733,974
美国专利6,740,488
美国专利6,740,488
美国专利6,818,807
美国专利6,825,400
美国专利6,893,826
美国专利6,900,014
美国专利7,002,058
美国申请60/685584
美国公开20040018518
美国公开2006068398
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序列表
<110>Malven,Marianne
     Rinehart,Jennifer
     Taylor,Nancy
     Dickinson,Ellen
<120>大豆事件MON89788和检测它的方法
<130>MONS:113WO
<140>未知
<141>2006-05-26
<150>60/685,584
<150>2005-05-27
<160>22
<170>3.3
<210>1
<211>20
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<222>(1)..(20)
<223>大豆基因组和转基因的嵌合分子
<400>1
tatcaagctc caaacactga                20
<210>2
<211>20
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<222>(1)..(20)
<223>大豆基因组序列和转基因的嵌合分子
<400>2
taataacgct cagactctag                20
<210>3
<211>1222
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<222>(1)..(1222)
<223>大豆基因组DNA和转基因插入物的嵌合分子
<400>3
cctgtacttc ccaaaacttc gcttccctga cccatcatat ccaggactgg acgattggct   60
tgattgatac cagatgggtg agtcgagtcc acctcggtag cggcatttat ggcaacgatt   120
gcagccacgt tgacctccat cattttttct catgctcatc atggcctcca tcatggtggt   180
catttggtct ttcatggcct ccatgtcggc cttcatctgc tcttgaactt catctatctc   240
actcatgatt ctagccttgg cacgtgtttg gtaagggtac cgtaaagcgc gttcgttctt   300
ttttattact atgattacat tttgacgatg atgatgattg taggaaagaa tgaaatgagt   360
aatgaaacaa ctaaataaac gtgaatgcat gacaatgata agttgctgaa gtattataaa   420
tttacatagg acattcagtg gaacgtaggg tcgaatcaaa tcctatttca ttaaaaacaa   480
tattgttcat cttgacagag ccaaagcata actagaaata caacatggac acatcagcga   540
ttcctaatta tgtgggtcat tagttcgacc atgtgttggc agtaacttga aagactatga   600
acttcatcgg gagcagagta tgtgtcagtc accgccttgg ctctggctaa caaccttggg   660
atctcttggc tctcatttag agtaagagca aatttgtcca tccatttcat ggcttcttta   720
tgcaataact ctatcacccc ttctcttgct tccctttcaa cctgcaaggt cgacactttt   780
gcctgttcgt cttctagcct tcgcccatga ctagcagcta ggttcacctt ctcttcatat   840
tggtcaatga ttatcaacat attttctttt gttttgctca actgttctct caaacttctc   900
ttcgatctct gacaactctt taacttatcc tctaacatca ggttttccat acttgatttg   960
tccctcttgg cttttctaag tttgagctcg ttactgctgc cccacaaagc ccctcgaaac   1020
ttgttcctgc tccactcttc cttttgggct tttttgtttc ccgctctagc gcttcaatcg   1080
tggttatcaa gctccaaaca ctgatagttt aaactgaagg cgggaaacga caatctgatc   1140
cccatcaagc tctagctaga gcggccgcgt tatcaagctt ctgcaggtcc tgctcgagtg   1200
gaagctaatt ctcagtccaa ag                                            1222
<210>4
<211>675
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<222>(1)..(675)
<223>大豆基因组DNA和转基因插入物的嵌合DNA分子
<400>4
gccaattgat tgacaacatg catcaatcga cctgcagcca ctcgaagcgg ccgcatcgat    60
cgtgaagttt ctcatctaag cccccatttg gacgtgaatg tagacacgtc gaaataaaga   120
tttccgaatt agaataattt gtttattgct ttcgcctata aatacgacgg atcgtaattt   180
gtcgttttat caaaatgtac tttcatttta taataacgct cagactctag tgactaccac   240
cttcactctc ctcaagcatt tcagcctctt ccccgctcag actccttagc tttgggagcc   300
aaattatccc ttacgttctc gacttcaacc atatgtgata gctgcctatg ataccatggc   360
tacttcccct tagttcttta tctttccttt ccgctttatt ccatgcctta ccgatcctct   420
gaagtgtctt tgcattagct tcattgaaac ctcacgcgat gaaaggtgtg atggtctcct   480
ccgatggcgc acttctcata gggtaaccta attgtcttac gaccaacata ggattataat   540
taatacaacc cctcgtccct ataaaaggga catttggaaa tccttcacat aagcataaca   600
ctcctacccc tctttctttc cactgtggga accaactaat ggacgctcct atcatgcctg   660
ccaagagttc ttccc                                                    675
<210>5
<211>21
<212>DNA
<213>Glycine max
<400>5
cctgtacttc ccaaaacttc g                                              21
<210>6
<211>22
<212>DNA
<213>Figwort mosaic virus
<400>6
ctttccactg agaattagct cc                                             22
<210>7
<211>24
<212>DNA
<213>Petunia hybrida
<400>7
gccaattgat tgacaacatg catc                                           24
<210>8
<211>20
<212>DNA
<213>Glycine max
<400>8
gggaagaact cttggcaggc                                                 20
<210>9
<211>6466
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<222>(1)..(6466)
<223>大豆基因组DNA和转基因插入物的嵌合DNA分子
<400>9
tgggggctgc ctgtacttcc caaaacttcg cttccctgac ccatcatatc caggactgga   60
cgattggctt gattgatacc agatgggtga gtcgagtcca cctcggtagc ggcatttatg   120
gcaacgattg cagccacgtt gacctccatc attttttctc atgctcatca tggcctccat   180
catggtggtc atttggtctt tcatggcctc catgtcggcc ttcatctgct cttgaacttc   240
atctatctca ctcatgattc tagccttggc acgtgtttgg taagggtacc gtaaagcgcg   300
ttcgttcttt tttattacta tgattacatt ttgacgatga tgatgattgt aggaaagaat   360
gaaatgagta atgaaacaac taaataaacg tgaatgcatg acaatgataa gttgctgaag   420
tattataaat ttacatagga cattcagtgg aacgtagggt cgaatcaaat cctatttcat   480
taaaaacaat attgttcatc ttgacagagc caaagcataa ctagaaatac aacatggaca   540
catcagcgat tcctaattat gtgggtcatt agttcgacca tgtgttggca gtaacttgaa   600
agactatgaa cttcatcggg agcagagtat gtgtcagtca ccgccttggc tctggctaac   660
aaccttggga tctcttggct ctcatttaga gtaagagcaa atttgtccat ccatttcatg   720
gcttctttat gcaataactc tatcacccct tctcttgctt ccctttcaac ctgcaaggtc   780
gacacttttg cctgttcgtc ttctagcctt cgcccatgac tagcagctag gttcaccttc   840
tcttcatatt ggtcaatgat tatcaacata ttttcttttg ttttgctcaa ctgttctctc   900
aaacttctct tcgatctctg acaactcttt aacttatcct ctaacatcag gttttccata   960
cttgatttgt ccctcttggc ttttctaagt ttgagctcgt tactgctgcc ccacaaagcc   1020
cctcgaaact tgttcctgct ccactcttcc ttttgggctt ttttgtttcc cgctctagcg   1080
cttcaatcgt ggttatcaag ctccaaacac tgatagttta aactgaaggc gggaaacgac   1140
aatctgatcc ccatcaagct ctagctagag cggccgcgtt atcaagcttc tgcaggtcct   1200
gctcgagtgg aagctaattc tcagtccaaa gcctcaacaa ggtcagggta cagagtctcc   1260
aaaccattag ccaaaagcta caggagatca atgaagaatc ttcaatcaaa gtaaactact   1320
gttccagcac atgcatcatg gtcagtaagt ttcagaaaaa gacatccacc gaagacttaa   1380
agttagtggg catctttgaa agtaatcttg tcaacatcga gcagctggct tgtggggacc 1440
agacaaaaaa ggaatggtgc agaattgtta ggcgcaccta ccaaaagcat ctttgccttt 1500
attgcaaaga taaagcagat tcctctagta caagtgggga acaaaataac gtggaaaaga 1560
gctgtcctga cagcccactc actaatgcgt atgacgaacg cagtgacgac cacaaaagaa 1620
ttagcttgag ctcaggattt agcagcattc cagattgggt tcaatcaaca aggtacgagc 1680
catatcactt tattcaaatt ggtatcgcca aaaccaagaa ggaactccca tcctcaaagg 1740
tttgtaagga agaattcgat atcaagcttg atatcggaag tttctctctt gagggaggtt 1800
gctcgtggaa tgggacacat atggttgtta taataaacca tttccattgt catgagattt 1860
tgaggttaat atatacttta cttgttcatt attttatttg gtgtttgaat aaatgatata 1920
aatggctctt gataatctgc attcattgag atatcaaata tttactctag agaagagtgt 1980
catatagatt gatggtccac aatcaatgaa atttttggga gacgaacatg tataaccatt 2040
tgcttgaata accttaatta aaaggtgtga ttaaatgatg tttgtaacat gtagtactaa 2100
acattcataa aacacaacca acccaagagg tattgagtat tcacggctaa acaggggcat 2160
aatggtaatt taaagaatga tattatttta tgttaaaccc taacattggt ttcggattca 2220
acgctataaa taaaaccact ctcgttgctg attccattta tcgttcttat tgaccctagc 2280
cgctacacac ttttctgcga tatctctgag gtaagcgtta acgtaccctt agatcgttct 2340
ttttcttttt cgtctgctga tcgttgctca tattatttcg atgattgttg gattcgatgc 2400
tctttgttga ttgatcgttc tgaaaattct gatctgttgt ttagatttta tcgattgtta 2460
atatcaacgt ttcactgctt ctaaacgata atttattcat gaaactattt tcccattctg 2520
atcgatcttg ttttgagatt ttaatttgtt cgattgattg ttggttggtg gatctatata 2580
cgagtgaact tgttgatttg cgtatttaag atgtatgtcg atttgaattg tgattgggta 2640
attctggagt agcataacaa atccagtgtt ccctttttct aagggtaatt ctcggattgt 2700
ttgctttata tctcttgaaa ttgccgattt gattgaattt agctcgctta gctcagatga 2760
tagagcacca caatttttgt ggtagaaatc ggtttgactc cgatagcggc tttttactat 2820
gattgttttg tgttaaagat gattttcata atggttatat atgtctactg tttttattga 2880
ttcaatattt gattgttctt ttttttgcag atttgttgac cagagatcta ccatggcgca 2940
agttagcaga atctgcaatg gtgtgcagaa cccatctctt atctccaatc tctcgaaatc 3000
cagtcaacgc aaatctccct tatcggtttc tctgaagacg cagcagcatc cacgagctta 3060
tccgatttcg tcgtcgtggg gattgaagaa gagtgggatg acgttaattg gctctgagct 3120
tcgtcctctt aaggtcatgt cttctgtttc cacggcgtgc atgcttcacg gtgcaagcag 3180
ccgtccagca actgctcgta agtcctctgg tctttctgga accgtccgta ttccaggtga 3240
caagtctatc tcccacaggt ccttcatgtt tggaggtctc gctagcggtg aaactcgtat 3300
caccggtctt ttggaaggtg aagatgttat caacactggt aaggctatgc aagctatggg 3360
tgccagaatc cgtaaggaag gtgatacttg gatcattgat ggtgttggta acggtggact 3420
ccttgctcct gaggctcctc tcgatttcgg taacgctgca actggttgcc gtttgactat 3480
gggtcttgtt ggtgtttacg atttcgatag cactttcatt ggtgacgctt ctctcactaa 3540
gcgtccaatg ggtcgtgtgt tgaacccact tcgcgaaatg ggtgtgcagg tgaagtctga 3600
agacggtgat cgtcttccag ttaccttgcg tggaccaaag actccaacgc caatcaccta 3660
cagggtacct atggcttccg ctcaagtgaa gtccgctgtt ctgcttgctg gtctcaacac 3720
cccaggtatc accactgtta tcgagccaat catgactcgt gaccacactg aaaagatgct 3780
tcaaggtttt ggtgctaacc ttaccgttga gactgatgct gacggtgtgc gtaccatccg 3840
tcttgaaggt cgtggtaagc tcaccggtca agtgattgat gttccaggtg atccatcctc 3900
tactgctttc ccattggttg ctgccttgct tgttccaggt tccgacgtca ccatccttaa 3960
cgttttgatg aacccaaccc gtactggtct catcttgact ctgcaggaaa tgggtgccga 4020
catcgaagtg atcaacccac gtcttgctgg tggagaagac gtggctgact tgcgtgttcg 4080
ttcttctact ttgaagggtg ttactgttcc agaagaccgt gctccttcta tgatcgacga 4140
gtatccaatt ctcgctgttg cagctgcatt cgctgaaggt gctaccgtta tgaacggttt 4200
ggaagaactc cgtgttaagg aaagcgaccg tctttctgct gtcgcaaacg gtctcaagct 4260
caacggtgtt gattgcgatg aaggtgagac ttctctcgtc gtgcgtggtc gtcctgacgg 4320
taagggtctc ggtaacgctt ctggagcagc tgtcgctacc cacctcgatc accgtatcgc 4380
tatgagcttc ctcgttatgg gtctcgtttc tgaaaaccct gttactgttg atgatgctac 4440
tatgatcgct actagcttcc cagagttcat ggatttgatg gctggtcttg gagctaagat 4500
cgaactctcc gacactaagg ctgcttgatg agctcaagaa ttcgagctcg gtaccggatc 4560
ctctagctag agctttcgtt cgtatcatcg gtttcgacaa cgttcgtcaa gttcaatgca 4620
tcagtttcat tgcgcacaca ccagaatcct actgagtttg agtattatgg cattgggaaa 4680
actgtttttc ttgtaccatt tgttgtgctt gtaatttact gtgtttttta ttcggttttc 4740
gctatcgaac tgtgaaatgg aaatggatgg agaagagtta atgaatgata tggtcctttt 4800
gttcattctc aaattaatat tatttgtttt ttctcttatt tgttgtgtgt tgaatttgaa 4860
attataagag atatgcaaac attttgtttt gagtaaaaat gtgtcaaatc gtggcctcta 4920
atgaccgaag ttaatatgag gagtaaaaca cttgtagttg taccattatg cttattcact 4980
aggcaacaaa tatattttca gacctagaaa agctgcaaat gttactgaat acaagtatgt 5040
cctcttgtgt tttagacatt tatgaacttt cctttatgta attttccaga atccttgtca 5100
gattctaatc attgctttat aattatagtt atactcatgg atttgtagtt gagtatgaaa 5160
atatttttta atgcatttta tgacttgcca attgattgac aacatgcatc aatcgacctg 5220
cagccactcg aagcggccgc atcgatcgtg aagtttctca tctaagcccc catttggacg 5280
tgaatgtaga cacgtcgaaa taaagatttc cgaattagaa taatttgttt attgctttcg 5340
cctataaata cgacggatcg taatttgtcg ttttatcaaa atgtactttc attttataat 5400
aacgctcaga ctctagtgac taccaccttc actctcctca agcatttcag cctcttcccc 5460
gctcagactc cttagctttg ggagccaaat tatcccttac gttctcgact tcaaccatat 5520
gtgatagctg cctatgatac catggctact tccccttagt tctttatctt tcctttccgc 5580
tttattccat gccttaccga tcctctgaag tgtctttgca ttagcttcat tgaaacctca 5640
cgcgatgaaa ggtgtgatgg tctcctccga tggcgcactt ctcatagggt aacctaattg 5700
tcttacgacc aacataggat tataattaat acaacccctc gtccctataa aagggacatt 5760
tggaaatcct tcacataagc ataacactcc tacccctctt tctttccact gtgggaacca 5820
actaatggac gctcctatca tgcctgccaa gagttcttcc caatttgcct cgtcctttcc 5880
tgagcacatg cgatgacctt gtatggggta gacagatcta ctttcatgat tgaagacgtg 5940
ggataccaac cacacataaa gagcaggcgc acaacagaaa atcctcgtag tgctcttctt 6000
gcatcttaag tcaaatgtat catacactta tgctaaaaca acaatgatcg ggctttcctt 6060
gctatggtga taagcaagaa aagcatcgat tgctactaga tccaccaact cgtctacatt 6120
cgaaaatagt actatcccaa acactagcag tgctaatacg tcgatgaatg atgcccactc 6180
tccttggctg gccagagttt ccgccttctc ctccaatcac ttccttggta ttccccctac 6240
cctattccta ctttgcttca ctcagtctaa ttctcatttc gagatcttga caactcctgc 6300
tattctcgcc atagaaggat agtacccaga aaaaaggtat ggcttccttc ctcctatcgg 6360
gcatcctaag atcccttcga actcctctat ggttggtgct aactgaaagt ccccaaaagt 6420
gaagcatctg agtgattggt catagtattg ggtgagagat gcgatg                6466
<210>10
<211>22
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<222>(1)..(22)
<223>合成的引物分子
<400>10
ccttttgggc ttttttgttt cc                                              22
<210>11
<211>20
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<222>(1)..(20)
<223>合成的引物分子
<400>11
cgtttcccgc cttcagttta                                                 20
<210>12
<211>20
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<222>(1)..(20)
<223>合成的引物分子
<400>12
tgtgtggtgt gacccattgg                                                 20
<210>13
<211>25
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<222>(1)..(25)
<223>合成的引物分子
<400>13
cctcaattgg gagatactgc actta                                           25
<210>14
<211>29
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<222>(1)..(29)
<223>合成的引物分子
<400>14
gtagtcacta gggtcagtaa agaatgtga                                       29
<210>15
<211>18
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<222>(1)..(18)
<223>合成的引物分子
<400>15
ttatcaagct ccaaacac                                                   18
<210>16
<211>22
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<222>(1)..(22)
<223>合成的引物分子
<400>16
tgagctcaaa gatatcaaca tg                                              22
<210>17
<211>22
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<222>(1)..(22)
<223>合成的引物分子
<400>17
agttaaatca tagttaataa tc                                              22
<210>18
<211>20
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<222>(1)..(20)
<223>合成的引物分子
<400>18
tcccgctcta gcgcttcaat                                             20
<210>19
<211>19
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<222>(1)..(19)
<223>合成的引物分子
<400>19
tcgagcagga cctgcagaa                                              19
<210>20
<211>24
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<222>(1)..(24)
<223>合成的引物分子
<400>20
ctgaaggcgg gaaacgacaa tctg                                        24
<210>21
<211>1103
<212>DNA
<213>Glycine max
<400>21
tgggggctgc ctgtacttcc caaaacttcg cttccctgac ccatcatatc caggactgga  60
cgattggctt gattgatacc agatgggtga gtcgagtcca cctcggtagc ggcatttatg  120
gcaacgattg cagccacgtt gacctccatc attttttctc atgctcatca tggcctccat  180
catggtggtc atttggtctt tcatggcctc catgtcggcc ttcatctgct cttgaacttc  240
atctatctca ctcatgattc tagccttggc acgtgtttgg taagggtacc gtaaagcgcg  300
ttcgttcttt tttattacta tgattacatt ttgacgatga tgatgattgt aggaaagaat  360
gaaatgagta atgaaacaac taaataaacg tgaatgcatg acaatgataa gttgctgaag  420
tattataaat ttacatagga cattcagtgg aacgtagggt cgaatcaaat cctatttcat  480
taaaaacaat attgttcatc ttgacagagc caaagcataa ctagaaatac aacatggaca  540
catcagcgat tcctaattat gtgggtcatt agttcgacca tgtgttggca gtaacttgaa  600
agactatgaa cttcatcggg agcagagtat gtgtcagtca ccgccttggc tctggctaac  660
aaccttggga tctcttggct ctcatttaga gtaagagcaa atttgtccat ccatttcatg  720
gcttctttat gcaataactc tatcacccct tctcttgctt ccctttcaac ctgcaaggtc  780
gacacttttg cctgttcgtc ttctagcctt cgcccatgac tagcagctag gttcaccttc  840
tcttcatatt ggtcaatgat tatcaacata ttttcttttg ttttgctcaa ctgttctctc  900
aaacttctct tcgatctctg acaactcttt aacttatcct ctaacatcag gttttccata  960
cttgatttgt ccctcttggc ttttctaagt ttgagctcgt tactgctgcc ccacaaagcc  1020
cctcgaaact tgttcctgct ccactcttcc ttttgggctt ttttgtttcc cgctctagcg  1080
cttcaatcgt ggttatcaag ctc                                         1103
<210>22
<211>1060
<212>DNA
<213>Glycine max
<400>22
cagactctag tgactaccac cttcactctc ctcaagcatt tcagcctctt ccccgctcag  60
actccttagc tttgggagcc aaattatccc ttacgttctc gacttcaacc atatgtgata  120
gctgcctatg ataccatggc tacttcccct tagttcttta tctttccttt ccgctttatt  180
ccatgcctta ccgatcctct gaagtgtctt tgcattagct tcattgaaac ctcacgcgat  240
gaaaggtgtg atggtctcct ccgatggcgc acttctcata gggtaaccta attgtcttac  300
gaccaacata ggattataat taatacaacc cctcgtccct ataaaaggga catttggaaa  360
tccttcacat aagcataaca ctcctacccc tctttctttc cactgtggga accaactaat  420
ggacgctcct atcatgcctg ccaagagttc ttcccaattt gcctcgtcct ttcctgagca  480
catgcgatga ccttgtatgg ggtagacaga tctactttca tgattgaaga cgtgggatac  540
caaccacaca taaagagcag gcgcacaaca gaaaatcctc gtagtgctct tcttgcatct  600
taagtcaaat gtatcataca cttatgctaa aacaacaatg atcgggcttt ccttgctatg  660
gtgataagca agaaaagcat cgattgctac tagatccacc aactcgtcta cattcgaaaa  720
tagtactatc ccaaacacta gcagtgctaa tacgtcgatg aatgatgccc actctccttg  780
gctggccaga gtttccgcct tctcctccaa tcacttcctt ggtattcccc ctaccctatt  840
cctactttgc ttcactcagt ctaattctca tttcgagatc ttgacaactc ctgctattct  900
cgccatagaa ggatagtacc cagaaaaaag gtatggcttc cttcctccta tcgggcatcc  960
taagatccct tcgaactcct ctatggttgg tgctaactga aagtccccaa aagtgaagca  1020
tctgagtgat tggtcatagt attgggtgag agatgcgatg                        1060

Claims (25)

1. 一种核酸序列,包含SEQ ID NO:1或SEQ ID NO:2的序列。
2. 包含事件MON89788的大豆植物或其部分,其中包含事件MON89788的代表性大豆种子已经以ATCC登记号PTA-6708保藏。
3. 权利要求2的植物的种子,其中所述种子包含事件MON89788。
4. 从权利要求3的种子产生的大豆商品产品。
5. 权利要求5的大豆商品产品,进一步限定为粗粉、粉、压片或油。
6. 权利要求2的大豆植物部分,限定为细胞、花粉、胚珠、花、芽、根或叶。
7. 权利要求2的大豆植物,进一步限定为包含所述事件MON89788的大豆植物的任何世代的子代植物。
8. 权利要求2的大豆植物,其中所述植物的基因组包含选自由SEQID NO:1、SEQ ID NO:2、SEQ ID NO:21和SEQ ID NO:22构成的组的至少一种DNA分子。
9. 权利要求2的大豆植物,其中当在DNA扩增方法中测试时所述植物的基因组产生对事件MON89788为诊断性的扩增子,所述扩增子包含SEQ ID NO:1或SEQ ID NO:2。
10. 权利要求3的种子,其中当在DNA扩增方法中测试时所述种子的DNA产生对事件MON89788为诊断性的扩增子,所述扩增子包含SEQ ID NO:1或SEQ ID NO:2。
11. 权利要求5的粗粉、粉、压片或油,进一步限定为包含核酸,所述核酸当在DNA扩增方法中测试时产生对事件MON89788为诊断性的扩增子,所述扩增子包含SEQ ID NO:1或SEQ ID NO:2。
12. 一种DNA多核苷酸引物分子,其包含SEQ ID NO:3或其互补物的至少11个连续核苷酸,其在DNA扩增方法中对于产生对事件MON89788为诊断性的扩增子是有用的。
13. 一种分离的DNA多核苷酸引物分子,其包含SEQ ID NO:4或其互补物的至少11个连续核苷酸,其在DNA扩增方法中对于产生对事件MON89788为诊断性的扩增子是有用的。
14. 一种特异于事件MON89788的DNA检测试剂盒,其至少包含核酸,所述核酸包含与SEQ ID NO:3或SEQ ID NO:4同源或互补的11个或更多个连续核苷酸。
15. 一种产生对草甘膦除草剂耐受的大豆植物的方法,包括向所述植物的基因组中引入事件MON89788。
16. 权利要求15的方法,限定为包含步骤:
(a)杂交包含事件MON89788的第一大豆植物与缺乏事件MON89788的第二大豆植物来产生子代植物;和
(b)至少选择包含所述事件MON89788并对草甘膦耐受的第一子代植物。
17. 权利要求16的方法,进一步包括自交所述第一子代植物来产生第二代子代植物,并选择至少对于所述事件MON89788是纯合的第一植物。
18. 一种检测样品中与大豆事件MON89788相应的DNA的存在的方法,所述方法包括:
(a)用引物集接触包含大豆DNA的样品,所述引物集当在使用来自大豆事件MON89788的基因组DNA的核酸扩增反应中使用时,产生对大豆事件MON89788为诊断性的扩增子;和
(b)进行核酸扩增反应,从而产生诊断性扩增子;和
(c)检测所述诊断性扩增子。
19. 一种检测样品中与事件MON89788相应的核酸的存在的方法,所述方法包括:
(a)获得大豆DNA的样品;和
(b)分析所述样品中来自事件MON89788的DNA序列的存在。
20. 权利要求19的方法,其中分析所述DNA样品包括检测SEQ IDNO:1、SEQ ID NO:2或其互补物的至少一种的核酸序列的存在。
21. 一种大豆植物,其包含草甘膦耐受性性状,所述性状与包含SEQ ID NO:1或SEQ ID NO:2的核酸分子是遗传连锁的。
22. 一种产生大豆商品产品的方法,包括
(a)获得权利要求2的大豆植物或其部分;和
(b)从所述大豆植物或其部分产生大豆商品产品。
23. 权利要求22的方法,其中所述商品产品被限定为粗粉、粉、压片、蛋白分离物或油。
24. 一种在包含了包含事件MON89788的大豆植物的田地中控制杂草生长的方法,所述方法包括用有效控制杂草生长的数量的草甘膦处理所述田地,其中所述大豆植物展现对草甘膦的耐受性。
25. 权利要求24的方法,其中处理所述田地在V1到R4生长期进行。
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US20190185875A1 (en) 2019-06-20
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