CN1753998B - 棉花事件mon88913及其组合物和检测方法 - Google Patents
棉花事件mon88913及其组合物和检测方法 Download PDFInfo
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- CN1753998B CN1753998B CN200480004124.1A CN200480004124A CN1753998B CN 1753998 B CN1753998 B CN 1753998B CN 200480004124 A CN200480004124 A CN 200480004124A CN 1753998 B CN1753998 B CN 1753998B
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Abstract
本发明提供棉花植物事件MON 88913的组合物和种子。也提供了基于DNA序列测定棉花植物事件MON 88913的存在的分析方法以及该DNA序列作为分子标记在DNA检测方法中的用途。
Description
本申请请求享受于2003年2月12提出的美国临时申请60/447,184的利益，该申请的全部内容在此引用作为参考。
技术领域
本发明涉及到植物分子生物学领域。更具体地说，本发明涉及草甘膦耐受的棉花事件MON 88913，以及在植物样品及其组合物中分析检测棉花事件MON 88913DNA存在的方法。
背景技术
棉花是世界上很多地区的重要的纤维作物。生物技术的方法已经应用到棉花上用于改善其农艺性状和产品的质量。向棉花植物中引入转基因的方法已经在美国专利5,004,863中得到证明。棉花生产中一个这样的重要的农艺性状是除草剂耐受性，具体地说，对草甘膦除草剂的耐受性。这个性状引入到了棉花植物中并且是现在用于棉花生产中的成功产品。目前商业化的Roundup 
棉花事件(1445)在四叶龄期间表现出对的活性组分的草甘膦的极好的耐受性(Nida等，J.Agric.Food Chem.44：1960-1966，1996；Nida etal.，J.Agric.Food Chem.44：1967-1974，1996)。然而，由于在某些环境条件下，超过四叶龄时进行的叶面施用必须受到限制，这归因于在雄性生殖组织中耐受性不足。雄性生殖耐受性的缺失看来似乎是在关键组织中CP4EPSPS的不充分表达的结果，这些组织对草甘膦高度敏感，并且草甘膦在这些严重衰败的组织(strong sinktissues)中大量积累(Pline等，Weed Sci.50：438-447，2002)。存在着对比Roundup
棉花1445具有更高的草甘膦耐受的棉花植物的需要。
为了确定有性繁殖的后代是否含有目的转基因，能检测特定事件的存在是有利的。此外，检测特定事件的方法将有利于遵守例如进入市场前许可所要求的规定、或来自重组体农作物的食品标记的规定。通过任何已知的诸如聚合酶链式反应(PCR)或使用核酸探针的DNA杂交法的核酸检测方法，可检测转基因的存在。这些检测方法 通常集中于常用的遗传元件，如启动子、3′转录终止子、标记基因等。结果，这些方法不能用于区别不同事件之间的差别，具体是那些使用相同的DNA构建体的产物，除非已知基因组染色体DNA临近于所插入的DNA(″侧翼基因组DNA″)。用于草甘膦耐受的棉花事件1445的事件特异性DNA检测方法已经得以阐述(US 20020120964，此处全部引用作为参考)。
本发明涉及到草甘膦耐受的棉花事件MON 88913、其所含有的组合物、以及涉及用于在棉花事件MON 88913及其后代中检测转基因/基因组插入区域的方法。
发明内容
本发明涉及到被命名为MON 88913的转基因棉花事件，其具有保藏号为PTA-4854的存放于美国典型培养物保藏中心(ATCC)的种子。本发明的另一方面包括棉花事件MON88913的后代植物或种子，或植物和种子的可再生部分。本发明也包括棉花事件MON 88913的植物部分，包括但不局限于花粉、胚珠、花、棉桃、棉绒、芽、根和叶。本发明涉及具有草甘膦耐受性表型和MON 88913的新的遗传组合物的棉花植物。
本发明的一个方面提供了DNA组合物和用于检测来自棉花事件MON 88913的转基因/基因组连接区域存在的方法。提供了分离的DNA分子，它包含至少一个选自SEQ ID NO：1和SEQ ID NO：2的转基因/基因组连接DNA分子及其互补序列，其中连接分子跨越了插入位点，所述插入位点包含插入到棉花基因组和基因组DNA的异源DNA，所述棉花基因组和基团组DNA来自棉花事件MON88913中插入位点两侧的棉花细胞。含有这些分子的棉籽及其植物材料也是本发明的一个方面。
提供了作为5′转基因/基因组区域SEQ ID NO：3或其互补物的分离的新DNA分子，其中这个DNA分子在棉花事件MON 88913中是新的。在其基因组中含有SEQ ID NO：3的棉花植物和种子是本发明的一个方面。根据本发明的另一方面，提供了作为3′转基因/基因组区域SEQ ID NO：4或其互补序列的分离的新DNA分子，其中这个DNA分子在棉花事件MON 88913中新的。在其基因组中含有SEQ ID NO：4的棉花植物和种子是本发明的一个方面。
根据本发明的另一方面，提供了用于DNA扩增方法的两条DNA分子，其中第一个DNA分子包含SEQ ID NO：3的DNA分子的转基因区域上的任意部分的至少长11个或更多个连续的多核苷酸，以及SEQ ID NO：3的棉花基因组5′侧翼区域的任意部分的相似长度的DNA分子，其中一起应用时，这些DNA分子可用作生产扩增子的DNA扩增方法中的DNA引物组。在DNA扩增方法中使用DNA引物组所产生的扩增子，可用于诊断棉花事件MON 88913。通过同源于或互补于SEQ ID NO：3的任意部分的DNA引物，从MON 88913DNA生产的任何扩增子，是本发明的一方面。
根据本发明的另一方面，提供两条用于DNA扩增方法的DNA分子，其中第一个DNA分子包含SEQ ID NO：4的DNA分子的转基因区域上的任意部分的至少长11个或更多个连续的多核苷酸，以及SEQ ID NO：4的棉花基因组3′侧翼区域的任意部分的相似长度的DNA分子，其中这些DNA分子可用作生产扩增子的DNA扩增方法中的DNA引物组。在DNA扩增方法中使用DNA引物组所产生的扩增子，可用于诊断棉花事件MON 88913。通过同源于或互补于SEQIDNO：4的任意部分的DNA引物，从MON 88913DNA生产的任何扩增子，是本发明的一个方面。
根据本发明的另一方面，提供在样品中检测特异性地对应于棉花事件MON88913DNA的DNA存在的方法。所述方法包括：(a)将包含DNA的样品与引物组进行接触，当该引物组与来自棉花事件MON 88913的基因组DNA一起被用于核酸扩增反应时，产生用于诊断棉花事件MON 88913的扩增子；(b)进行核酸扩增反应，由此产生扩增子；和(c)检测扩增子。
根据本发明的另一方面，提供在样品中检测特异性地对应于棉花事件MON88913DNA的DNA存在的方法。所述方法包括：(a)将含有DNA的样品同含有SEQ ID NO：1或SEQID NO：2的DNA探针相接触，该探针在严谨的杂交条件下与来自棉花事件MON 88913的基因组DNA杂交，而在严谨的杂交条件下不与对照棉花植物DNA杂交；(b)将样品和探针置于严谨的杂交条件下；和(c)检测探针与棉花事件MON 88913DNA的杂交。
根据本发明的另一方面，提供生产耐受草甘膦施用的棉花植物的方法，该方法包括：(a)将包含本发明的可赋予对草甘膦施用的耐受性的表达盒的第一亲本棉花事件MON88913，与缺乏草甘膦耐受性的第二亲本进行有性杂交，由此产生的大量的后代植物；和(b)选择耐受草甘膦施用的后代。该方法可任择地包括使后代植物同第二亲本棉花植物的回交和选择草甘膦耐受性的后代以产生耐受草甘膦施用的纯育的棉花变种的步骤。
根据本发明的另一方面，提供用于确定棉花事件MON 88913后代的接合性的方法，包括：(a)将包含棉花DNA的样品与包含SEQ IDNO：21、SEQ ID NO：22、SEQ ID NO：23、SEQ IDNO：24、和SEQID NO：25的引物组进行接触，当所述引物组与来自棉花事件MON88913的基因组DNA一起被用于核酸扩增反应时，产生用于诊断棉花事件MON 88913的第一扩增子；和(b)进行核酸扩增反应，由此产生第一个扩增子；和(c)检测第一个扩增子；和(d)将包含棉花DNA的样品与引物组进行接触，当所述引物组与来自棉花的基因组DNA一起被用于核酸扩增反应时，产生包含天然棉花基因组DNA的第二扩增子，所述天然棉花基因组DNA同源于被鉴定为棉花事件MON 88913的转基因插入的棉花基因组区；和(e)进行核酸扩增反应，由此产生第二扩增子；和(f)检测第二扩增子；和(g)比较样品中的第一和第二扩增子，其中两个扩增子的存在表明该样品对转基因插入是杂合的。
检测接合性的方法包括将棉花DNA样品与包含SEQ ID NO：21、SEQ ID NO：22、SEQID NO：23、SEQ ID NO：24和SEQ ID NO：25的引物和探针进行接触；使用终点 
PCR条件；和检测扩增子产物。
控制棉花事件MON 88913的作物或田地中杂草的方法，包括向MON 88913棉田中施用含有有效量草甘膦的除草剂的步骤。
本发明的前述和其他方面将在以下详述和附图中变得更加显然。
附图说明
图1.pMON51915的质粒图谱。
图2.在棉花事件MON 88913中插入物的基因组构成。
图3.MON 88913的5′DNA连接序列(SEQ ID NO：1)和3′DNA连接序列(SEQ ID NO：2)。
图4.MON 88913的5′转基因/基因组DNA区域(SEQID NO：3)。
图5.MON 88913的3′转基因/基因组DNA区域(SEQID NO：4)。
具体实施方式
本发明涉及耐受草甘膦的棉花事件MON 88913、其中所包含的组合物和检测棉花事件MON 88913及其后代中转基因/基因组插入区域的方法。提供以下定义和方法以更好地详细说明本发明，并指导本领域技术人员实施本发明。除非另外指出，本领域技术人员根据相关领域中的常规用法来理解术语和缩略词。分子生物学的普通术语的定义也可在Rieger等，Glossary ofGenetics：Classical andMolecu lar，5th edition，Springer-Verlag：New York，1991；和Lewin，Genes V，Oxford University Press：New York，1994中找到。如37CFR§1.822所列出的，使用于用于DNA碱基命名法。
如此处所用的，术语“棉花”意指陆地棉(Gossypiumhirsutum)并且包含了所有的能够同棉花事件MON 88913交配的植物品种。本发明的植物是棉花植物，更具体地说，是棉花植物MON88913。
如此处所用的，术语“包括”意指“包含但不限于”。
如此处所用的，术语“农作物”意指栽培植物或植物的部分，如种植于田地、小区、田垄(row)、温室、平地、或容器中的植物。
″草甘膦″是指N-膦酰甲基甘氨酸(N-phosphonomethylglycine)和其盐。N-膦酰甲基甘氨酸是一个众所周知的对广谱的植物品种具有活性的除草剂。草甘膦是 
(Monsanto Co.)的活性成分，是一个在环境中具有理想的短半衰期的安全除草剂。草甘膦是 除草剂(Monsanto Co.)的活性成分。使用“草甘膦除草剂”进行的处理是指利用 
Roundup 
Roundup 
除草剂或任何含草甘膦的其他除草剂配制剂进行的处理。商业化的草甘膦配制剂包括但不限于MonsantoCom pany出售的如
ULTRA、 ULTRAMAX、 
WEATHERMAX、ROUNDUP CT、 
EXTRA、 
BIACTIVE、
BIOFORCE、 
和 
除草剂，其含有草甘膦作为它的盐；Monsanto Company出售的 DRY和
其含有草甘膦作为它的铵盐；MonsantoCom pany出售的 
GEOFORCE，其含有草甘膦作为它的铵盐；Syngenta Crop Protection出售的
除草剂，其含有草甘膦作为它的三甲磺酰盐。当施用到植物表面时，草甘膦可进入植物全身。由于它抑制可提供用于合成芳族氨基酸前体的莽草酸途径，草甘膦具有植物毒性。草甘膦抑制了植物体内发现的磷酸烯醇式丙酮酰莽草酸合成酶(EPSPS)。草甘膦耐受性可以通过表达对草甘膦严谨性较低并因此在草甘膦存在下可以保留其催化活性的细菌EPSPS变体和植物EPSPS变体来实现(美国专利5,633,435、5,094,945、4,535,060、和6,040,497)。
转基因“事件”通过异源DNA如含有目的基因的核苷酸构建体(pMON51915，图1)转化植物细胞，再生由转基因插入植物细胞的基因组而产生的植物群体，并且挑选以插入到特殊基因组位点为特征的特殊植物而得以制备。术语“事件”指最初的转化体植物和含有异源DNA的转化体后代。术语“事件”也包括通过在事件和另一个植物之间进行有性异型杂交而生产的后代，其中该后代含有异源DNA。即使在与回归亲本进行重复回交后，来自于转化的亲本事件的插入的DNA和侧翼基因组DNA也还存在于杂交后代中的同一染色体位点。术语“事件”也指来自于含有插入DNA和与插入DNA直接相邻的侧翼基因组序列的DNA的原始转化体，可以预计这段DNA将被转移到后代中，并且作为一个含有插入的DNA的亲本系与不含有插入的DNA的亲本系进行有性杂交的结果，所述后代接收了包含目的转基因的插入DNA(如原始转化体和由自交产生的后代)。
已知在植物中外源基因的表达受到它们所在染色体上位置的影响，这可能是由于染色质的结构(如异染色质)或靠近整合位点的转录调控元件(如增强子)(Weising等，Ann.Rev.Genet 22：421- 477，1988)。因此，常常需要筛选大量的转基因植物来用于鉴定以所导入的目的基因的最佳表达为特征的事件。例如，在植物和其他生物体中观察到导入的转基因在不同事件中的表达水平有着广泛的差异。表达的时间或空间模式也有不同，例如，在不同的转基因植物组织内转基因相对表达的差异，这与根据导入基因构建体的转录调控元件的推测的表达模式不符合。因此，通常生产成百上千个不同的事件并从这些事件中筛选具有期望的转基因表达水平和用于商业目的模式的单个事件。使用常规育种方法通过有性杂交，具有期望转基因表达的水平或模式可用于将转基因基因渗入其他遗传背景中。所述杂交的后代保持了原始转化体的转基因表达特性。这个策略可用于在大量的品种中确保可靠的基因表达，以使其更好地适应当地生长条件和市场要求。
草甘膦耐受棉花植物可以通过将由通过pMON51915中含有的植物表达盒转化而衍生的转基因棉花植物细胞发育而来的棉花植物组成的并且耐受草甘膦施用的第一亲本棉花植物，与缺少对草甘膦除草剂的耐受性的第二亲本植物进行有性杂交，由此产生大量的第一后代植物；随后选择耐受草甘膦的第一后代植物；自交第一代后代植物，由此产生大量的第二代后代植物；随后从第二后代植物中选择耐受草甘膦的植物。这些步骤中还包括使第一代草甘膦耐受性后代植物或第二代草甘膦耐受性植物后代与第二亲本植物或第三亲本棉花植物回交，因此产生耐受草甘膦除草剂施用的棉花植物。本发明中，这些转基因棉花植物也被命名为棉花事件MON 88913并在此可以被称为MON 88913。
也可以理解的是也能够将两个不同的转基因植物交配而产生含有两个独立地分离添加的外源基因的后代。适当的后代的自交能够产生对于两个所添加的外源基因纯合的植物。也可预期通过非转基因植物对亲本植物进行回交和异型杂交，其称为无性繁殖。通常用于不同性状和农作物的其他育种方法的描述，可在几篇参考文献的一篇中找到，如Fehr，in Breeding Methods for Cultivar Development，WilcoxJ.ed.，American Society ofAgronomy，Madison WI(1987)。
“探针”是一段分离的连接有常规可检测的标记或报告分子，如放射性同位素、配体、化学发光试剂或酶的核酸。这样的探针可以互补于目标核酸链，就本发明而言，互补于来自MON 88913的 基因组DNA链，无论它来自MON 88913植物还是来自包含MON88913DNA的样品。根据本发明的探针不仅包括脱氧核糖核酸或核糖核酸，也包括特异性地结合目标DNA序列和用于检测目的DNA序列存在的聚酰胺和其他探针材料。
DNA引物是分离的多核酸，它们通过核酸杂交与互补的目标DNA链退火形成引物和目标DNA链的杂交子，随后经聚合酶如DNA聚合酶沿着目标DNA链延长。本发明中的DNA引物对或引物组是指至少两个用于通过如聚合酶链式反应(PCR)或其他常规核酸扩增方法来扩增目标核酸序列的DNA引物分子。
探针和引物一般是11个或更长的多核苷酸，通常是18个或更长的多核苷酸、24个或更长的多核苷酸、或30个或更长的多核苷酸。选择具有足够的长度在高严谨杂交条件下特异性地杂交目标序列的探针和引物。优选地，尽管保持同目标序列杂交的能力并且不同于目标序列的探针可以通过传统方法设计得到，然而根据本发明的探针和引物具有与目标序列完全的序列相似性。
制备与使用探针和引物的方法描述在，如MolecularCloning：A LaboratoryManual，2nd ed.，vol.1-3，ed.Sambrook等，Cold Spring Harbor Laboratory Press，ColdSpring Harbor，NY，1989(在下文，″Sambrook等，1989″)；Current Protocols inMolecularBiology，ed.Ausubel等，Greene Publishing and Wiley-Interscience，NewYork，1992(定期更新)(在下文，″Ausubel等，1992″)；和Innis等，PCR Protocols：A Guideto Methods and Applications，AcademicPress：San Diego，1990中。PCR DNA引物对能从已知序列衍生得到，例如，通过使用为此目的设计的计算机程序，如Primer(Version 0.5，
1991，Whitehead Institute for Biomedical Research，Cambridge，MA)。
根据此处公开的在基因组DNA和转基因的插入序列侧翼的引物和探针，通过常规方法，如通过分离来自MON 88913基因组的DNA、对转基因/基因组区进行重新克隆和对所述的DNA分子进行测序，可用于确认(或者，如果需要，修正)所公开的DNA序列。
本发明的核酸探针和引物在严谨条件下同目标DNA分子杂交。任何常规的核酸杂交或扩增的方法都可以用于在样品中鉴定来自 转基因事件的DNA存在。在某些情况下，多核酸分子或其片段能特异性地杂交其它核酸分子。如此处所用的，如果两个分子能形成反向双链的核酸结构，则两个多核酸分子被称为能彼此特异性地结合。如果两个核酸分子显示出完全互补，一个核酸分子被称为另一个核酸分子的互补物。如此处所用的，当一个核酸分子的每个核苷酸互补于另一个核酸分子的核苷酸，这些分子被称为表现出完全互补。当两个分子相互杂交能保持足够的稳定以使它们在至少是常规的“低严谨”条件下互相退火，则这两个分子被称为“最低限度互补”。类似的，两个分子相互杂交能保持足够的稳定以使它们在常规的“高严谨”条件下互相退火，这些分子被称为“互补”。常规严谨条件如Sambrook等，1989，和Haymes等，Nucleic Acid Hybridization，A Practical Approach，IRLPress，Washington，DC(1985)所述。因此允许相对于完全互补有偏离，只要所述偏离没有完全排除分子形成双链结构的能力。为了使核酸分子用作引物或探针，仅仅需要核酸分子在序列上是充分互补的，以能够在所用的特定溶剂和盐浓度下形成稳定的双链结构。
如此处所用的，基本上同源的DNA序列是指在高严谨条件下，可与跟它比较的目标DNA分子的互补序列特异地杂交的DNA分子序列。促进DNA杂交的合适的严谨条件，如在约45℃下的6.0×氯化钠/柠檬酸钠(SSC)，随后在50℃以2.0×SSC洗涤，是本领域技术人员熟知的或者可以在Current Protocols in Molecular Biology，JohnWiley&Sons，N.Y.(1989)，6.3.1-6.3.6查到。例如，洗涤步骤中的盐浓度可选自低严谨的50℃下的2.0×SSC到高严谨的50℃下的0.2×SSC。此外，洗涤步骤的温度也可以从大约22℃的室温的低严谨条件升高到大约65℃的高严谨条件。温度和盐浓度都是可以变化的，或温度或盐浓度保持恒定而改变另一个参数。在优选的实施方案中，在中度严谨的条件下例如约2.0×SSC和约65℃下，本发明的多核酸分子特异地杂交如SEQ ID NO：3或4所列的一个或多个核酸分子或其互补序列或二者之一的片断。在一个特别优选的实施方案中，在高严谨的条件下，本发明的核酸特异地杂交如SEQ ID NO：3或4所列的一个或多个核酸分子或互补序列或二者之一的片断。在本发明的一方面中，本发明优选的标记核酸分子具有如SEQ ID NO：1或2所列的一个或多个核酸序列或其互补序列或二者之一的片断。在本发明的 另一方面中，本发明优选的标记核酸分子共有与SEQ ID NO：1或2所列的核酸分子或其互补序列或二者之一的片断具有序列同一性的基本部分，其中序列同一性介于80％和100％或90％和100％之间。在本发明的更进一方面中，本发明优选的标记核酸分子共有与SEQ IDNO：1或2所列的核酸分子或其互补分子或二者之一的片断95％和100％的序列同一性。SEQ ID NO：1或SEQ ID NO：2可以在植物育种方法中用作鉴定遗传杂交的后代的标记，这类似于简单序列重复DNA标记分析所描述的方法，″DNA markers：Protocols，applications，and overviews：(1997)173-185，Cregan，等，eds.，Wiley-Liss NY；在此全部引用作为参考。探针对目标DNA分子的杂交可通过本领域技术员已知的任一方法得以检测，这些方法包括但不限于荧光标签法、放射性标签法、基于抗体的标签法和化学发光标签法。
关于利用特定的扩增引物对进行的目标核苷酸序列的扩增(如，通过PCR)，“严谨条件”指的是允许引物对仅与目标核酸序列杂交的条件，其中具有相应野生型序列(或其互补序列)的引物在DNA热扩增反应中将结合并优选产生一个独特的扩增产物-扩增子。
术语“特异于(目标序列)”是指在严谨杂交条件下探针或引物仅同包含有目标序列的样品中的目标序列杂交。
正如在此所用的，″扩增的DNA″或″扩增子″是指针对多核酸模板一部分的目标多核酸分子的多核酸扩增方法的产物。例如，为了确定棉花植物是否是含有来自本发明的棉花事件MON 88913植物的转基因事件基因组DNA进行有性杂交的结果，将从棉花植物组织样品提取的DNA经过多核酸扩增方法，使用包含来源于紧邻插入异源DNA的插入位点的MON88913基因组的DNA序列的一条引物和来源于插入的异源DNA(转基因DNA)的第二条引物的引物对，以生产用于诊断MON 88913事件DNA的存在的增强子。诊断性扩增子具有一定的长度并具有用于诊断事件的DNA序列。扩增子的长度范围可在引物对加一个核苷酸碱基对、优选地加大约五十个核苷酸碱基对(bps)、更优选地加大约二百五十个核苷酸碱基对、并且更加优选的大约二四百五十个或更多个核苷酸碱基对之间变化。或者，引物对可来源于插入的异源DNA两边的基因组序列，由此产生包含全部插入物多核酸序列的扩增子(如，分离自SEQ ID NO：3的基因组 部分的正向引物，分离自SEQ ID NO：4的基因组部分的反向引物，后者扩增包含了两个pMON51915DNA片断的表达盒的DNA分子所述片断被插入MON 88913基因组中，该插入物包含8512bp的插入物，图2)。来自植物基因组序列的引物对的成员可以与插入的DNA序列相距一定的距离，该距离的范围可以在从一个核苷酸碱基对到最高大约两万个核苷酸碱基对的范围内变化。使用术语″扩增子″特别排除了在DNA热扩增反应中形成的引物二聚体。
多核酸扩增可以通过本领域已知的各种多核酸扩增方法来实现，包括聚合酶链式反应(PCR)。扩增方法是本领域已知的，并在尤其是在美国专利4,683,195和4,683,202和PCR Protocols：A Guide toMethods and Applications，ed.Innis 等，A cademic Press，San Diego，1990得以描述。PCR扩增方法已发展成可扩增最多22kb(千碱基)的基因组DNA和最多42kb的噬菌体DNA(Cheng等，Proc.Natl.Acad.Sci.USA 91：5695-5699，1994)。这些方法以及本领域已知的其他方法可用于实施本发明。使用源于本文中提供的序列的引物，通过扩增来自MON 88913种子或由保存于ATCC并具有保藏号PTA-4854的种子发育的植物的DNA分子，随后对PCR扩增子或其克隆的DNA片断进行标准DNA测序，可检验异源DNA插入序列或来自MON 8891的侧翼基因组DNA序列(且如果需要得以校正)。基于DNA扩增方法的DNA检测试剂盒含有特异性地扩增诊断性扩增子的DNA引物。该试剂盒提供基于检测方法的、终点
的、或任何本领域已知的检测诊断性扩增子的方法的琼脂糖。包含与SEQ IDNO：3或SEQID NO：4的任何一部分同源或互补的DNA引物的试剂盒是本发明的一个目的。
可以通过多种技术来检测通过这些方法产生的扩增子。一个所述的方法是遗传彼特分析(Nikiforov，等.Nucleic Acid Res.22：4167-4175，1994)，其中设计可重叠基因组序列和插入的DNA序列的相邻侧翼的DNA寡核苷酸。将寡核苷酸固定在微量滴定板的孔中。在对目的区域进行PCR后(使用插入序列中的一个引物和基因组序列的相邻侧翼的一个引物)，可以将单链PCR产物同固定的寡核苷酸杂交并作为模板用于利用DNA聚合酶和特异于下一个期望碱基的标记双脱氧核苷酸三磷酸盐(ddNTPs)所进行的单碱基延伸反应。读数器可以是荧光的或基于ELISA的。信号表明转基因/基因组序列的存在归因于成功的扩增、杂交和单碱基延伸。
另一个方法是Winge(Innov.Pharma.Tech.00：18-24，2000)描述的高温测序(pyrosequencing)技术。在这个方法中，设计重叠相邻的基因组DNA和插入DNA连接区的寡核苷酸。使寡核苷酸与来自目的区域的单链PCR产物(一引物位于插入序列，另一引物位于侧翼基因组序列)杂交，并和DNA聚合酶、ATP、硫酸化酶、荧光素酶、三磷酸腺苷双磷酸酶、酰苷5′磷酰硫酸盐以及荧光素一起温育。单独加入脱氧核苷三磷酸(dNTPs)并且掺入导致了被测定的光信号。光信号表明转基因/基因组序列的存在归因于成功的扩增、杂交和单碱基或多碱基延伸。
如Chen等描述的荧光偏振(Genome Res.9：492-498，1999)是一种可用于检测本发明的扩增子的方法。在使用此方法时，涉及重叠基因组侧翼和插入DNA的连接区的寡核苷酸。将寡核苷酸与来自目的区域的单链PCR产物(一个引物位于插入序列，另一个引物位于侧翼基因组序列)杂交，并和DNA聚合酶以及荧光标记的ddNTP一起温育。单碱基延伸导致ddNTP的掺入。使用荧光计根据偏振的变化可以测量掺入。偏振的变化表明转基因/基因组序列的存在归因于成功的扩增、杂交和单碱基延伸。
Tyangi等(Nature Biotech.14：303-308，1996)描述了分子信标(molecularBeacon)可用于序列检测。简要地，设计重叠侧翼基因组和插入DNA连接区的FRET寡核苷酸探针。FRET探针的独特结构使其含有在很接近时保持荧光和淬灭部分的二级结构。将FRET探针和PCR引物(一个引物位于插入序列中，另一引物位于侧翼基因 组序列中)在热稳定聚合酶和dNTPs存在下循环。伴随着成功的PCR扩增，FRET探针与目标序列的杂交导致探针二级结构的去除以及荧光和猝灭部分的空间分离。产生了荧光信号。荧光信号表明转基因/基因组序列的存在，归因于成功的扩增和杂交。
使用此处公开的组合物和在DNA检测领域中熟知的方法可以研制出DNA检测试剂盒。该试剂盒可用于鉴定样品中棉花事件MON 88913DNA，并能应用于培育含有MON88913DNA的棉花植物的方法。试剂盒含有可用作引物和探针的DNA序列，并且该序列同源于或互补于SEQ ID NO：3或SEQ ID NO：4的任何部分、或同源于或互补于pMON51915的任一转基因遗传元件中包含的DNA，所述pMON51915已经被插入MON 88913DNA中(图2)。这些DNA序列可用于DNA扩增方法(PCR)，或用作多核酸杂交方法中的探针，如Southern分析、northern分析的探针。含有MON 88913DNA(图2)的转基因遗传元件包括第一个表达盒，该表达盒包含以拟南芥(Arabidopsis)延伸因子1α(At.Efla)启动子作为嵌合元件构建的玄参嵌合体启动子(FMV35S/Efla，美国专利6,462,258，SEQ ID NO：28，在此全部引入作为参考)，并且其有效连接拟南芥延伸因子1-α的翻译前导序列(leader)和内含子(如Axelos等描述的Genbank登录号X16430，Mol.Gen.Genet.219：106-112，1989)、有效连接拟南芥EPSPS的叶绿体转运肽(TS-At.EPSPS：CTP2，Klee等，Mol.Gen.Genet.210：47-442，1987)、有效连接来自土壤杆菌属菌株CP4的耐受草甘膦的5-烯醇式丙酮酸莽草酸-3-磷酸合酶(EPSPS)(aroA：CP4，美国专利5,633,435)、有效连接来自豌豆的核酮糖1，5-二磷酸羧化酶E9的3′终止区域(T-Ps.RbcS2：E9，Coruzzi，等，EMBO J.3：1671-1679，1984)，而第二个表达盒含有包括Act8基因(SEQ ID NO：29，美国专利6,462,258)的第一个内含子的CaMV35S-Act8启动子，并且其有效连接拟南芥EPSPS叶绿体转运肽(TS-At.EPSPS：CTP2)、有效连接来自土壤杆菌属菌株CP4的耐受草甘膦的EPSPS(aroA：CP4，美国专利5,633,435，在此全部引用作为参考)、有效连接来自豌豆的核酮糖1，5-二磷酸羧化酶E9的3′终止区域。
下列实施例包括了用于证明本发明的某些优选的实施方案的实施例。本领域技术人员应当理解，实施例中公开的技术代表发明 者发现的在本发明实施中有效的技术，因此可认为其组成了用于实施本发明的优选模式。然而，根据本公开内容，本领域的技术人员应当理解，可在不偏离本发明的精神和范围的情况下，在公开的具体实施方案中进行多种改变，而仍可获得相同或相似的结果。
实施例
实施例1
使用于源于pMON51915(图1)的DNA片段，通过农杆菌介导的转化棉花细胞来生产转基因棉花事件MON 88913。使用三亲本交配方法将植物转化构建体pMON51915交配到农杆菌(Ditta等，Proc.Natl.Acad.Sci.77：7347-7351，1980)中。根据下述的方法利用转基因实施棉花细胞的转化：美国专利5,004,863、美国专利5,159,135、美国专利5,518,908，在此全部引入作为参考。棉花转化基本上按WO/0036911或U.S.Patent 5,846,797中所述的方法进行实施，在此全部引入作为参考。对这些方法的改良包括但不局限于下列实施例。Coker130种子经表面消毒并黑暗发芽。从发芽的籽苗上切下长约1-1.5厘米的胚轴外植体。将经转化含有pMON51915的根癌农杆菌菌株ABI(Agrobacterium tumefaciens strain ABI)在不含抗生素的LB培养基(Luria broth)中在28℃生长16小时，然后稀释成约2×108细菌/毫升(ml)。将胚轴外植体浸入农杆菌接种体中2-5分钟，然后在MS+1.9mg/l KNO3+3％葡萄糖(TRM)上以每平板30外植体于24℃在暗处共培养约45小时。将外植体转移到含有150mg/l头孢霉素和300EM草甘膦的TRM上培养四个培养周期，每个培养周期大约为6周。在第三或第四培养周期结束时，从原始外植体上分离胚胎发生愈伤组织(embryogenic calli)并放置到同样的培养基上。通过简单地在液体TRM+3％葡萄糖中悬浮胚胎发生愈伤组织进行一次亚培养，然后把悬浮液倒在‘TRM’+150mg/l头孢霉素+300μM草甘膦平板上。在液体亚培养后3-8周收集体细胞胚，然后在含0.5％葡萄糖的Stewart和Hsu培养基上生长。让来源于体细胞胚的小苗在含有以40mM NO3/10mMNH4+2％蔗糖改良的Stewart&Hsu培养基的Magenta盒中成熟到约4-7cm(3-6片叶)。随后将这些植物移植到盆栽土中，以4″盆、100％湿度、每天光照16小时处理4-6天，随后以50％湿度处理5-10天。
pMON51915的DNA片段含有两个插入到MON 88913基因组(图2)中的转基因表达盒，该表达盒总体上赋予了MON 88913和其后代以草甘膦耐受性。
MON 88913植物和种子具有可再生部分。种子的可再生部分包括但不限于胚芽、子叶和芽或根的分生组织。植物的可再生部分包括但不限于叶片、叶柄、胚轴、茎部分和顶点以及根分生组织。本发明也包括棉花事件MON 88913的植物部分，其包括但不限于花粉、胚珠、花、园荚、纤维、芽、根和叶。本发明也包括MON 88913种子中的可提取组分，其包括但不限于蛋白质、粗粉、面粉、果壳、油和棉绒纤维。
实施例2
草甘膦耐受性棉花事件MON 88913选自很多耐受草甘膦营养性和生殖性伤害的转基因棉花事件。商品级品质的转基因事件的成功生产目前需要生产大量的事件。在本发明中，MON 88913是通过大量不同的包括pMON51915的DNA构建体转化的约1000R0事件中的一个事件。通过一系列的分子分析和草甘膦耐受性筛选从许多事件中选择MON 88913事件。
在温室草甘膦耐受性测试中，对植物的营养性和生殖性耐受性进行评分，从而筛选事件。将来自每个事件的十五到二十五个R1 种子种植于含有Metro-Mix 350培养基的15个槽盘(cell tray)中，该盘含有泥炭、蛭石、营养物、润湿剂和加工过的树皮和灰烬的混和物。培养基中含有的追肥是Osmocote 14-14-14、Osmocote Plus 15-9-12和MicroMax微量元素。所有植物都在温室内生长。生长季节期间白天平均温度是32摄氏度(℃)，而夜晚平均温度是24℃。光周期设定为具有最大光强度的16小时光照和8小时黑暗。生长循环期间的平均相对湿度是45％。随后，植物在4叶期和8叶期连续以48oz/A(oz＝盎司，A＝英亩)喷洒Roundup 
(含草甘膦的除草剂)。在四叶期施用草甘膦七天后，评价植物受到的植物性伤害且收集分离草甘膦耐受表型的。使用这些数据来确证事件转基因插入物是遵循孟德尔遗传模式的单显性基因的模式。具有良好的营养性耐性的事件随后被移植到含有上述相同的Metro-Mix 350培养基的10英寸盘中并长至成熟。 由于需要调节植物高度，以PixPlus(BASF，Research Triangle Park，NC)处理植物。在移植后三个月，在最初的五个结果枝的最初结果位置对植物的棉桃保持力(retention)进行绘图。该植物绘图的保持力的最大值是5(五个棉桃保留下来)。这提供了一个迅速、间接的植物生育力的测定方法。经过温室筛选，目前的商业化事件(RR棉花1445)的平均保持力少于1.0。采集平均棉桃保持力值高于或等于三的事件，并用于进一步的筛选。具有棉桃保持力高于或等于二的事件具有作为新的草甘膦耐受性植物筛选的值。
通过DNA印迹来分析符合 
Ultra营养性和生殖性的耐受标准的事件的拷贝数目。对在最初的温室实验中表现良好的耐受性的单拷贝事件进一步在：1)以高草甘膦率进行的额外温室耐受实验、2)重复的田间实验中和通过3)额外的分子筛选进行表征。使用纯合体植物实施温室耐受性测试。在所有的实验中含有作为对照的目前商业化Roundup 棉花事件的1445事件(商品标准)。将种子种植于15个槽盘并在四叶期用64oz/A Roundup 
处理而在八叶期用96oz/A Roundup 
处理。随后将植物移植到10英寸的盆中，而在中季对四株植物在首次五个结果枝的首次结果位置进行绘图。收集所有事件的季节末期的数据，包括重量、棉桃数、棉桃尺寸和棉桃保持力。
使用田间测试来筛选显示最好的生长率、果实保持力和产量的事件。按随机分区方案安排田间实验，进行三次重复和三次处理。将事件种植在两排、30英尺的小块土地里。处理由不喷洒的、在4、6、10、14节点期喷洒64oz/A(1.5lb ae/A)Roundup 
和在4、6、10、14节点期喷洒96oz/A(1.5lb ae/A)Roundup 
组成。每块地在十株植物上完成中季绘图。收集在首次五个结果节点的第一和第二结果位置的棉桃保持力数据，该数据给出0-10的数值范围。在0-10分值上，棉桃保持力值等于或大于3的植物被评定为新的草甘膦耐受性的植物筛选。
比较MON 88913和RR棉花1445的棉绒产量(磅/亩、1磅/亩)的田间测试(10个位置)显示MON 88913基本上提供了防止草甘膦(酸当量磅/亩、lb ae/A)对产量的影响(表1)。产量是棉桃保持力的衡量标准，为了获得草甘膦耐受性而工程化的棉花植物，其在第 一和第二结果位置保留了基本数量的棉桃，这将保持相对于非草甘膦耐受性棉花植物在产量上的优势。根据要被控制的杂草种类和杂草发育时期的不同，含有有效剂量的草甘膦的除草剂，其用于控制MON88913田间中的对照杂草，大约包含4oz/A，并可大于128oz/A。草甘膦可以与其他除草剂混合以增强除草剂对某些杂草种类的活性。 表1.在草甘膦处理后MON 88913和1445的棉绒产量的比较。 10个区域的产量(lb/A)
实施例3
通过使用CTAB方法(Rogers等，Plant Mol.Biol.5：69-76，1985)或根据生产商的说明书的DneasyTM96植物试剂盒(目录号69181，Qiagen Inc.，Valencia，CA)对用于所有PCR反应和Southernblot分析的棉花基因组DNA进行分离。收集2-4叶期的叶片组织。从各株植物收集最小的真叶并立即干冰冷冻。使用例如以下方法提取DNA。使用塑料珠在液氮中磨碎组织，向0.75克(g)组织中加入5ml提取缓冲液并在55℃温育45分钟。CTAB提取缓冲液由100mM TrispH8.0、1.4M NaCl、20mM EDTA、添加了5μl(微升)β-巯基乙醇的2％CTAB、5μlRNA酶和1％PVPP组成。随后用等体积的氯仿(5ml)抽提样品，然后在室温以3700转/分钟离心15分钟。将水相转移到新离心管，并用等体积的异丙醇沉淀DNA。以3700转/分钟离心15分钟后，用70％的乙醇洗涤沉淀、空气干燥并重悬于250μl水中。
使用TAIL-PCR(Liu等，Plant Journal 8：457-463，1995)获得用于MON 88913事件的邻近于转基因插入的棉花基因组DNA。使用GenomeWalker试剂盒(CloneTechLaboratories，Palo Alto，CA)按生产方法实施基因组DNA的延伸。简单地，将使用前述CTAB方法所分离的DNA(～5μg)通过多种限制性内切酶(EcoRV，Scal)在100μl的总体积中于37℃消化过夜。使用QLAquick PCR纯化柱(cat#28104，Qiagen，Inc.)除去限制性内切酶。接头(adaptor)分子连接法是已在 生产方法中描述的方法。使用用于初级反应的FMV-1引物(SEQ IDNO：5)和AP1引物(CloneTech Laboratories)，以及用于次级反应的巢式FMV-2引物(SEQ ID NO：6)和AP2引物(CloneTech Laboratories)来扩增DNA。
使用反向PCR来分离MON 88913的3′转基因/基因组DNA。使用三个限制性酶BclI、NcoI和HindII消化总基因组DNA(～10μg)。在37℃消化过夜后，经QIAquick PCR纯化柱对DNA进行纯化。用50μl水从柱子上洗脱DNA并稀释到1ml。将稀释的洗脱液(85μl)与10μl缓冲液(10×)和5μlT4连接酶混合以环化片段。在16℃温育过夜后，将连接酶在70℃下热失活。使用一系列巢式引物通过PCR扩增样品。PCR的引物组合包括：用于BclI和NcoI样品的引物对8099-E9-1/E9-2(SEQ ID NO：7/SEQ ID NO：8)和用于HindIII样品的引物对8099-E9-1/Act8反向(SEQ ID NO：7/SEQ IDNO：9)；用于BclI和NcoI样品的引物对8099-E9-2/E9-1(SEQIDNO：10/SEQ ID NO：11)和用于HindIII样品的引物对8099-E9-2/Act8(SEQ ID NO：10/SEQID NO：12)；用于BclI和NcoI样品的引物对8099-E9-3/E9-1(SEQ ID NO：13/SEQ ID NO：11)和用于HindIII样品的引物对8099-E9-3/Act8(SEQ ID NO：13/SEQ ID NO：12)。PCR条件包括：初级PCR＝7个循环的94℃2秒钟、72℃10分钟；37个循环的94℃2秒钟、67℃10分钟；1个循环的67℃10分钟；第二级和第三级PCR＝5个循环的94℃2秒钟、72℃10分钟；24个循环的94℃2秒钟、67℃10分钟；1个循环67℃10分钟。
或者，可以通过MON 88913事件的3′末端的PCR进行DNA扩增，其条件包括：7个循环的94℃25秒钟、72℃3分钟；37个循环的94℃25秒钟、67℃3分钟；1个循环的67℃7分钟。所有后续的扩增按下列条件操作：7个循环的94℃2秒钟、72℃4分钟；37个循环的94℃2秒钟、67℃4分钟；1个循环的67℃7分钟。所有的扩增子在0.8％琼脂糖胶上通过溴化乙锭染色得以显现。通过直接使用QIAquick PCR纯化试剂盒(cat&num；28104，QiagenInc.)纯化PCR样品，或使用QIAq uick Gel Extraction试剂盒(cat#28704，Qiagen Inc.)通过从凝胶上提取合适的片断来制备DNA，用于测序。
设计了一系列的DNA引物，对MON 88913的转基因插入物和邻近的侧翼基因组区域进行测序。通过五个重叠片断，设计可允许扩增全部的转基因和基因组侧翼序列的DNA引物。设计可允许单独扩增各个EPSPS-CTP2/aroA-CP4/RbcS2：E9区域的专一性引物(uniqueprimer)。对于所有用于测序的片断，重复扩增了三次。DNA引物对组合被用作5′转基因/基因组区域(SEQ ID NO：14和SEQ IDNO：15)、3′转基因/基因组区域((SEQ ID NO：16和SEQ IDNO：17)和插入遗传元件(SEQ ID NO：18和SEQ ID NO：11；SEQ ID NO：19和SEQ ID NO：15；SEQ ID NO：20和SEQ ID NO：11)的测序引物。总基因组DNA被用于所有的PCR反应。所有的扩增子在0.8％的琼脂糖胶上经溴化乙锭染色得以显示。通过直接使用QIAquick PCRPurification试剂盒(cat&num；28104，Qiagen Inc.)纯化PCR样品，或使用QIAquick GelExtraction试剂盒(cat#28704，Qiagen Inc.)从胶上提取合适的片断，来制备DNA，用以测序。使用DNA序列分析仪(ABI PrismTM 377，PE Biosystems，Foster City，CA)和DNASTAR序列分析软件(DNASTAR Inc.，Madison，WI)来生产DNA序列。
使用TOPO TA 
试剂盒(Invitrogen)，将来自MON 88913转基因/基因组插入物的侧翼区域的DNA片段进行亚克隆。图4显示5′转基因/基因组区域的DNA序列，图5显示3′转基因/基因组区域。在图4和5显示的DNA序列中，转基因插入序列以斜体子表示。
实施例4
使用DNA事件引物对来生产诊断棉花事件MON 88913基因组的扩增子。诊断MON88913基因组的扩增子包含至少一个连接序列、SEQ ID NO：1或SEQ ID NO：2。可产生诊断MON 88913的扩增子的引物对，当用于表2中列出的方法时，引物对包括但不限于5′扩增子序列的SEQ ID NO：14和SEQ ID NO：15，以及3′扩增子序列的SEQ ID NO：16和SEQ ID NO：17。除了这些引物外，同源于或互补于SEQ ID NO：3或SEQ ID NO：4的任何引物对，其可在DNA扩增反应中产生用于诊断MON 88913的扩增子，也是本发明的 一个方面。含有可用于DNA扩增反应中产生用于诊断MON 88913的扩增子的SEQ ID NO：3或其互补序列上至少11个连续核苷酸的任何单个的分离的DNA多核酸引物分子也是本发明的一个方面。含有可用于DNA扩增反应中产生用于诊断MON 88913的扩增子的SEQ IDNO：4或其互补序列上至少11个连续核苷酸的任何一个分离的DNA多核酸引物分子也是本发明的一方面。表2和表3显示用于分析的扩增条件的实例。然而，使用DNA引物的方法的任何改良都在本领域常规技术范围内，所述DNA引物同源于或互补于SEQ ID NO：3或SEQ ID NO：4或MON 88913的转基因插入物中包含的遗传元件的DNA序列，并可生产用于诊断MON 88913的扩增子。诊断性扩增子包含同源于或互补于至少一个转基因/基因组连接DNA(SEQ ID NO：1或SEQ ID NO：2)或其基本部分的DNA分子。
对事件MON 88913植物组织样品的分析应包括来自事件MON 88913的阳性组织对照、来自非事件MON 88913的棉花植物的阴性对照和不含棉花基因组DNA的阴性对照。通过DNA扩增方法领域的技术人员，可从SEQ ID NO：3和SEQ ID NO：4中选择额外的引物序列，以及通过表2和表3中所示的方法，所选择的用于生产扩增子的条件可以不同，但产生用于诊断事件MON 88913的扩增子。根据对表2和3的方法作出修改的这些DNA引物序列的用途被包括在本发明的范围内。本发明的一方面是通过来源于SEQ ID NO：3或SEQ ID NO：4的至少一条DNA引物序列所生产的扩增子，所述扩增子用于诊断MON 88913。
本发明的一个方面是含有至少一个源于SEQ ID NO：3或SEQ ID NO：4的DNA引物的
DNA检测试剂盒，当所述引物被用于DNA扩增反应中时，可生产用于诊断MON 88913的扩增
子。本发明的一方面是通过至少一条来源于pMON51915的任何遗传元件的引物序列所生产
的扩增子，所述扩增子用于诊断MON 88913。棉花植物或种子是本发明的一个方面，其中当
其基因组在DNA扩增方法中进行测试时，产生包含SEQ ID NO：1或SEQ ID NO：2的扩增子。对
MON 88913扩增子的分析可以通过使用Stratagene Robocycler、MJEngine、Perkin-Elmer
9700或如表3中所示的Eppendorf MastercyclerGradient thermocycler或通过本领域技
术人员已知的方法和设备进行。 表2.用于鉴定MON 88913的5′转基因插入/基因组连接区
域的PCR程序和反应混合物条件。
| 步骤 | 试剂 | 量 | 注释 |
| 1 | 不含核酸酶的水 | 加到终体积20μl | - |
| 2 | 10×反应缓冲液(含MgCl2) | 2.0μl | 1×缓冲液终浓度的，1.5mM的MgCl2终浓度 |
| 3 | 10mM dATP、dCTP、dGTP和dTTP的溶液 | 0.4μl | 200μM终浓度的每种dNTP |
| 4 | 事件引物(SEQ ID NO：14)(重悬浮于1×TE缓冲液或不含核酸酶的水直至10μM的浓度) | 0.4μl | 0.2μM终浓度 |
| 5 | 事件引物(SEQ ID NO：15)(重悬浮于1×TE缓冲液或不含核酸酶的水直至10μM的浓度) | 0.4μl | 0.2μM终浓度 |
| 6 | RNA酶、无DNA酶(500ng/μl) | 0.1μl | 50ng/反应 |
| 7 | REDTaqDNA聚合酶(1单位/μl) | 1.0μl(推荐在下一步骤 前更换移液管) | 1单位/反应 |
| 8 | 提取的DNA(模板)：·将要被分析的样品＊单个叶片＊叶片组(最大值50叶片/组)·阴性对照·阴性对照·阳性对照 | ·10-200ng基因组DNA·200ng基因组DNA·50ng棉花基因组DNA (非MON88913)·无模板DNA·50ng MON88913基因 组DNA | - |
| 9 | 轻柔混和并往每个反应液面加入I-2滴矿物油 |
表3.推荐的不同的热循环仪的PCR参数
使用下列循环参数在Stratagene Robocycler、MJ Engine、Engine、Perkin-Elmer
9700、或Eppendorf Mastercycler Gradient热循环仪中进行DNA扩增。MJ Engine或
Eppendorf MastercyclerGradient热循环仪应当以计算出的模式运行。Perkin-Elmer
9700thermocycler以最大的斜率速度运行。
| 循环数 | 设置：Stratagene Robocycler |
| 1 | 94℃3分钟 |
| 38 | 94℃1分钟 60℃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 |
| 38 | 94℃10秒 60℃30秒 72℃1分钟 |
| 1 | 72℃10分钟 |
| 循环数 | 装置：Eppendorf Mastercycler Gradient |
| 1 | 94℃3分钟 |
| 38 | 94℃15秒 60℃15秒 72℃1分钟30秒 |
| 1 | 72℃10分钟 |
实施例5
将MON 88913基因组DNA和对照棉花基因组DNA(每个～15μg)经不同的限制性内切酶(140U)在含有15μl相应的产品缓冲液(NEB、Beverely、MA)的150μl总体积中消化。将限制性内切酶， 如BgII、BamHI、NcoI、HindIII、和BcII用于MON 88913的Southern分析中。内切酶消化在合适的温度下进行至少6小时。温育后，使用3M乙酸钠和2.5倍体积的乙醇沉淀DNA。随后，DNA经70％乙醇洗涤、干燥并重悬于40μl的TBE中。在样品中加入加样缓冲液(0.2×)，并在琼脂糖凝胶(0.8％)上以30伏电压进行电泳16-18小时。凝胶经溴化乙锭染色，然后用脱嘌呤溶液(0.125N HCl)处理10分钟、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丁三醇胺碱、1.5M氯化钠)处理30分钟。使用Turboblotter(Schleicherand Schuell，Dassel，Germany)将DNA转移到Hybond-N膜上4-6小时，然后经UV光线固定到膜上。
让膜在20m l DIG Easy Hyb溶液(Roche MolecularBiochemicals，Indianaapolis，IN；cat.#1603558)中在45℃进行预杂交2-4小时。通过使用Radprime DNA标记试剂盒(Invitrogen，Carlsbad，CA；cat.#18428-011)制备同SEQ ID NO：1、或SEQ ID NO：2、或SEQID 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)除去未掺入的核苷酸。用含有1百万计数每毫升的终浓度的10ml变性探针的预热的DIGEasy Hyb溶液代替预杂交液。在45℃杂交斑点16-18小时。
在45℃用低严谨溶液(5×SSC，0.1×SDS)洗涤斑点，然后在65℃用高严谨溶液(0.1×SSC，0.1％SDS)反复洗涤。将斑点在荧光屏(Amersham Biosciences，Piscataway，NJ)前曝光2小时以上，并使用Data Storm 860仪(Amersham Biosciences)读出曝光值。
实施例6
在表4和表5所描述的用于实施例条件的接合性分析中描述了用于鉴定含有事件MON88913的纯合子棉花后代杂合的方法。用于接合性分析的引物是引物SQ1099(SEQ IDNO：21)、SQ1100(SEQ ID NO：22)、SQ1353(SEQ ID NO：23)、6FAMTM标记引物(SEQID NO：24)以及VICTM标记引物(SEQ ID NO：25)，其中6FAM和VIC是连接到DNA引物的Applied Biosystems(Foster City，CA)上的荧光染色产物。
当SEQ ID NO：21、SEQ ID NO：22和SEQ ID NO：23用 于这些反应方法中时，会产生
用于非转基因棉花的DNA扩增子、用于含有事件MON 88913的杂合棉花的两个DNA扩增子以
及用于不同于任何非MON 88913棉花的纯合MON 88913棉花的扩增子。这个分析的对照应包
括含有事件MON 88913DNA的纯合和杂合棉花的阳性对照、非转基因棉花的阴性对照以及不
含模板DNA的阴性对照。该分析通过使用Stratagene Robocycler、MJ Engine、Perkin-
Elmer 9700或Eppendorf Mastercycler Gradient热循环仪得以最优化。本领域技术人员
已知的可鉴定通过MON88913事件植物产生的杂交后代的接合性的其他方法和装置包括在
本技术领域的范围内。 表4.接合性分析反应溶液
| 步骤 | 试剂 | 量 | 注释 |
| 1 | 不含核酸酶的水 | 加到10μl终体积 | - |
| 2 | 2×Universal Master Mix(Applied Biosystemscat.#4304437) | 5μl | 1×终浓度 |
| 3 | 引物SQ 1099、SQ1100、SQ1353(重悬于不含核酸酶的水直至20μM终浓度) | 0.5μl | 0.25μM终浓度 |
| 4 | 引物6FAMTM(重悬于不含核酸酶的水直至10μM的终浓度) | 0.2μl | 0.4μM终浓度 |
| 5 | 引物VICTM(无核酸酶水重悬至10μM终浓度) | 0.2μl | 0.15μM终浓度 |
| 6 | REDTaq DNA聚合酶(1单位/μl) | 1.0μl(推荐在下一步骤 前更换移液管) | 1单位/反应 |
| 7 | 提取的DNA(模板)：·待分析的样品(单个叶片)·阴性对照·阴性对照·阳性对照·阳性对照 | 3.0μl4-80ng基因组DNA4ng非转基因棉花基因组DNA无DNA模板 (悬浮DNA的溶液)4ng来自已知事件MON 88913杂合子棉花的基因组DNA4ng来自已知事件MON 88913纯合子棉花的基因组DNA | 以水稀释 |
| 8 | 轻柔混合，在各个反应液顶端加入1-2滴矿物油 |
表5.接合性分析热循环仪条件
使用下列循环参数在Stratagene Robocycler、MJ Engine、Engine、Perkin-Elmer
9700或Eppendorf Mastercycler Gradient热循环仪中进行DNA扩增。当在MJ Engine或
Eppendorf MastercyclerGradient热循环仪中进行PCR时，热循环仪应当以已经计算出的
模式运行。当在Perkin-Elmer 9700热循环仪中进行PCR时，热循环仪应当以最大的斜率速
度运行
| 循环数 | 设置：Stratagene Robocycler |
| 1 | 94℃ 3分钟 |
| 38 | 94℃ 1分钟 60℃ 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 |
| 38 | 94℃ 30秒 60℃30秒 72℃1分钟30秒 |
| 1 | 72℃ 10分钟 |
| 循环数 | 设置：Eppendorf Mastercycler Gradient |
| 1 | 94℃ 3分钟 |
| 38 | 94℃ 15秒 60℃15秒 72℃ 1分钟30秒 |
| 1 | 72℃ 10分钟 |
实施例7
使用终点 
的方法对棉花基因组DNA样品进行分 析。使用含有事件引物SEQ ID NO：21、SEQ ID NO：22和6-FAM探针SEQ ID NO：24的引物组A与含有事件引物SEQID NO：26、SEQID NO：27和6-FAM探针SEQ ID NO：28的引物组B，来生产用于诊断MON 88913基因组DNA的扩增子。该方法使用96孔或342孔规格和Applied Biosystems GeneAmp PCRSystem 9700或MJResearch DNA Engine PT-22。根据前述从棉花组织样品提取的DNA应该在每个PCR反应5-10ng的范围内。每个反应含有10μl终体积，由0.5μl等浓度的事件引物(20μM)、5.0μl的2信的通用主混合物、0.2μl的6-FAM探针(10μM)、3μl DNA样品(5-10ng)并加水到10μl组成。热循环参数是1个循环的50℃2分钟，1个循环的95℃10分钟，10个循环的95℃15秒、64℃1分钟，然后是-1℃/循环，30个循环的95℃15秒、54℃1分钟，然后保持在10℃。产生的扩增子经微量培养板读数仪，如TECAN Safire(Durham，NC)根据生产商描述的条件得以检测。DNA检测领域中已知的其他装备和分析方法能用于检测本发明的扩增子。
以上公开的和权利要求书中引用的棉花MON 88913的种子，即MonsantaTechnology LLC的保藏物，已根据布达佩斯条约保藏在美国典型培养物保藏中心(ATCC)，10801University Boulevard，Manassas，Va.20110。ATCC保藏号是PTA-4854。保藏物将在存放处保养30年、或在最后请求后延长5年，或在专利的有效使用期限，无论那一个更长久，并根据需要在此期限内进行替代。
已经图解和详述了本发明的原理，对于本领域技术人员，在不偏离这些原理下可在排列和细节上对本发明进行修改应该是显而易见的。我们要求保护在附加的权利要求书的精神和范围内的所有改良。
本说明书中引用的所有出版物和公开专利文件都在此引用作为参考，其程度等同于具体并分别指出各个出版物或专利申请受到引用作为参考。
Claims (6)
1.DNA分子，其是5’转基因-基因组区域SEQ ID NO:3或其互补序列或3’转基因-基因组区域SEQ ID NO:4或其互补序列。
2.生产耐受草甘膦除草剂施用的棉花植物的方法，包括：
(a)将第一草甘膦耐受的亲本植物与缺乏草甘膦耐受性的第二亲本棉花植物进行有性杂交，由此产生大量的第一后代植物，所述第一草甘膦耐受的亲本植物是含权利要求1所述的DNA的转基因棉花；和
(b)选择耐受草甘膦的第一后代植物。
3.权利要求2的方法，进一步包括对耐受草甘膦的第一后代植物进行自交，由此产生大量的第二后代植物，和选择耐受草甘膦的第二后代植物的步骤。
4.检测含有DNA的棉花植物样品中对应于含权利要求1的DNA的所述转基因棉花的DNA存在的方法，该方法包括：
(a)将该样品与探针进行接触，该探针在严谨杂交条件下与来自含权利要求1所述的DNA的转基因棉花的基因组DNA杂交，而在严谨杂交条件下不与对照棉花植物基因组DNA杂交，其中该探针与SEQ ID NO:1或SEQ ID NO:2相同或互补；
(b)将样品和探针置于严谨杂交条件下；和
(c)检测探针与DNA的杂交。
5.权利要求4的方法，其中所述样品包括含DNA的棉花植物部分，所述棉花植物部分选自由花粉、胚珠、花、棉桃、芽、根、叶和种子构成的组。
6.在棉花农作物中控制杂草的方法，所述棉花农作物为含权利要求1所述的DNA的转基因棉花，该方法包括对所述棉花农作物施用有效剂量的含草甘膦的除草剂的步骤。
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| UY28769A1 (es) | 2004-03-30 | 2005-09-30 | Monsanto Technology Llc | Métodos para controlar agentes patógenos en plantas usando n-fosfonometilglicina |
| CN102792969B (zh) | 2005-03-04 | 2015-01-21 | 孟山都技术公司 | 减轻用除草剂草甘膦制剂处理的草甘膦耐受性转基因棉花植物内的坏死 |
| PT1885176T (pt) * | 2005-05-27 | 2016-11-28 | Monsanto Technology Llc | Evento mon89788 de soja e métodos para a sua deteção |
| EP1922409B1 (en) * | 2005-08-08 | 2017-11-08 | Bayer CropScience NV | Herbicide tolerant cotton plants and methods for identifying same |
| US7388134B1 (en) | 2005-10-27 | 2008-06-17 | Monsanto Technology Llc | Cotton variety 370001G |
| US7250563B1 (en) | 2005-10-27 | 2007-07-31 | Monsanto Technology Llc | Cotton variety 170001G |
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| US7253345B1 (en) | 2005-10-27 | 2007-08-07 | Monsanto Technology Llc | Cotton variety 450001G |
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| US7626093B2 (en) * | 2006-03-14 | 2009-12-01 | Bayer Cropscience Ag | Cotton cultivar 04Y341 |
| CA2651428A1 (en) * | 2006-05-12 | 2007-11-22 | Commonwealth Scientific And Industrial Research Organisation | Enzymes for degrading herbicides |
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