CN102858996B - 原种事件ee-gm3以及用于鉴定生物样品中的该事件的方法和试剂盒 - Google Patents
原种事件ee-gm3以及用于鉴定生物样品中的该事件的方法和试剂盒 Download PDFInfo
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Abstract
本发明提供了特定的转基因大豆植物、植物材料和种子，其特征在于此类产物在大豆基因组的特定位置上具有特定的除草剂抗性转化事件。还提供了使得能够在生物样品中快速且明确地鉴定所述事件的工具。
Description
交叉参考相关申请
本申请要求2010年7月23日提交的美国临时专利申请No.61/367,227；2009年11月23目提交的美国临时专利申请No.61/263,690；和2009年11月23日提交的欧洲申请No.EP09014564.0的优先权，将所述每一个美国临时专利申请和欧洲专利申请的公开内容通过引用整体并入本文。
发明领域
本发明涉及转基因大豆植物、植物材料和种子，其特征在于包含特定的转化事件，具体地在于编码赋予除草剂抗性的蛋白质的基因在大豆基因组的特定位置上的存在。本发明的大豆植物将耐除草剂表型与在除草剂不存在的情况下等同于非转化大豆品系的不同遗传背景中的农艺性状、遗传稳定性和功能性组合。本发明还提供了用于鉴定包含特定转化事件EE-GM3的植物材料在生物样品中存在的方法和试剂盒。
发明背景
植物中转基因的表型表达可通过基因本身的结构和通过其在植物基因组中的位置来确定。同时转基因或“外源DNA”在基因组的不同位置上的存在可以以不同方式影响植物的总体表型。通过基因操作将具有商业吸引力的性状在农业或工业上成功能地引入植物视不同的因素而定可以是冗长的过程。遗传转化的植物的实际转化和再生只是一系列选择步骤的第一步，所述一系列选择步骤包括广泛的遗传表征、繁育以及田间试验的评估，最终导致原种事件(eliteevent)的选择。
基于关于新型食品/饲料、GMO和非-GMO产物的分离以及专利材料(proprietarymaterial)的鉴定的讨论，原种事件的明确鉴定变得日益重要。理想地，这样的鉴定方法快速且简单，无需大量实验室装置。此外，所述方法应当提供使得能够明确鉴定原种事件而无需专家解释，但必要时经历得起专家的细查的结果。本文中描述了用于鉴定生物样品中的原种事件EE-GM3的特殊工具。
在本发明中，EE-GM3已在耐除草剂大豆(大豆(Glycinemax))的开发中被鉴定为来自转基因大豆植物群体的良种事件，所述耐除草剂大豆包含与赋予对4-羟基苯丙酮酸二氧合酶(HPPD)抑制剂的抗性的基因组合的编码草甘膦抗性的基因，所述基因各自于植物可表达启动子的控制之下。
本领域已公开了包含耐除草剂基因的大豆植物。然而，现有技术公开内容没有一个教导或提出本发明。
在本领域中已知在大豆植物中获得具有可接受的农艺性状、无产量抑制现象(yielddrag)以及提供足够的除草剂抗性(当然针对2个不同种类的除草剂)的商业耐除草剂原种转化事件绝不简单容易。
事实上，已报导市场上发布的具有耐除草剂的第一大豆植物(事件40-3-2)与(近-)等基因系(isogenicline)相比较具有显著的产量抑制现象(Elmore等(2001)Agron.J.93:408-412)。
同样地，OptimumGAT(TM)大豆经产生将对草甘膦的抗性与对ALS除草剂的抗性组合，但它们的开发者已报导此类大豆本身不满足草甘膦抗性的标准(无与另一种草甘膦抗性大豆事件(例如事件40-3-2)的组合(参见例如，www.bloomberg.com/apps/news?pid=newsaixhive&sid=ad4L0hH9MKWE))。
本发明的优选实施方案的概述
本发明涉及转基因大豆植物、其种子、细胞或组织，所述转基因大豆植物、其种子、细胞或组织包含稳定地整合入其基因组的表达盒(所述表达盒包含含有2mEPSPS基因的编码序列的耐除草剂基因和含有HPPD-PfW336的编码序列的另一种耐除草剂基因(如本文实施例1.1中描述和SEQIDNo1中所示的两个基因))，所述转基因大豆植物或其种子、细胞或组织耐受基于草甘膦以及HPPD抑制剂除草剂例如异噁唑草酮，并且在除草剂不存在的情况下，具有大体上与非转基因等基因系相当的农艺性状。在施用一种或多种提供了针对其的抗性的除草剂后，植物将具有与非转基因植物相比较更优的农艺表型。
根据本发明，大豆植物或其种子、细胞或组织包括原种事件EE-GM3。
更具体地，本发明涉及转基因大豆、其种子、细胞或组织，其基因组DNA的特征在于这样的事实：当在本文中描述的PCR鉴定方案中进行分析时，使用分别针对EE-GM3的和包含耐除草剂基因的外源DNA的5’或3’侧翼区域的两个引物，产生对于EE-GM3有特异性的片段。引物可分别针对SEQIDNO：2内的5'侧翼区域和包含耐除草剂基因的外源DNA的5'侧翼区域。引物还可分别针对SEQIDNO：3内的3'侧翼区域和包含耐除草剂基因的外源DNA的3'侧翼区域，例如分别包含SEQIDNO：5和SEQIDNO：4或SEQIDNo.：7的核苷酸序列或(基本上)由所述核苷酸序列组成的引物，并且产生100至800bp的DNA片段，例如约263bp或706bp的片段。
包含本发明的原种事件的参照种子已在登录号NCIMB41659下保藏在NCIMB。本发明的一个实施方案是以NCIMB登录号NCIMB41659保藏的包含原种事件EE-GM3的种子，所述种子将生长成耐受除草剂，特别地耐受草甘膦和/或HPPD抑制剂例如异噁唑草酮的大豆植物。NCIMB保藏号NCIMB41659的种子是由至少约95%的包含本发明的原种事件的转基因种子组成的对于转移的DNA是纯合的种子批，所述转基因种子将生长成耐除草剂植物，其中所述植物耐受草甘膦和/或异噁唑草酮。可播种可获自保藏的种子或从所述种子获得的种子或后代种子(例如，在与其它具有不同遗传背景的大豆植物杂交后)，可用本文中描述的草甘膦或异噁唑草酮处理正在生长的植物以获得100%的耐受草甘膦或异噁唑草酮的植物，所述植物包含本发明的原种事件。本发明还涉及来自包含本发明的原种事件的植物的细胞、组织、子代和后代，所述植物可从保藏在NCIMB的具有登录号NCIMB41659的种子生长而来。本发明还涉及可从包含本发明的原种事件的大豆植物(例如从保藏在NCIMB的具有登录号NCIMB41659的种子生长而来的植物)获得(例如通过所述大豆植物的繁殖和/或与其繁育)的植物。本发明还涉及包含原种事件EE-GM3的大豆植物。
本发明还涉及用于鉴定包含原种事件EE-GM3的转基因、其细胞或组织的方法，所述方法基于鉴定表征DNA序列或由这样的DNA序列编码的氨基酸序列在转基因植物、细胞或组织中的存在。根据本发明的优选实施方案中，这样的表征DNA序列为包含事件的插入位点的15bp或至少15bp、优选20bp或至少20bp，最优选30bp或更多碱基对的序列，即包含耐除草剂基因的插入的外源DNA的一部分和与其邻接的大豆基因组的一部分(5'或3'侧翼序区域)，从而允许特异性鉴定原种事件。本发明还涉及包含本文中鉴定的事件EE-GM3的植物。
本发明还涉及用于鉴定生物样品中的原种事件EE-GM3的方法，所述方法基于特异性识别EE-GM3的包含耐除草剂基因的外源DNA的5'和/或3'侧翼序列的引物或探针。
更具体地，本发明涉及这样的方法，所述方法包括使用利用至少两个引物的聚合酶链式反应扩增存在于生物样品中的核酸，优选以获得100至800bp的DNA片段，所述引物之一识别EE-GM3的包含耐除草剂基因的外源DNA的5’或3’侧翼区域，另一个引物识别包含耐除草剂基因的外源DNA内的序列。引物可分别识别EE-GM3的5'侧翼区域内的序列(SEQIDNo.2，从位置1至位置1451)或EE-GM3的3'侧翼区域内的序列(SEQIDNo3的位置241至位置1408的互补序列)和包含耐除草剂基因的外源DNA内的序列(SEQIDNo2的位置1452至1843或SEQIDNo3的位置1至位置240的互补序列)。识别3'侧翼区域的引物可包含SEQIDNo.5的核苷酸序列，并且识别包含耐除草剂基因的外源DNA内的序列的引物可包含本文中描述的SEQIDNo.4或SEQIDNo.7的核苷酸序列。本发明还涉及特定引物和使用此类引物扩增的特定DNA，如本文中所描述的。
本发明更具体地涉及用于鉴定生物样品中的原种事件EE-GM3的方法，所述方法包括使用聚合酶链式反应，利用至少两个分别包含SEQIDNo.4和SEQIDNo.5的核苷酸序列或(基本上)由所述核苷酸序列组成的引物(以获得约263bp的DNA片段)或利用两个分别包含SEQIDNo.5和SEQIDNo.7的核苷酸序列或(基本上)由所述核苷酸序列组成的引物(以获得约706bp的DNA片段)扩增存在于生物样品中的核酸的序列。同样地，包含由此鉴定的原种事件EE-GM3的植物包括在本发明中。
本发明还涉及本文中描述的EE-GM3的特异性侧翼序列，所述序列可用于开发鉴定生物样品中的EE-GM3的特异性鉴定方法。此类特异性侧翼序列还可在鉴定测定中用作参照对照材料。更具体地，本发明涉及EE-GM3的5'和/或3'侧翼区域，所述区域可用于开发本文中进一步描述的特异性引物和探针。还适合用作参照材料的是优选约150-850bp的核酸分子，所述核酸分子包含可利用包含SEQIDNo.7和SEQIDNo.5的核苷酸序列或SEQIDNo.4和SEQIDNo.5的核苷酸序列或(基本上)由所述核苷酸序列组成的引物扩增的序列。
本发明还涉及基于此类特异性引物或探针的使用鉴定EE-GM3在生物样品中的存在的鉴定方法。引物可包含与引物(所述引物包含选自SEQIDNo2的核苷酸序列的17至约200个连续核苷酸的核苷酸序列，例如选自SEQIDNo2的核苷酸1452至核苷酸1843的核苷酸序列的互补序列或SEQIDNo3的核苷酸1至核苷酸240的核苷酸序列的17至约200个连续核苷酸的核苷酸序列或由或基本上由所述核苷酸序列组成)组合的选自SEQIDNo2的核苷酸1至核苷酸1451的核苷酸序列或SEQID3的核苷酸241至核苷酸1408的核苷酸序列的互补序列的17至约200个连续核苷酸的核苷酸序列或由或基本由所述核苷酸序列组成。引物还可包含位于它们最3'末端的这些核苷酸序列，并且还包含无关序列或来源于提及的核苷酸序列但包含错配的序列。
本发明还涉及用于鉴定生物样品中的原种事件EE-GM3的试剂盒，所述试剂盒包括至少一种特异性识别EE-GM3的包含耐除草剂基因的外源DNA的5’或3’侧翼区域的引物或探针。
本发明的试剂盒，除了特异性识别EE-GM3的5’或3’侧翼区域的引物外，还可包括特异性识别EE-GM3的包含耐除草剂基因的外源DNA内的序列的第二引物以用于PCR鉴定方案。本发明的试剂盒可包括至少两个特定引物，所述引物之一特异性识别EE-GM3的5’侧翼区域内的序列，另一个引物识别包含耐除草剂基因的外源DNA内的序列。识别3'侧翼区域的引物可包含SEQIDNo.5的核苷酸序列并且识别包含耐除草剂基因的转基因或外源DNA的引物可包含SEQIDNo.4或7的核苷酸序列，或本文中描述的任何其它引物或引物组合。
本发明还涉及用于鉴定生物样品中的原种事件EE-GM3的试剂盒，所述试剂盒包括包含SEQIDNo.5和SEQIDNo.4的核苷酸序列或(基本上)由所述核苷酸序列组成的PCR引物，以用于本文中描述的EE-GM3PCR鉴定方案。
本发明还涉及用于鉴定生物样品中的原种事件EE-GM3的试剂盒，所述试剂盒包括特异性探针，所述探针包含对应于与EE-GM3特异性区域具有80%至100%的序列同一性的序列(或与所述序列互补)的序列或(基本上)由所述序列组成。优选地，所述探针的序列对应于包含EE-GM3的5’或3’侧翼区域的部分的特异性区域。最优选地，所述特异性探针包含与SEQIDNo2的核苷酸1431至1472之间的序列具有80%至100%的序列同一性的序列或与IDNo.3的核苷酸220至260之间的序列具有80%至100%的序列同一性的序列或(基本上)由所述序列组成(或与所述序列互补)。
根据本发明的另一个方面，公开了DNA序列，所述DNA序列包含事件的插入位点和足够长度的大豆基因组DNA和包含耐除草剂基因(转基因)的外源DNA的多核苷酸，以用作用于检测EE-GM3的引物或探针以及表征包含事件EE-GM3的植物。此类序列可分别在连接位点的每一侧包含大豆基因组DNA的至少9个核苷酸和EE-GM3的包含耐除草剂基因的外源DNA的相似数目的核苷酸。最优选地，此类DNA序列包含大豆基因组DNA的至少9个核苷酸和与SEQIDNO：2或SEQIDNO：3中的插入位点邻接的包含耐除草剂基因的外源DNA的相似数目的核苷酸。在本发明的一个方面，提供了包含此类特定DNA序列的大豆植物。
本发明包括的方法和试剂盒可用于不同目的例如但不限于下列目的：鉴定EE-GM3在植物、植物材料或产物例如但不限于包含植物材料或来源于植物材料的食品或饲料产品(新鲜的或加工的)中的存在或测定其(下限)阈值；此外或可选择地，为了分离转基因与非转基因材料，可将本发明的方法和试剂盒用于鉴定转基因植物材料；此外或可选择地，可将本发明的方法和试剂盒用于测定包含EE-GM3的植物材料的质量(即，纯材料百分比)。
本发明还涉及与EE-GM3的5'和/或3'侧翼区域，以及涉及从EE-GM3的5'和/或3'侧翼序列开发的特异性引物和探针。
本发明还涉及从包含原种事件EE-GM3的植物获得的基因组DNA。此类基因组DNA可在本文中描述的鉴定测定中用作参照对照材料。
本文中还提供了转基因耐除草剂大豆植物或其细胞、部分、种子或后代，其各自包含至少一个原种事件，所述原种事件包含外源DNA，所述外源DNA包含：
i)包含来自玉蜀黍的在植物可表达启动子的控制下编码耐草甘膦的EPSPS酶的修饰epsps基因的第一嵌合基因，和
ii)包含来自荧光假单胞菌(Pseudomonasfluorescen)的在植物可表达的启动子控制之下编码耐HPPD抑制剂除草剂的酶的修饰hppd基因的第二嵌合基因。
在一个实施方案中，所述原种事件包含紧邻所述外源DNA的上游和与其邻接的SEQIDNo2的核苷酸1至1451以及紧邻所述外源DNA的下游和与其邻接的SEQIDNo3的核苷酸241至1408。
在其它实施方案中，所述原种事件可通过与从已在保藏号NCIMB41659下保藏在NCIMB的包含所述事件的参照种子生长而来的大豆植物繁育获得。
在另一个实施方案中，所述大豆植物、其细胞、部分、种子或后代的基因组DNA，当使用原种事件鉴定方案，利用两个分别包含SEQIDNo4和SEQIDNo5的核苷酸序列分析其所述原种事件时，产生(约)263bp的DNA片段。
本文中还提供了用于鉴定生物样品中的耐草甘膦和/或HPPD抑制剂除草剂(例如异噁唑草酮)的转基因大豆植物或其细胞、部分、种子或后代的方法，所述方法包括使用聚合酶链式反应，利用至少两个引物从存在于生物样品中的核酸扩增100至500bp的DNA片段，所述引物之一识别上面指定的原种事件的5'侧翼区域，所述5'侧翼区域包含SEQIDNo2的核苷酸1至核苷酸1451的核苷酸序列，或识别所述原种事件的3'侧翼区域，所述3'侧翼区域包含SEQIDNo3的核苷酸241至核苷酸1408的互补序列的核苷酸序列，所述引物的另一个引物识别外源DNA内的序列，所述外源DNA包含SEQIDNo2的核苷酸1452至核苷酸1843的互补序列的核苷酸序列或SEQIDNo3的核苷酸1至核苷酸240的核苷酸序列。
本文中还提供了用于鉴定生物样品中的耐草甘膦和/或HPPD抑制剂除草剂(例如异噁唑草酮)的转基因大豆植物或其细胞、部分、种子或后代的试剂盒，所述试剂盒包括一种识别上文中指定的原种事件的5'侧翼区域的引物，所述5'侧翼区域包含SEQIDNo2的核苷酸1至核苷酸1451的核苷酸序列，或一种识别所述原种事件的3'侧翼区域的引物，所述3'侧翼区域包含SEQIDNo3的核苷酸241至核苷酸3408的互补序列的核苷酸序列，和一种识别所述外源DNA内的序列的引物，所述外源DNA包含SEQIDNo.2的核苷酸1452至核苷酸1843的互补序列的核苷酸序列或SEQIDNo3的核苷酸1至核苷酸240的核苷酸序列。
在本发明的一个实施方案中，如本文中所用的原种事件EE-GM3的外源DNA包含SEQIDNo11的核苷酸位置1452至核苷酸位置16638的核苷酸序列或其互补序列，或包含与SEQIDNo11的核苷酸位置1452至核苷酸位置16638的核苷酸序列或其互补序列具有至少95%、98%、99%或99.5%的序列同一性的序列。
本文中还提供了在它们的基因组中包含核酸分子的大豆植物、植物细胞、组织或种子，所述核酸分子包含与SEQIDNo.11的核苷酸位置1452至核苷酸位置16638的核苷酸序列或其互补序列具有至少97%、98%或至少99%的序列同一性的核苷酸序列或与SEQIDNo.11或其互补序列具有至少97%、98%或至少99%的序列同一性的核苷酸序列。
本发明的一个实施方案提供在它们的基因组中包含核酸分子的大豆植物、植物细胞、组织或种子，所述核酸分子与SEQIDNo1的核苷酸序列或其互补序列杂交，或与SEQIDNo.11的核苷酸位置1452至核苷酸位置16638的核苷酸序列或其互补序列杂交，或与SEQIDNo.11的核苷酸序列或其互补序列杂交。
本文中还提供了分离的核酸分子，所述核酸分子包含与SEQIDNo.11的核苷酸位置1452至核苷酸位置16638的核苷酸序列或其互补序列具有至少99%的序列同一性的核苷酸序列，或与SEQIDNo.11或其互补序列具有至少99%的序列同一性的核苷酸序列，或分离的核酸分子包含与SEQIDNo.11的核苷酸位置1452至核苷酸位置16638的核苷酸序列或其互补序列杂交的核苷酸序列或与SEQIDNo.11的核苷酸序列或其互补序列杂交的核苷酸序列。
附图简述
可结合附图(通过引用并入本文)理解无意将本发明限定于描述的特定实施方案的下列实施例，其中：
图1：引用的核苷酸序列与引物之间的关系的示意图。黑色条：外源DNA；阴影条：植物来源的DNA；格纹箭头(a)：嵌合HPPDPfW366编码基因(关于嵌合基因的组成参见表1)；阴影箭头(b)：嵌合2mEPSPS编码基因(关于嵌合基因的组成参见表1)；黑色箭头：寡核苷酸引物，条下的数字表示核苷酸位置；(c)是指显示的核苷酸序列的互补序列；注意：示意图未按规定比例绘制。
图2：通过针对EE-GM3开发的PCR鉴定方案获得的结果。凝胶的上样序列：泳道1：分子量标准(100bp梯带)；泳道2和3：来自包含转基因事件EE-GM3的大豆植物的DNA样品；泳道4-7：来自未包含原种事件EE-GM3但包含相同的耐除草剂基因(其它转化事件)的转基因大豆植物的DNA样品；泳道8：来自野生型大豆的DNA样品；泳道9：无模板DNA对照；泳道10：分子量标准。
图3：通过针对EE-GM3开发的接合性评分PCR方案获得的结果。凝胶的上样序列：泳道1：分子量标准(100bp梯带)；泳道2和5：来自包含以纯合子形式存在的转基因事件EE-GM3的大豆植物的DNA样品；泳道3、8和9：来自包含以杂合子形式存在的转基因事件EE-GM3的大豆植物的DNA样品；泳道4、6和7：来自不成对的(azygous)大豆的DNA样品；泳道10：无模板DNA对照；泳道11：分子量标准。
本发明的优选实施方案的详细描述
重组DNA分子在植物基因组中的组合通常由细胞或组织的转化产生。整合的具体位点通常归因于随机整合。
作为用重组DNA或“转化DNA”转化植物细胞或组织的结果而被引入植物基因组的和源自这样的转化DNA的DNA在下文中称为包含一个或多个“转基因”的“外源DNA”。EE-GM3的转基因是草甘膦和HPPD抑制剂耐除草剂基因。在本发明的说明书中“植物DNA”将指源自已被转化的植物的DNA。植物DNA通常见于对应的野生型植物的相同基因座中。外源DNA的特征可在于重组DNA分子在植物基因组中整合的位点上的定位和构型。其中已插入了重组DNA的植物基因组中的位置也称为“插入位点”或“靶位点”。可将重组DNA至称为“插入前植物DNA”的植物基因组的区域中的插入与植物DNA的缺失(称为“靶位点缺失”)结合。如本文中所用，“侧翼区域”或“侧翼序列”是指与引入的DNA不同的至少20bp，优选至少50bp和达到5000bp的DNA的序列，优选来自植物基因组的恰好位于外源DNA的上游并且与其邻接或恰好位于外源DNA的下游并且与其邻接的DNA。导致外源DNA的随机整合的转化方法将导致具有不同侧翼区域的转化体，所述侧翼区域对于每一个转化体是特征性的和独特的。当通过常规杂交将重组DNA引入植物时，其在植物基因组中的插入位点或其侧翼区域将通常不被改变。
如本文中所用，“分离的核酸(序列)”或“分离的DNA(序列)”是指不再存在于其所分离自的天然环境中的核酸或DNA(序列)，例如，在另一种细菌宿主或植物基因组中的核酸序列，或融合至来自另一种来源的DNA或核酸的核酸或DNA，例如当包含在处于植物可表达启动子的控制之下的嵌合基因中时。
事件定义为(人工)基因座，所述基因座，作为基因工程的结果，携带包含至少一个拷贝的目标基因的外源DNA或转基因。事件的常见等位基因状态为外源DNA的存在或不存在。事件的表型特征在于转基因的表达。在基因水平上，事件是植物的基因组成的部分。在分子水平上，事件的特征在于限制性图谱(例如，通过Southern印迹确定的)，在于转基因的上游和/或下游侧翼序列、分子标志的定位和/转基因的分子构型。通常地，用包含至少一个目标基因的DNA转化植物产生包含许多分离事件的转化体群体，所述每一个转化体是独特的。事件的特征在于外源DNA和侧翼序列的至少一个序列。
如本文中所用，原种事件是基于转基因的表达和稳定性以及其与包含其的植物的最佳农艺特征的相容性，从通过利用相同转化DNA的转化获得的一组事件选择的事件。因此用于原种事件选择的标准是如下的一项或多项，优选两项或更多项，有利地所有项：
a)外源DNA的存在不削弱植物的其它期望的特征，例如与农艺性状或商业价值相关的特征；
b)事件的特征在于明确确定的分子构型，所述分子构型可稳定地遗传并且可针对其开发用于身份控制(identitycontrol)的适当工具；
c)目标基因在携有所述事件的植物可能在正常农艺应用中所暴露的许多环境条件下，以商业可接受的水平在事件的杂合子(或半合子)和纯合子条件中都显示正确的、适当的和稳定的空间和时间表型表达。
优选地，将外源DNA与植物基因组中的位置连接，所述位置使得能够容易地进行至期望的商业遗传背景中的基因渗入。
通过在不同的相关遗传背景中进行原种事件的基因渗入，然后观察对1、2个或所有标准例如上述a)、b)和c)的顺应性来将事件的状态确认作为原种事件。
因此“原种事件”是指满足上述标准的包含外源DNA的基因座。植物、植物材料或后代例如种子可在其基因组中包含一个或多个原种事件。
开发用于鉴定原种事件或包含原种事件的植物或植物材料或包含含有原种事件的植物材料的产物的工具基于原种事件的特定基因组特征，例如包含外源DNA的基因组区域的特定限制性图谱、分子标志或外源DNA的侧翼区域的序列。
一旦已对外源DNA的一个或两个侧翼区域进行了测序，则可利用分子生物学技术开发特异性识别样品的核酸(DNA或RNA)中的该(这些)序列的引物和探针。例如，可开发PCR法来鉴定生物样品(例如植物、植物材料或包含植物材料的产物的样品)中的原种事件。这样的PCR基于至少两个特异性“引物”，一个识别原种事件的5’或3’侧翼区域内的序列并且另一个识别外源DNA内的序列。引物优选具有15至35个核苷酸的序列，所述序列在最优化的PCR条件下分别“特异性识别”原种事件的5’或3’侧翼区域和原种事件的外源DNA内的序列，以便从包含原种事件的核酸样品扩增特异性片段(“整合片段”或鉴别扩增子(discriminatingamplicon))。这意味着在最优化的PCR条件下只有靶向整合片段而无植物基因组或外源DNA中的其它序列被扩增。
适用于本发明的PCR引物可以是下列序列：:
-长度范围为17nt至约200nt的寡核苷酸，其在它们的3'末端上包含选自5'侧翼序列(SEQIDNo2的核苷酸1至核苷酸1451)中的植物DNA的至少17个连续核苷酸，优选20个连续核苷酸的核苷酸序列(识别5'侧翼序列的引物)；或
-长度范围为17nt至约200nt的寡核苷酸，其在它们的3'末端上包含选自3'侧翼序列(SEQIDNo3的核苷酸241至核苷酸1408的互补序列)中的植物DNA的至少17个连续核苷酸，优选20个连续核苷酸的核苷酸序列(识别3'侧翼序列的引物)；或
-长度范围为17nt至约200nt的寡核苷酸，其在它们的3'末端上包含选自插入的DNA序列(SEQIDNo2的核苷酸1452至核苷酸1843的互补序列)的至少17个连续核苷酸，优选20个连续核苷酸的核苷酸序列(识别外源DNA的引物)；或
-长度范围为17nt至约200nt的寡核苷酸，其包含选自插入的DNA序列(SEQIDNo3的核苷酸1至核苷酸240)的至少17个连续核苷酸，优选20个连续核苷酸的核苷酸序列；或
-长度范围为17nt至约200nt的寡核苷酸，其包含选自插入的DNA片段或其互补序列(SEQIDNo1或SEQIDNo11的核苷酸位置1452至16638)的核苷酸序列的至少17个连续核苷酸，优选20个连续核苷酸的核苷酸序列。
所述引物当然可以比提及的17个连续核苷酸长，可以例如为20、21、30、35、50、75、100、150、200nt长或甚至更长。引物可完全由选自提及的侧翼序列和外源DNA序列的核苷酸序列的核苷酸序列组成。然而，引物在它们的5'末端(即定位在3'的17个连续核苷酸之外的)上的核苷酸序列不太重要。因此，引物的5'序列可视情况而定包含选自侧翼序列或外源DNA的核苷酸序列或由所述序列组成，但可包含几个(例如，1、2、5或10)错配。引物的5'序列甚至可完全为与侧翼序列或外源DNA无关的核苷酸序列，例如代表一个或多个限制性内切酶识别位点的核苷酸序列。此类无关序列或具有错配的侧翼DNA序列应当优选不长于100，更优选不长于50或甚至25个核苷酸。
此外，适当的引物可在它们的3'末端包含横跨植物DNA源性序列与外源DNA序列之间的连接区(位于SEQIDNo2的核苷酸1451-1452和SEQIDNo3的核苷酸240-241上)的核苷酸序列或基本上由所述核苷酸序列组成，只要提及的定位在3'的17个连续核苷酸不仅仅来源于SEQIDNo2或3中的外源DNA或植物源性序列。
对于本领域技术人员也立即明白的是恰当选择的PCR引物不应当包含彼此互补的序列。
为了本发明的目的，“SEQIDNo：X中所示的核苷酸序列的互补序列”是可通过按照沙加夫法则用它们的互补核苷酸替代核苷酸并且以5'至3'方向，即以所示的核苷酸序列的相反方向读取序列从所示核苷酸序列衍生的核苷酸序列。
适用于引物的实例是SEQIDNo5(3'侧翼序列识别引物)、SEQIDNo4(与3'侧翼序列识别引物一起使用的外源DNA识别引物)或SEQIDNo7(与3'侧翼序列识别引物一起使用的外源DNA识别引物)的寡核苷酸序列。
合适的寡核苷酸引物的其它实例在它们的3'末端包含下列序列或(基本上)由此类序列组成：
a.5'侧翼序列识别引物：
-SEQIDNo2的核苷酸264至核苷酸283的核苷酸序列
-SEQIDNo2的核苷酸266至核苷酸285的核苷酸序列
-SEQIDNo2的核苷酸1240至核苷酸1259的核苷酸序列
-SEQIDNo2的核苷酸265至核苷酸285的核苷酸序列
-SEQIDNo2的核苷酸265至核苷酸283的核苷酸序列
-SEQIDNo2的核苷酸1239至核苷酸1259的核苷酸序列
-SEQIDNo2的核苷酸1241至核苷酸1259的核苷酸序列
-SEQIDNo2的核苷酸1244至核苷酸1263的核苷酸序列
-SEQIDNo2的核苷酸1248至核苷酸1267的核苷酸序列
-SEQIDNo2的核苷酸1250至核苷酸1269的核苷酸序列
-SEQIDNo2的核苷酸262至核苷酸279的核苷酸序列
-SEQIDNo2的核苷酸263至核苷酸279的核苷酸序列
-SEQIDNo2的核苷酸264至核苷酸285的核苷酸序列
-SEQIDNo2的核苷酸266至核苷酸283的核苷酸序列
-SEQIDNo2的核苷酸1238至核苷酸1259的核苷酸序列
-SEQIDNo2的核苷酸1242至核苷酸1259的核苷酸序列
-SEQIDNo2的核苷酸1243至核苷酸1263的核苷酸序列
-SEQIDNo2的核苷酸1245至核苷酸1263的核苷酸序列
-SEQIDNo2的核苷酸1247至核苷酸1267的核苷酸序列
-SEQIDNo2的核苷酸1249至核苷酸1269的核苷酸序列
-SEQIDNo2的核苷酸1249至核苷酸1267的核苷酸序列
-SEQIDNo2的核苷酸263至核苷酸285的核苷酸序列
-SEQIDNo2的核苷酸267至核苷酸283的核苷酸序列
-SEQIDNo2的核苷酸1242至核苷酸1263的核苷酸序列
-SEQIDNo2的核苷酸1243至核苷酸1259的核苷酸序列-
-SEQIDNo2的核苷酸1246至核苷酸1267的核苷酸序列
-SEQIDNo2的核苷酸1246至核苷酸1263的核苷酸序列
-SEQIDNo2的核苷酸1248至核苷酸1269的核苷酸序列
-SEQIDNo2的核苷酸1250至核苷酸1271的核苷酸序列
-SEQIDNo2的核苷酸1250至核苷酸1267的核苷酸序列
-SEQIDNo2的核苷酸1241至核苷酸1263的核苷酸序列
-SEQIDNo2的核苷酸1245至核苷酸1267的核苷酸序列
-SEQIDNo2的核苷酸1247至核苷酸1269的核苷酸序列
-SEQIDNo2的核苷酸1247至核苷酸1263的核苷酸序列
-SEQIDNo2的核苷酸1249至核苷酸1271的核苷酸序列
-SEQIDNo2的核苷酸1242至核苷酸1261的核苷酸序列
-SEQIDNo2的核苷酸1241至核苷酸1261的核苷酸序列
-SEQIDNo2的核苷酸1243至核苷酸1261的核苷酸序列
-SEQIDNo2的核苷酸1240至核苷酸1261的核苷酸序列
-SEQIDNo2的核苷酸1244至核苷酸1261的核苷酸序列
-SEQIDNo2的核苷酸1239至核苷酸1261的核苷酸序列
-SEQIDNo2的核苷酸1245至核苷酸1261的核苷酸序列
b.与5'侧翼序列识别引物一起使用的外源DNA序列识别引物：
-SEQIDNo2的核苷酸1732至核苷酸1751的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1735至核苷酸1754的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1731至核苷酸1750的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1732至核苷酸1750的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1732至核苷酸1752的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1731至核苷酸1749的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1732至核苷酸1749的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1731至核苷酸1751的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1732至核苷酸1753的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1731至核苷酸1748的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1732至核苷酸1748的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1735至核苷酸1751的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1731至核苷酸1752的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1732至核苷酸1754的核苷酸序列的互补序列
SEQIDNo2的核苷酸1731至核苷酸1747的核苷酸序列的互补序列
SEQIDNo2的核苷酸1731至核苷酸1753的核苷酸序列的互补序列
SEQIDNo2的核苷酸1727至核苷酸1746的核苷酸序列的互补序列
SEQIDNo2的核苷酸1727至核苷酸1745的核苷酸序列的互补序列
SEQIDNo2的核苷酸1727至核苷酸1747的核苷酸序列的互补序列
SEQIDNo2的核苷酸1727至核苷酸1744的核苷酸序列的互补序列
SEQIDNo2的核苷酸1727至核苷酸1748的核苷酸序列的互补序列
SEQIDNo2的核苷酸1727至核苷酸1749的核苷酸序列的互补序列
SEQIDNo2的核苷酸1726至核苷酸1745的核苷酸序列的互补序列
SEQIDNo2的核苷酸1726至核苷酸1744的核苷酸序列的互补序列
SEQIDNo2的核苷酸1726至核苷酸1746的核苷酸序列的互补序列
SEQIDNo2的核苷酸1726至核苷酸1747的核苷酸序列的互补序列
SEQIDNo2的核苷酸1726至核苷酸1748的核苷酸序列的互补序列
SEQIDNo2的核苷酸1724至核苷酸1744的核苷酸序列的互补序列
SEQIDNo2的核苷酸1724至核苷酸1745的核苷酸序列的互补序列
SEQIDNo2的核苷酸1724至核苷酸1746的核苷酸序列的互补序列
SEQIDNo2的核苷酸1461至核苷酸1478的核苷酸序列的互补序列
SEQIDNo2的核苷酸1670至核苷酸1686的核苷酸序列的互补序列
SEQIDNo2的核苷酸1469至核苷酸1486的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1508至核苷酸1527的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1667至核苷酸1686的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1670至核苷酸1687的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1673至核苷酸1689的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1688至核苷酸1704的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1688至核苷酸1705的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1692至核苷酸1709的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1467至核苷酸1486的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1481至核苷酸1497的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1481至核苷酸1498的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1491至核苷酸1507的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1491至核苷酸1508的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1672至核苷酸1688的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1673至核苷酸1690的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1673至核苷酸1691的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1688至核苷酸1706的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1691至核苷酸1707的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1691至核苷酸1708的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1469至核苷酸1487的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1481至核苷酸1499的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1489至核苷酸1505的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1489至核苷酸1506的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1489至核苷酸1507的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1489至核苷酸1508的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1666至核苷酸1686的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1667至核苷酸1687的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1670至核苷酸1688的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1672至核苷酸1689的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1688至核苷酸1707的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1691至核苷酸1709的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1692至核苷酸1710的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1481至核苷酸1500的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1491至核苷酸1509的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1670至核苷酸1689的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1672至核苷酸1690的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1672至核苷酸1691的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1687至核苷酸1705的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1687至核苷酸1706的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1691至核苷酸1710的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1472至核苷酸1488的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1488至核苷酸1507的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1491至核苷酸1510的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1495至核苷酸1512的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1495至核苷酸1513的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1495至核苷酸1514的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1673至核苷酸1692的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1678至核苷酸1694的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1678至核苷酸1695的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1678至核苷酸1696的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1687至核苷酸1703的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1687至核苷酸1704的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1692至核苷酸1711的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1469至核苷酸1488的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1488至核苷酸1506的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1491至核苷酸1511的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1670至核苷酸1690的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1678至核苷酸1697的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1688至核苷酸1709的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1467至核苷酸1487的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1488至核苷酸1508的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1495至核苷酸1511的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1491至核苷酸1512的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1666至核苷酸1687的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1667至核苷酸1688的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1672至核苷酸1692的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1673至核苷酸1693的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1687至核苷酸1707的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1472至核苷酸1490的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1472至核苷酸1491的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1481至核苷酸1501的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1489至核苷酸1509的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1495至核苷酸1515的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1670至核苷酸1691的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1673至核苷酸1694的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1678至核苷酸1698的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1469至核苷酸1489的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1667至核苷酸1689的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1687至核苷酸1708的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1688至核苷酸1710的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1691至核苷酸1711的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1472至核苷酸1492的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1489至核苷酸1510的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1666至核苷酸1688的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1687至核苷酸1709的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1692至核苷酸1712的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1467至核苷酸1488的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1469至核苷酸1490的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1488至核苷酸1509的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1489至核苷酸1511的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1678至核苷酸1699的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1472至核苷酸1493的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1472至核苷酸1494的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1481至核苷酸1502的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1670至核苷酸1692的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1469至核苷酸1491的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1488至核苷酸1510的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1691至核苷酸1712的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1692至核苷酸1713的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1692至核苷酸1714的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1467至核苷酸1489的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1678至核苷酸1700的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1481至核苷酸1503的核苷酸序列的互补序列
-SEQIDNo2的核苷酸1691至核苷酸1713的核苷酸序列的互补序列
c.3'侧翼序列识别引物：
-SEQIDNo3的核苷酸828至核苷酸847的核苷酸序列的互补序列
-SEQIDNo3的核苷酸830至核苷酸849的核苷酸序列的互补序列
-SEQIDNo3的核苷酸828至核苷酸846的核苷酸序列的互补序列
-SEQIDNo3的核苷酸828至核苷酸848的核苷酸序列的互补序列
-SEQIDNo3的核苷酸830至核苷酸848的核苷酸序列的互补序列
-SEQIDNo3的核苷酸830至核苷酸850的核苷酸序列的互补序列
-SEQIDNo3的核苷酸828至核苷酸845的核苷酸序列的互补序列
-SEQIDNo3的核苷酸830至核苷酸847的核苷酸序列的互补序列
-SEQIDNo3的核苷酸828至核苷酸849的核苷酸序列的互补序列
-SEQIDNo3的核苷酸830至核苷酸851的核苷酸序列的互补序列
-SEQIDNo3的核苷酸828至核苷酸844的核苷酸序列的互补序列
-SEQIDNo3的核苷酸830至核苷酸846的核苷酸序列的互补序列
-SEQIDNo3的核苷酸828至核苷酸850的核苷酸序列的互补序列
-SEQIDNo3的核苷酸830至核苷酸852的核苷酸序列的互补序列
-SEQIDNo3的核苷酸992至核苷酸1009的核苷酸序列的互补序列
-SEQIDNo3的核苷酸731至核苷酸752的核苷酸序列的互补序列
-SEQIDNo3的核苷酸776至核苷酸795的核苷酸序列的互补序列
-SEQIDNo3的核苷酸731至核苷酸753的核苷酸序列的互补序列
-SEQIDNo3的核苷酸776至核苷酸794的核苷酸序列的互补序列
-SEQIDNo3的核苷酸776至核苷酸796的核苷酸序列的互补序列
-SEQIDNo3的核苷酸776至核苷酸793的核苷酸序列的互补序列
-SEQIDNo3的核苷酸776至核苷酸797的核苷酸序列的互补序列
-SEQIDNo3的核苷酸776至核苷酸792的核苷酸序列的互补序列
-SEQIDNo3的核苷酸776至核苷酸798的核苷酸序列的互补序列
-SEQIDNo3的核苷酸733至核苷酸752的核苷酸序列的互补序列
-SEQIDNo3的核苷酸733至核苷酸753的核苷酸序列的互补序列
-SEQIDNo3的核苷酸733至核苷酸754的核苷酸序列的互补序列
-SEQIDNo3的核苷酸733至核苷酸755的核苷酸序列的互补序列
-SEQIDNo3的核苷酸838至核苷酸854的核苷酸序列的互补序列
-SEQIDNo3的核苷酸246至核苷酸263的核苷酸序列的互补序列
-SEQIDNo3的核苷酸838至核苷酸855的核苷酸序列的互补序列
-SEQIDNo3的核苷酸245至核苷酸264的核苷酸序列的互补序列
d.与3'侧翼序列识别引物一起使用的外源DNA序列识别引物：
-SEQIDNo3的核苷酸173至核苷酸192的核苷酸序列
-SEQIDNo3的核苷酸22至核苷酸41的核苷酸序列
-SEQIDNo3的核苷酸172至核苷酸192的核苷酸序列
-SEQIDNo3的核苷酸174至核苷酸192的核苷酸序列
-SEQIDNo3的核苷酸191至核苷酸210的核苷酸序列
-SEQIDNo3的核苷酸171至核苷酸192的核苷酸序列
-SEQIDNo3的核苷酸175至核苷酸192的核苷酸序列
-SEQIDNo3的核苷酸190至核苷酸210的核苷酸序列
-SEQIDNo3的核苷酸192至核苷酸210的核苷酸序列
-SEQIDNo3的核苷酸176至核苷酸192的核苷酸序列
-SEQIDNo3的核苷酸189至核苷酸210的核苷酸序列
-SEQIDNo3的核苷酸193至核苷酸210的核苷酸序列
-SEQIDNo3的核苷酸188至核苷酸210的核苷酸序列
-SEQIDNo3的核苷酸194至核苷酸210的核苷酸序列
-SEQIDNo3的核苷酸199至核苷酸218的核苷酸序列
-SEQIDNo3的核苷酸200至核苷酸218的核苷酸序列
-SEQIDNo3的核苷酸197至核苷酸218的核苷酸序列
-SEQIDNo3的核苷酸201至核苷酸218的核苷酸序列
-SEQIDNo3的核苷酸201至核苷酸220的核苷酸序列
-SEQIDNo3的核苷酸200至核苷酸220的核苷酸序列
-SEQIDNo3的核苷酸199至核苷酸220的核苷酸序列
-SEQIDNo3的核苷酸200至核苷酸221的核苷酸序列
-SEQIDNo3的核苷酸199至核苷酸221的核苷酸序列
-SEQIDNo3的核苷酸150至核苷酸172的核苷酸序列
如本文中所用，“SEQIDNo.Z的位置X至位置Y的核苷酸序列”表示包含两个核苷酸终点的核苷酸序列。
优选地，扩增片段具有50至500个核苷酸的长度，例如，100至350个核苷酸的长度。特异性引物可具有分别与原种事件以及原种事件的外源DNA的5’或3’侧翼区域内的序列具有80至100%的同一性的序列，只要错配仍然允许在最优化的PCR条件下利用这些引物特异性鉴定所述原种事件。然而可允许的错配的范围可由本领域技术人员凭经验容易地确定或对于本领域技术人员来说是已知的。
整合片段的检测可以以各种方式进行，例如通过凝胶分析后进行大小评估。还可对整合片段进行直接测序。用于检测扩增的DNA片段的其它序列特异性方法在本领域也是已知的。
由于引物的序列及其在基因组中的相对位置对于原种事件是独特的，因此整合片段的扩增将只在包含原种事件(的核酸)的生物样品中发生。优选地，当进行PCR以鉴定原种事件EE-GM3在未知样品中的存在时，将对照包括在一组引物中，可利用所述引物扩增所述事件的植物物种的“管家基因”内的片段。管家基因是在大多数细胞类型中表达并且与对于所有细胞是共同的基础代谢活性有关的基因。优选地，从管家基因扩增的片段大于扩增的整合片段。取决于待分析的样品，可包括其它对照。
标准PCR方案描述于本领域中，例如描述于“PCR扩增手册”(RocheMolecularBiochemicals,第2版,1999)和其它参考资料中。在针对每一个原种事件的“PCR(或聚合酶链式反应)鉴定方案”中指定了用于PCR的最佳条件，包括特异性引物的序列。然而应理解，针对具体的实验条件可能需要调整PCR鉴定方案的许多参数，或略微改变所述参数以获得相似的结果。例如，使用不同的方法制备DNA可能需要调整例如所使用的引物、聚合酶的量和退火条件。类似地，其它引物的选择可确定用于PCR鉴定方案的其它最佳条件。然而这些调整对于本领域技术人员来说是显然的，并且在现有PCR应用手册例如上文中引用的手册中进行进一步详细说明。
或者，特异性引物可用于扩增可用作用于鉴定生物样品中的EE-GM3的“特异性探针”的整合片段。在允许探针与核酸中的其对应片段杂交的条件下将生物样品的核酸与探针接触，导致核酸/探针杂交体的形成。可检测(例如通过核酸或探针的标记)该杂交体的形成，其中该杂交体的形成表示存在EE-GM3。基于与特异性探针(在固相载体上或在溶液中)杂交的此类鉴定方法已在本领域中进行了描述。特异性探针优选是在最优化条件下与原种事件的5’或3’侧翼区域内的区域(优选还包含与其邻接的外源DNA的部分(在下文中称为“特异性区域”))特异性杂交的序列。优选，特异性探针包含与特异性区域的核苷酸序列具有至少80%、优选80至85%、更优选85至90%、特别优选90至95%、最优选95%至100%的同一性(或互补)的50至500bp，优选100至350bp的序列。优选地，所述特异性探针将包含与原种事件特异性区域具有同一性(或互补)的约15至约100个连续核苷酸的序列。
适合作为用于检测原种事件EE-GM3的PCR引物的寡核苷酸还可用于开发测定包含所述原种事件的植物的接合性状态的基于PCR的方案。为此目的，以使它们彼此相向并且具有位于引物之间的插入位点的方式设计识别整合之前的野生型基因座的两个引物。这些引物可以是分别特异性识别SEQIDNO2或3内包含的5'和3'侧翼序列的引物。这些引物还可以是特异性识别5'或3'侧翼序列的引物。对于本发明，识别整合之前的野生型基因座的特别合适的引物是包含SEQIDNo4和SEQID6的核苷酸序列或(基本上)由所述序列组成的引物。该组引物与与转化DNA序列互补的第三引物(例如包含SEQIDNo7的核苷酸序列或(基本上)由所述核苷酸序列组成的引物)一起允许EE-GM3特异性基因座以及野生型基因座的同时诊断PCR扩增。如果植物对于转基因座或相应的野生型基因座是纯合的，那么诊断PCR将对于转基因或野生型基因座产生通常，优选通常在长度上是单一的PCR产物。如果植物对于转基因基因座是半合子，两基因座特异性PCR产物将出现，反映了转基因和野生型基因座的扩增。
此外，还可使用本文中提供的原种事件特异性序列信息来开发对于原种事件EE-GM3是特异的检测方法，所述方法与基于PCR的扩增法不同。此类替代检测方法包括基于特定核酸结构的侵入切割(invasivecleavage)的线性信号放大检测法，也称为InvaderTM技术(如例如美国专利5,985,557“InvasiveCleavageofNucleicAcids”，6,001,567“DetectionofNucleicAcidsequencesbyInvaderDirectedCleavage”中所描述的，通过引用并入本文)。为了此目的，将靶序列与包含SEQIDNo2的核苷酸1452至核苷酸1469的核苷酸序列或其互补序列的标记的第一核酸寡核苷酸或包含SEQIDNo3的核苷酸223至核苷酸240的核苷酸序列或其互补序列的所述标记核酸探针杂交，并且还可与包含SEQIDNo2的核苷酸1434至核苷酸1451的核苷酸序列或其互补序列的第二核酸寡核苷酸或包含SEQIDNo3的核苷酸241至核苷酸258的核苷酸序列或其互补序列的所述标记核酸探针杂交，其中所述第一寡核苷酸与第二寡核苷酸重叠至少一个寡核苷酸。通过该杂交产生的双链体或三链体结构允许利用酶进行选择性探针切割，从而使靶序列保持完整。随后可能地通过导致进一步信号放大的中间步骤检测切割的标记探针。
如本文中所用，“试剂盒”是指为了进行本发明的方法，更具体地，鉴定生物样品中的原种事件EE-GM3或确定包含EE-GM3的植物材料的接合性状态而使用的一组试剂。更具体地，本发明的试剂盒的优选实施方案包括至少一个或两个如上所述用于鉴定原种事件的特异性引物，或用于确定接合性状态的三个特异性引物。任选地，试剂盒还可包括本文中描述的用于PCR鉴定方案的任何其它试剂。或者，根据本发明的另一个实施方案，试剂盒可包括上述特异性探针，其与生物样品的核酸特异性杂交以鉴定EE-GM3存在于其中。任选地，试剂盒还可包括用于使用特异性探针鉴定生物样品中的EE-GM3的任何其它试剂(例如但不限于杂交缓冲液、标记物)。
为了进行质量控制(例如，种子批的纯度)、检测原种事件在植物材料或包含或来源于植物材料的材料(例如但不限于食品或饲料产品)中的存在或不存在，可使用本发明的试剂盒，并且特别地可调整其组分。
如本文中所用，关于核苷酸序列(DNA或RNA)的“序列同一性”是指具有相同核苷酸的位置的数目除以两个序列的较短者中的核苷酸序列的数目。利用Wilbur和Lipmann算法(Wilbur和Lipmann,1983,Proc.Nat.Acad.Sci.USA80:726)，使用20个核苷酸的窗口大小、4个核苷酸的字长以及为4的空位罚分进行两个核苷酸序列的比对。可以例如使用GeneticsComputerGroup的序列分析软件包(GCG，UniversityofWisconsinBiotechnologyCenter)方便地进行序列数据的计算机辅助分析和解释，包括上文中描述的序列比对。当此类序列具有至少约75%、特别地至少约80%、更特别地至少约85%、更特别地至少约90%、特别地至少约95%、更特别地至少约98%或至少约99%的序列同一性时，序列被标示为“基本上相似”。很清楚，当RNA序列被认为与DNA基本上相似或与DNA序列具有一定程度的序列同一性时，DNA序列中的胸苷(T)被认为等同于RNA序列中的尿嘧啶(U)。同样地，很清楚小的差异或突变可随着时间过去在DNA序列中出现并且对于本发明的事件特异性引物或探针可允许一些错配，这样在本文中在本发明的任何实施方案中对于本发明的任何3'或5'侧翼DNA或任何插入物或外源DNA或任何引物或探针指定的任何DNA序列，也包括基本上与本文中提供的序列相似的序列，例如与本发明的任何3'或5'侧翼DNA、任何引物或探针或任何插入物或外源DNA的给定的序列杂交或与所述序列具有至少90%、95%、96%、97%、98%或至少99%的序列同一性的序列。
如本文中所用，术语“引物”包括能够以模板依赖性方法例如PCR引发新生核酸的合成的任何核酸。通常地，引物是10至30个核苷酸的寡核苷酸，但可使用更长的序列。可以以双链形式提供引物，尽管单链形式是优选的。探针可用作引物，但被设计用以结合靶DNA或RNA并且无需用于扩增过程中。
如本文中所用，当指特异性引物时，术语“识别”是指特异性引物在方法中所示的条件(例如PCR鉴定方案的条件)下与原始事件的核酸序列特异性杂交的事实，其中通过阳性对照和阴性对照的存在确定特异性。
如本文中所用，术语“杂交”当指特异性探针时，是指探针在标准严格条件下结合原种事件的核酸序列特异性区域的事实。如本文中所用，标准严格条件是指本文中描述的用于杂交的条件或指如由Sambrook等,1989(MolecularCloning：ALaboratoryManual,第2版,ColdSpringHarborLaboratoryPress,NY)描述的常规杂交条件，所述杂交条件例如可包括下列步骤：1)将植物基因组DNA片段固定在滤膜上，2)将滤膜在42℃下于50%甲酰胺、5XSSPE、2XDenhardt's试剂和0.1%SDS中预杂交1或2小时，或在68℃下于6XSSC、2XDenhardt's试剂和0.1%SDS中预杂交1至2小时，3)添加已被标记的杂交探针，4)温育16至24小时，5)在室温下于1XSSC、0.1%SDS中洗涤滤膜20分钟，6)在68℃下于0.2XSSC,0.1%SDS中洗涤滤膜3次，每次20分钟，和7)使用增感屏，将滤膜在-70℃下暴露于X-射线胶片24至48小时。
如本文中所用，生物样品是植物、植物材料或包含植物材料的产物的样品。术语“植物”意欲包括处于任何成熟阶段的大豆(大豆)植物组织以及获自或来源于任何这样的植物的任何细胞、组织或器官，包括但不限于任何种子、叶、茎、花、根、单细胞、配子、细胞培养物、组织培养物或原生质体。如本文中所用，“植物材料”是指获自或来源于植物的材料。包含植物材料的产物涉及食品、饲料或使用植物材料产生的或可被植物材料污染的其它产物。应理解，在本发明的说明书中，测试此类生物样品的对于EE-GM3有特异性的核酸的存在，所述核酸的存在暗示着核酸在样品中的存在。因此本文中提及的用于鉴定生物样品中的原种事件EE-GM3的方法涉及鉴定生物样品中的包含原种事件的核酸。
如本文中所用，“包含”将解释为指定提及的所述特征、整数、步骤、试剂或组分的存在，但不排除一个或多个特征、整数、步骤或组合分或其组群的存在或添加。因此，例如，包含核苷酸或氨基酸的序列的核酸或蛋白质可包含比实际上引述的核苷酸或氨基酸更多的核苷酸或氨基酸，即包括在更大的核酸或蛋白质中。包含功能或结构上确定的DNA序列的嵌合基因可包含额外的DNA序列，例如启动子和转录终止序列。
本发明还涉及在包含该事件的大豆植物、从这类植物获得的包含原种事件EE-GM3的后代植物和种子开发原种事件EE-GM3，和涉及从包含该事件的植物产生的植物细胞或植物材料。可如实施例1中所述获得包含原种事件EE-GM3的植物。本发明还涉及在保藏号NCIMB41659下保藏在NCIMB下的包含原种事件EE-GM3的种子或来源于其的包含原种事件EE-GM3的衍生物。如本文中所用，“(种子的)衍生物”是指可从这样的种子生长而来的植物，由杂交或回交而产生的后代，以及所述植物的植物细胞、器官、部分、组织、组织培养物、原生质体和植物材料。
可按照实施例2中针对EE-GM3所描述的PCR鉴定方案鉴定包含EE-GM3的大豆植物或植物材料。简而言之，使用特异性识别EE-GM3的5’或3’侧翼序列内的序列的引物例如具有SEQIDNO：5的序列的引物和识别外源DNA内的序列的引物例如具有SEQIDNO：4的序列的引物通过PCR扩增存在于生物样品中的大豆基因组DNA。扩增内源大豆序列的部分的DNA引物用作PCR扩增的阳性对照。当进行PCR扩增时，材料产生预期大小的片段，材料包含来自具有原种事件EE-GM3的大豆植物的植物材料。
具有EE-GM3的植物的特征在于它们的草甘膦抗性，以及在于它们对HPPD抑制剂例如异噁唑草酮的抗性。具有EE-GM3的植物的特征也在于在除草剂应用不存在的情况下具有可与商购获得的大豆品种相比较的农艺特征。已观察到，外源DNA在本文中描述的大豆植物基因组的插入区域中的存在赋予了包含该事件的植物特别令人感兴趣的表型和分子特征。
本发明的一个实施方案提供了大豆植物中的可通过在大豆基因组的特定位置插入2个转基因获得的原种事件，所述原种事件赋予此类大豆植物对草甘膦和HPPD抑制剂除草剂例如异噁唑草酮的抗性，其中与等基因系相比较(如本文中所用，“等基因系”或“近等基因系”是具有相同遗传背景但不存在转基因的大豆品系，例如具有与用于转化的植物相同的遗传背景的植物，或已失去转基因的分离姊妹系)，此类原种事件对此类大豆的农艺性状不产生负面影响此类大豆植物的产量的任何影响。具体地，本发明提供了大豆植物中的原种事件，其中与等基因系相比较，所述原种事件在此类大豆植物的基因组中的插入或存在在此类大豆植物中不引起增加对疾病的易感性，不引起产量抑制现象，或不引起增强的倒伏。因此，本发明在大豆植物中提供了称为EE-GM3的原种事件，所述组合产生可耐受草甘膦和HPPD抑制剂除草剂(同时或单独地)的施用而与等基因系相比较不负面影响所述大豆植物的产量的大豆植物，所述大豆植物在它们的疾病易感性或倒伏方面不具有统计上显著的差异，如等基因大豆植物。这些特征使得现有原种事件对控制大豆田中的耐草甘膦的杂草极具吸引力，并且还可用于在大豆田中防止或延迟其它耐草甘膦的发展(例如，通过施用草甘膦和异噁唑草酮，确保至少2个作用模式施用于大豆田)。
本文中还提供了包含事件EE-GM3的大豆植物或其部分，其中包含事件EE-GM3的代表性大豆种子已于NCIMB登录号41659下保藏。本文中还提供了包含该事件的此类植物的种子以及从此类种子产生的大豆产物，其中所述大豆产物包含事件EE-GM3。此类大豆产物可以是膳食、磨碎的种子、粉末、薄片等或可包括所述形式。特别地，这样的大豆产物包含产生诊断事件EE-GM3的扩增子或对于所述事件是特异的扩增子，例如包含SEQIDNo.2或3的扩增子的核酸。本文中还提供了用于产生大豆产物的方法，包括获得包含事件EE-GM3的大豆种子，和从其产生这样的大豆产物。
本文中还提供了大豆植物，其为任何上述大豆植物的后代，并且包含事件EE-GM3。
本文中还提供了用于产生耐草甘膦和/或异噁唑草酮除草剂的大豆植物的方法，其包括：具体地通过将缺乏事件EE-GM3的第一大豆植物与包含EE-GM3的大豆植物杂交，然后选择耐草甘膦和/或异噁唑草酮的后代植物来将事件EE-GM3引入这样的植物的基因组。
本文中还提供了耐草甘膦和/或异噁唑草酮植物，所述植物明确地不具有产量抑制现象，具有可接受的农艺特征，包含2mEPSPS和HPPD蛋白，并且能够产生诊断事件EE-GM3的扩增子。这样本文中还提供了特定的分离的扩增子(DNA序列片段)，其可使用本文中描述的特定检测工具来获得，特别地在它们的序列中包括源于植物DNA的DNA片段和对于该植物是外源的或异源的DNA片段例如通过转化插入植物基因组中的DNA的扩增子，如本文中所定义的。
本文中还提供了用于控制包含事件EE-GM3的大豆植物的田中或待种植此类大豆植物的田中的杂草的方法，包括用有效量的基于异噁唑草酮的除草剂处理田，其中此类植物抗此类除草剂。
本文中还提供了包含SEQIDNo1的序列或基本上与其相似的序列的DNA，以及包含此类DNA序列的任何植物、细胞、组织或种子(特别是大豆的)，例如包含EE-GM3，特别地含有2个相邻区域(所述区域包含SEQIDNo1或(基本上)由所述序列组成)的DNA，或包含2个相邻区域(所述区域包含一些核苷酸改变、缺失或添加的SEQIDNo1或由所述序列组成)的DNA，例如包含两个位于彼此接近(例如，相隔200-500nt或短于2000或10.000nt)的缺失、或置换了4、6、8或10个核苷酸的SEQIDNo1的DNA的植物、细胞、组织或种子。在一个实施方案中，这包括SEQIDNo.11的核苷酸位置2257至核苷酸位置16601的DNA，其中2、4、6、8或10个核苷酸已被其它核苷酸置换，或其中2、4、6、8或10个核苷酸已缺失或添加，或SEQIDNo.11的核苷酸位置2257至核苷酸位置16601的DNA以及SEQIDNo.11的DNA，以及包含此类DNA序列的任一个的任何植物、细胞、组织或种子(特别是大豆的)。本文中还包括任何大豆植物、细胞、组织或种子，其包含SEQIDNo.11的DNA序列(对于常规大豆植物、种子、组织或细胞是异源的或外源的)或包含SEQIDNo.11的核苷酸位置2257至核苷酸位置16601的DNA序列，或包含与SEQIDNo.11的序列具有至少99%或99.5%的序列同一性的DNA序列，或包含与SEQIDNo.11的核苷酸位置2257至核苷酸位置16601的序列具有至少99%或99.5%的序列同一性的DNA序列。
本文中还提供了在其基因组中包含事件EE-GM3的转基因大豆植物、植物细胞、组织或种子，所述事件EE-GM3特征在于包含与SEQIDNo.2的核苷酸1441至核苷酸1462基本上相似的核苷酸序列的核酸分子，以及包含与SEQIDNo.3的核苷酸230至251或所述序列的互补序列基本上相似的核苷酸序列的核酸分子，或在其基因组中包含特征在于事件EE-GM3的大豆植物、植物细胞、组织或种子，所述事件EE-GM3特征在于包含与SEQIDNo.2基本上相似的核苷酸序列的核酸分子，以及包含与SEQIDNo.3或所述序列的互补序列基本上相似的核苷酸序列的核酸分子。
本文中甚至还提供了包含EE-GM3的大豆植物、细胞、组织或种子，其特征在于在其细胞的基因组中包含与SEQIDNo.2的核苷酸1431至1472具有至少80%、90%、95%或100%的序列同一性的核酸序列和与SEQIDNo.3的核苷酸220至261具有至少80%、90%、95%或100%的序列同一性的核酸序列或所述序列的互补序列。
如本文中所用，术语“异噁唑草酮”是指除草剂异噁唑草酮[即(5-环丙基-4-异噁唑基)[2-(甲磺酰基)-4-(三氟甲基)苯基]甲酮]，其活性代谢物二酮腈以及包含所述化合物的任何混合物或溶液。对于对本发明的事件的应用是有用的HPPD抑制性除草剂为二酮腈，例如2-氰基-3-环丙基-1-(2-甲磺酰-4-三氟甲基苯基)-丙烷-1,3-二酮和2-氰基-1-[4-(甲磺酰基)-2-三氟甲基苯基]-3-(1-甲基环丙基)丙烷-1,3-二酮；其它异噁唑类；和吡唑啉盐类，例如苯吡唑草酮[即[3-(4,5-二氢-3-异噁唑基)-2-甲基-4-(甲磺酰基)苯基](5-羟基-1-甲基-1H-吡唑-4-基)甲酮]，和磺酰草吡唑(pyrasulfotole)[(5-羟基-1,3-二甲基吡唑-4-基(2-甲磺酰-4-三氟甲基苯基)甲酮]；或盐酸苯偶氮吡胺[2-[4-(2,4-二氯苯甲酰基)-1,3-二甲基吡唑-5-基氧基]乙酰苯]。
在本发明的一个实施方案中，在种植大豆植物或播种之前，可用HPPD抑制剂除草剂例如异噁唑草酮('IFT')处理待种植包含EE-GM3事件的大豆植物的田，所述除草剂可清除田中可被HPPD抑制剂杀死的杂草，从而使得能够实施免耕，然后，随后在该相同的预处理的田上种植或播种大豆(使用HPPD抑制剂除草剂的烧毁应用)。IFT的残余活性还将保护正在出芽和生长的大豆植物在早期生长阶段免受杂草的竞争。一旦大豆植物具有一定的大小，并且杂草有趋势重现，则可将草甘膦或HPPD抑制剂-草甘膦混合物作为芽后除草剂在植物的顶部上方施用。
在本发明的另一个实施方案中，可在大豆植物出芽前但在种子播种后(可在播种前使用其它方法，通常地常规耕地实践例如耕犁、凿式耕犁(chisselploughing)或苗床准备使田间无杂草)，用HPPD抑制剂除草剂例如IFT处理其中播种了包含EE-GM3事件的种子的田，其中残余活性将使田间保持无被除草剂杀死的杂草，以便正在出芽和生长的大豆植物没有杂草竞争(HPPD抑制剂除草剂的芽后施用)。一旦大豆植物具有一定的大小，并且杂草有趋势重现，则可将草甘膦或HPPD抑制剂-草甘膦混合物作为芽后除草剂在植物的顶部上方施用。
在本发明的另一个实施方案中，可在大豆植物(已从播种的种子出芽)的顶部上方用HPPD抑制剂除草剂例如IFT处理包含EE-GM3事件的植物，这可清除田中可被HPPD抑制剂杀死的杂草，可将该应用与利用作为芽后除草剂的草甘膦的处理一起(例如，在喷雾罐混合物中)，在其之前或之后于植物的顶部上方施用(HPPD抑制剂除草剂(具有或不具有草甘膦)的芽后施用)。
同样地，根据本发明，可用下列杀虫剂、除草剂或杀真菌剂处理具有EE-GM3的大豆植物，或可用包含下列杀虫剂、除草剂或杀真菌剂的包衣包被具有EE-GM3的大豆种子：
大豆除草剂：
甲草胺(Alachlor)、苯达松(Bentazone)、氟乐灵(Trifluralin)、氯嘧磺隆(Chlorimuron-Ethyl)、氯酯磺草胺(Cloransulam-Methyl)、噁唑禾草灵(Fenoxaprop)、氟磺胺草醚(Fomesafen)、吡氟禾草灵(Fluazifop)、草甘膦、甲氧咪草烟(Imazamox)、灭草喹(Imazaquin)、咪草烟(Imazethapyr)、(S-)丙草胺(Metolachlor)、嗪草酮(Metribuzin)、二甲戊灵(Pendimethalin)、得杀草(Tepraloxydim)、异噁唑草酮。
大豆杀虫剂：
λ-三氟氯氰菊酯(cyhalothrin)、灭多威(Methomyl)、对硫磷(Parathion)、硫双威(Thiocarb)、吡虫啉(Imidacloprid)、氯虫酰肼(Clothianidin)、噻虫嗪(Thiamethoxam)、噻虫啉(Thiacloprid)、啶虫脒(Acetamiprid)、呋虫胺(Dinetofuran,)、氟虫酰胺(Flubendiamide)、Rynaxypyr、Cyazypyr、多杀菌素(Spinosad)、乙基多杀菌素(Spinotoram)、埃玛菌素(Emamectin-Benzoate)、氟虫腈(Fipronil)、乙虫腈(Ethiprole)、溴氰菊酯(Deltamethrin)、β-氟氯氰菊酯(-Cyfluthrin)、γ和λ三氟氯氰菊酯、4-[[(6-氯吡啶-3-基)甲基](2,2-二氟乙基)氨基]呋喃-2(5H)-酮、螺虫乙酯(Spirotetramat)、螺螨酯(Spinodiclofen)、杀铃脲(Triflumuron)、氟啶虫酰胺(Flonicamid)、硫双威(Thiodicarb)、β-氟氯氰菊酯。
大豆杀真菌剂：
腈嘧菌酯(Azoxystrobin)、环丙唑醇(Cyproconazole)、氟环唑(Epoxiconazole)、粉唑醇(Flutriafol)、吡唑醚菌酯(Pyraclostrobin)、戊唑醇(Tebuconazole)、布洛芬、丙硫菌唑(Prothioconazole)、氟醚唑(Tetraconazole)。
下列实施例描述了原种事件EE-GM3的开发和鉴定、包含该事件的不同大豆品系的开发以及用于特异性鉴定生物样品中的原种事件EE-GM3的工具的开发。
除非在实施例中另外指出，否则按照“SambrookJ和RussellDW(编著)(2001)MolecularCloning：ALaboratoryManual,第3版,ColdSpringHarborLaboratoryPress,NewYork”和“AusubelFA,BrentR,KingstonRE,MooreDD,SeidmanJG，SmithJA和StruhlK(编著)(2006)CurrentProtocolsinMolecularBiology.JohnWiley&Sons,NewYork”中描述的标准方案进行所有重组技术。
标准材料和参考资料描述于“CroyRDD(编)(1993)PlantMolecularBiologyLabFax,BIOSScientificPublishersLtd.,OxfordandBlackwellScientificPublications,Oxford”和“BrownTA,(1998)MolecularBiologyLabFax,第2版,AcademicPress,SanDiego”中。用于聚合酶链式反应(PCR)的标准材料和方法可见于“McPhersonMJandMΦllerSG(2000)PCR(TheBasics),BIOSScientificPublishersLtd.,Oxford”和“PCRApplicationsManual,第3版(2006),RocheDiagnosticsGmbH,Mannheim或www.roche-applied-science.com”中。
应当理解，下列实施例中的任何实验室方案例如PCR方案中的许多参数可能需要针对具体实验室条件进行调整，并且可略作改进以获得相似结果。例如，使用不同方法制备DNA或在PCR法中选择其它引物可决定用于PCR方案的其它最佳条件。然而这些调整对于本领域技术人员来说是显然的，并且在现有PCR应用手册中进行进一步说明。
在说明书和实施例中，参考下列序列：
SEQIDNo.1：载体pSF10的SalI片段核苷酸序列。
SEQIDNo.2：包含侧翼连接EE-GM3的包含耐除草剂基因的外源DNA的5′区域的核苷酸序列。
SEQIDNo.3：包含侧翼连接EE-GM3的包含耐除草剂基因的外源DNA的3′区域的核苷酸序列。
SEQIDNo.4：引物SOY028
SEQIDNo.5：引物SOY029
SEQIDNo.6：引物SMP187
SEQIDNo.7：引物STV019
SEQIDNo.8：扩增子的核苷酸序列
SEQIDNo.9：用于扩增对照片段的引物1(SOY01)
SEQIDNo.10：用于扩增对照片段的引物的2(SOY02)
SEQIDNo.11：EE-GM3的外源DNA和植物侧翼序列的核苷酸序列
SEQIDNo.12:引物SHA130
SEQIDNo.13:引物SHA178
实施例
1.利用耐除草剂基因转化大豆
1.1.包含2mEPSPS和HPPD-Pf-W336嵌合基因的外源DNA的描述
质粒pSF10为pUC19衍生的克隆载体，其包含位于约7.3kb的SalI片段上的嵌合2mepsps基因和嵌合hppd-Pf-W336。两个SalI限制性位点之间包含的DNA的完全说明示于下表1中。核苷酸序列示于SEQIDNo.1中。
表1.pSF10的SalI限制性位点之间包含的DNA的核苷酸位置(SEQIDNo1)
1.2.事件EE-GM3
通过至大豆类型Jack的细胞内的直接基因转移将约7.3kb的HPLC纯化的SalI线性化的pSF10片段(包含耐2mEPSPS草甘膦基因和耐HPPD抑制剂基因HPPD-Pf-W336)用于获得转化的大豆植物(Nickell,C.D.,G.R.Noel,D.J.Thomas和R.Waller.Registrationof'Jack'soybean.CropSci1365.30.1990)，然后将转化的植物细胞再生成转基因能育大豆植物。
1.2.1原种事件EE-GM3的鉴定
基于广泛选择法选择原种事件EE-GM3，所述选择法基于耐除草剂基因的良好表达和稳定性，并且评估其与最佳农艺特征例如株高、节距(heighttonode)、直立(stand)、活力(vigor)、种子产量的相容性。从使用相同嵌合基因获得的许多不同转化事件选择含有该事件的大豆植物。该事件的选择中使用的参数为：a)对田间试验中异噁唑草酮除草剂施用的可接受的抗性，b)对田间试验中草甘膦除草剂施用的可接受的抗性，c)对田间试验的异噁唑草酮和草甘膦除草剂的组合施用的可接受的抗性，d)耐除草剂转基因在大豆植物基因组的单个基因座上的插入，载体主链不存在，e)与用于转化的亲本植物相似的总体农艺学(成熟度、倒伏、疾病易感性等)，和f)无因转化DNA的插入引起的产量抑制现象(与在相同条件下生长的无事件的等基因系例如用于转化的植物品系相比较时)。
在T3代，选择大豆转化事件EE-GM3的纯合品系的种子产量。在亲本品种Jack适应的地区进行多位点重复农艺田间研究(Multi-locationreplicatedagronomicfieldstudy)。田间评估包括除草剂抗性和农艺性状。发现包含EE-GM3的植物的农艺性能与Jack相当(当不施用除草剂时)。
田间评估还显示携带EE-GM3事件的植物具有：
-与Jack相比较相似的植物形态学和种子特征，
-与Jack相比较对大豆疾病的反应无变化，和
-与Jack相比较在种子萌发或休眠上无变化。
将从温室中的初始转化体(T0)植物(用构建体转化的植物，以产生事件EE-GM3)收获的种子(T1或S1代)种植在田中。种植3个小区，以0、2或4kg/ha草甘膦进行喷施。从显示期望水平的耐除草剂草甘膦的抗性的植物收获种子。
将从田间生长的自花授粉T1植物收获的种子(T2代)按“株行(planttorow)”播种。行(完全或部分抗性)和行内个体植物(抗性或敏感性)的分离数据的卡方分析显示EE-GM3的单个插入的预期的孟德尔遗传。
继续选择和种子增加直至品系被确定为对于转化事件EE-GM3是纯合的，并且被选择用于在第4代进行核心种子生产。随后，将T5代种子用作开发不同品种的候选者。将第6代(T6代)中的植物在设计用以将事件移入与更广的商业大豆种质基础的基因渗入程序中与常规大豆繁育系(breedingline)杂交。将F1杂交植物(EE-GM3品系x常规品系)生长至成熟，种植F2种子。利用设计用以鉴定EE-GM3插入物的接合性的PCR引物分析901株F2植物的叶样品。观察到按照孟德尔法则进行的单插入分离的1:2:1的预期比率。
将选择的事件EE-GM3引入不同的商业遗传背景，比较不同位置上的田间试验的结果。使用不同的处理，用草甘膦除草剂和/或异噁唑草酮除草剂攻击植物。植物展现良好的除草剂抗性。将数百个不同的包含事件EE-GM3的大豆品种用于遗传研究，施用除草剂。随后在田间增加从该试验选择的品系，也用除草剂处理所述品系。从该试验，增加了50个选择的品系，也对这些品系进行除草剂处理。用异噁唑草酮和草甘膦喷施用的较后品系的植物毒性评分显示反应的差异性，但品系间的反应范围反映相似的差异性，如在相同处理和环境条件下生长的原始Jack背景中的EE-GM3事件的4个重复间观察到的。因此，对于包含EE-GM3的植物观察到广泛的种质间的对相关除草剂的抗性。
此外，包含事件EE-GM3的植物具有正常的叶、花和荚形态学、优良能育性，并且在多个遗传背景中不显示疾病或异常昆虫易感性。在至多个遗传背景的基因渗入过程中，未观察到异常问题或异常情况。
在一个季节中，设计10位置研究以比较包含转化事件EE-GM3的双耐除草剂大豆与转化亲本品种Jack和一些非转基因大豆品种的农艺性状。使用随机化完全区组设计，将EE-GM3植物种植在重复区，使用常规杂草控制或使用期望的除草剂、草甘膦和异噁唑草酮。以70克ai/Ha的目标速率(targetrate)用异噁唑草酮除草剂和以1060克ai/Ha的目标速率用草甘膦除草剂喷施具有包含转化事件EE-GM3的大豆植物的小区。在约V4-V5植物生长阶段以叶面喷雾的方式对这些植物施用除草剂。在早、中和晚季进行农艺学观察。Jack和非转基因品种区的植物密度(参数；直立计数(standcount))比事件EE-GM3区中的高一个标准差。早期直立坟数差异可能是种子批质量的结果，因为EE-GM3播种种子是在反季节苗圃中产生，而非转基因品种的种子是在紧接着的美国正常生产季节产生的。然而，达到50%的萌发和植物活力评级(plantvigorrating)的天数相同，这表明对于这些性能参数种子批是可比较的。在晚季直立计数中，Jack和非转基因品种与EE-GM3植物保持一个标准差的差异。EE-GM3事件植物的小区产量也比Jack的小区产量低一个标准差，这可能是由于EE-GM3事件小区的更低植物密度的结果。非转基因品种的产量比Jack高，由于在最近的品种中发现的产量潜能的提高，这是可预期的。
在一个试验中，在3个生长阶段：V4-5、R1和完全成熟进行植物健康评级。第一评级在期望的除草剂施用后不久进行。在最终的植物健康评级时，用两种除草剂喷施的含有EE-GM3的植物具有与未喷施的Jack植物或未喷施的包含EE-GM3的植物相同的评分。在由农艺员工进行评级中，喷施除草剂的植物在V4-5和R1植物生长阶段接受3-4(中度伤)的健康评级。未喷施的植物(未转化的Jack或包含EE-GM3的大豆植物)被评级为4.6-4.8(5的评级表示无损伤)。在最终植物健康评级时，所有小区接受为5的相同评级(无损伤)。
一个试验季是异常降雨之一并且在期望的除草剂施用后EE-GM3植物中的作物损伤比在其它季节中观察到的更明显。田间评估还包括监测适合性特征(fitnesscharacter)(生殖、疾病抗性、生殖力、种子散布、休眠、持久性(persistence))。对于生殖特征；至萌发的天数、达到50%的开花的天数和达到90%荚果成熟的天数，EE-GM3与Jack植物都不相同。在对疾病和虫害的天然浸染的反应中差异不显著。虽然EE-GM3产生比Jack少的最终产量，但未发现生殖力(100粒种子重量)的差异。种子分散参数(豆荚落粒(podshattering)和植物倒伏)的评估发现EE-GM3和Jack具有相同的豆荚落粒评分，但发现EE-GM3植物不太容易倒伏。从10个位置收获的种子的评估发现没有因萌发或休眠测试而产生的问题。
在这些试验中，在具有异常降雨的季中，无论杂草控制处理如何，EE-GM3植物的最终产量都低于Jack的产量一个标准差(可能是EE-GM3事件小区的更低植物密度的结果)。在异常潮湿的季节中，EE-GM3小区的作物损伤(10-30%的作物面积变白)据报导在草甘膦和异噁唑草酮除草剂的叶面施用后达到6周。然而，到成熟时，“无损伤”植物健康评级被赋予所有小区。在原种大豆品种背景中进行EE-GM3基因渗入的重复多位点田间试验，当与不包含转基因的近等基因姊妹系相比较时，预期显示包含事件EE-GM3的植物与近等基因系(在除草剂处理不存在的情况下)之间无产量差异。
此外，在重复田间试验中，当用IFT(70grai/ha和0.5%NIS,Agridex)进行芽前或芽后处理时，在包含EE-GM3的大豆植物中发现显著的作物抗性(低于10%的变白)，但当利用磺酰草吡唑(35grai/ha和0.5%NIS,Agridex)(另一种HPPD抑制剂除草剂)的芽后施用处理时，也在包含EE-GM3的大豆植物中发现显著的作物抗性(低于10%的变白)。
1.2.2原种事件EE-GM3的侧翼区域和外源DNA的鉴定
EE-GM3原种事件中的侧翼连接包含耐除草剂基因的外源DNA的区域的序列被测定为如下：
1.2.2.1.右(5')侧翼区域
对被鉴定为包含5'侧翼区域的片段进行测序，其核苷酸序列示于SEQIDNo.2中。核苷酸1与1451之间的序列对应于植物DNA，而核苷酸1452与1843之间的序列对应于外源DNA。
1.2.2.2.左(3')侧翼区域
对被鉴定为包含3'侧翼区域的片段进行测序，其核苷酸序列示于SEQIDNo.3中。核苷酸1与240之间的序列对应于外源DNA，而核苷酸241与1408之间的序列对应于植物DNA。
1.2.2.3.EE-GM3的包含耐除草剂基因的外源DNA
通过使用不同的分子技术，已确定原种事件EE-GM3的包含耐除草剂基因的外源DNA以头接头取向包含两个部分3'组蛋白At序列，后接2个几乎完全拷贝的以头接头取向排列的pSF10的SalI片段(参见图1)。
因此EE-GM3的包含耐除草剂基因的外源DNA按顺序包含下列序列：
-从核苷酸1至核苷酸199：对应于SEQID1的nt6760至nt6958的核苷酸序列的互补序列的核苷酸序列；
-从核苷酸200至核苷酸624：对应于SEQID1的nt6874至nt7298的核苷酸序列的核苷酸序列；
-从核苷酸625至核苷酸7909：对应于SEQID1的nt7至nt7291的核苷酸序列的核苷酸序列；
-从核苷酸7910至核苷酸15163：对应于SEQID1的nt12至nt7265的核苷酸序列的核苷酸序列；和
-从核苷酸15164至核苷酸15187：对应于SEQID3的nt217至nt240的核苷酸序列的核苷酸序列(该序列不对应于pSF10质粒DNA或野生型植物DNA，从而被称为填充DNA)。
SEQIDNo2的核苷酸1至1451的5'侧翼序列在该外源DNA之前紧邻其上游并且与外源DNA邻接，并且SEQIDNo3的核苷酸241至核苷酸1408的3'侧翼序列在外源DNA之后紧邻其下游离并且与外源DNA邻接。
EE-GM3中的外源DNA和侧翼DNA序列的确认完全DNA测序产生了SEQIDNo.11中报导的序列。在该序列中，插入的DNA是从核苷酸位置1452至核苷酸位置16638，以头接尾取向排列的2个来自pSF10的几乎完全的拷贝是从核苷酸位置2257至核苷酸位置16601。SEQIDNo.11的5'侧翼DNA序列是SEQIDNo.11的核苷酸位置1至核苷酸位置1451的序列，并且SEQIDNo.11的3'侧翼DNA序列是SEQIDNo.11的核苷酸位置16639至核苷酸位置17806的序列。
2.针对EE-GM3的聚合酶链式反应鉴定方案的开发
2.1.引物
开发识别原种事件内的序列的特异性引物。
开发识别EE-GM3的3'侧翼区域内的序列的引物。随后在外源DNA的序列内选择第二引物以便引物横跨约263个核苷酸的序列。发现下列引物在对EE-GM3DNA的PCR反应中产生特别清晰和可重现的结果：
SOY028：5'-ATC.gCT.TTA.ACg.TCC.CTC.Ag-3(SEQIDNo.：4)(靶：插入DNA)
SOY029：5'-CAA.ggC.CTC.gAg.ATT.ATC-3'(SEQIDNo.：5)(靶：植物DNA)
优选地将靶向内源序列的引物包含在PCR混合物中。这些引物用作未知样品和DNA阳性对照的内部对照。利用内源引物对的阳性结果(413bp的PCR扩增片段的存在)表明在基因组DNA制剂中存在丰富的优质DNA可用于产生PCR产物。选择内源引物以识别内源肌动蛋白大豆基因：
SOY015'-gTC.AgC.CAC.ACA.gTg.CCT.AT-3'(SEQIDNo.：9)
SOY025'-gTT.ACC.gTA.CAg.gTC.TTT.CC-3'(SEQIDNo.：10)
2.2.扩增片段
PCR反应中的预期扩增片段为：
对于引物对SOY01-SOY02：413bp(内源对照)
对于引物对SOY028-SOY029：263bp(EE-GM3原种事件)
2.3.模板DNA
按照Edwards等(NucleicAcidResearch,19,p1349,1991)从叶冲孔制备模板DNA。当使用利用其它方法制备的DNA时，应当利用不同量的模板进行测试运行。通常50ng的基因组模板DNA产生最佳结果。
2.4.指定的阳性和阴性对照
为了避免假阳性或阴性，决定应当将下列阳性和阴性对照包括在PCR运行中：
-MasterMix对照(DNA阴性对照)。这是其中未向反应物中添加DNA的PCR。当观察到预期的结果(无PCR产物)时，这表明PCR混合物未被靶DNA污染。
-DNA阳性对照(已知包含转基因序列的基因组DNA样品)。该阳性对照的成功扩增表明PCR在允许靶序列扩增的条件下运行。
-野生型DNA对照。这是其中提供的模板DNA是从非转基因植物制备的基因组DNA。当观察到预期的结果：无转基因PCR产物扩增但内源PCR产物扩增时，这表明在基因组DNA样品中不存在可检测的转基因背景扩增。
2.5.PCR条件
在下列条件下获得最佳结果(在描述中，用于最佳结果的各种条件意指提供此类条件的实例。很明显，本领域技术人员可改变条件、试剂和参数(例如使用其它Taq聚合酶)，并且获得期望的结果)：
用于25μl反应的PCR混合物包含：
20ng模板DNA
2.5μl10x扩增缓冲液(与Taq聚合酶一起由制造商提供的)
0.5μl10mMdNTP's
0.4μlSOY01(10皮摩尔/μl)
0.4μlSOY02(10皮摩尔/μl)
0.7μlSOY028(10皮摩尔/μl)
0.7μlSOY029(10皮摩尔/μl)
0.1μlTaqDNA聚合酶(5个单位/μl)
加水至25μl
遵照以获取最佳结果的热循环设置(thermocyclingprofile)如下：
在95℃下进行4分钟
随后：在95℃下进行1分钟
在57℃下进行1分钟
在72℃下进行2分钟
进行5个循环
随后：在92℃下进行30秒
在57℃下进行30秒
在72℃下进行1分钟
进行25个循环
随后：在72℃下进行10分钟
2.6.琼脂糖凝胶分析
为了最佳地显现PCR的结果，已确定应当将10至20μl的PCR样品施加在1.5%的琼脂糖凝胶(Tris-硼酸盐缓冲液)上，使用适当的分子量标准(例如100bp梯带Pharmacia)。
2.7.结果的验证
已确定，除非1)DNA阳性对照显示预期的PCR产物(转基因和内源片段)，2)DNA阴性对照对于PCR扩增呈阴性(无片段)和3)野生型DNA对照显示预期的结果(内源片段扩增)，否则来自单个PCR运行和单个PCR混合物内的转基因植物DNA样品的数据应当是不可接受的。
当按照上述针对EE-GM3的PCR鉴定方案进行时，显示可见量的具有期望大小的转基因和内源PCR产物的泳道表明基因组模板DNA从其制备的相应植物遗传了EE-GM3原种事件。未显示可见量的任一转基因PCR产物但显示可见量的内源PCR产物的泳道表明基因组模板DNA从其制备的相应植物不包含原种事件。未显示可见量的内源PCR产物和转基因PCR产物的泳道表明基因组DNA的质量和/或量不允许PCR产物产生。不能给这些植物评分。应当重复基因组DNA制备，并且必须进行新的PCR运行(利用适当的对照)。
2.8.使用鉴别PCR方案鉴定EE-GM3
在尝试筛选未知之前，必须利用适当的对照进行测试运行。开发的方案可能需要针对在实验室之间可能不同的组分(模板DNA制剂、TaqDNA聚合酶、引物的质量、dNTP、热循环仪等)进行最优化。
内源序列的扩增在所述方案中起着关键作用。必须获得扩增出等摩尔量的已知转基因基因组DNA模板中的内源序列和转基因序列的PCR和热循环条件。每当靶向内源片段不被扩增时，或每当靶向序列在相同溴化乙锭染色强度下不被扩增时，如通过琼脂糖凝胶电泳判断的，则可能需要PCR条件的最优化。
按照上述方案测试来自许多大豆植物的叶材料，其中一些包含EE-GM3。来自原种事件EE-GM3和来自大豆野生型的样品分别被用作阳性对照和阴性对照。
图2举例说明利用针对EE-GM3的原种事件PCR鉴定方案对许多大豆植物样品获得的结果。发现泳道2和3中的样品包含原种事件EE-GM3，因为检测到263bp的条带，然而泳道4至8中的样品不包含EE-GM3。泳道6和7包含来自使用相同耐除草剂嵌合基因获得的其它大豆转化事件的样品；泳道8包含来自野生型大豆植物的DNA，泳道9代表阴性对照(水)样品，泳道1和10代表分子量标准(100bp梯带)。
2.9.用于大批种子的EE-GM3检测的dPCR测定
建立鉴别PCR(dPCR)测定以检测大批种子中EE-GM3的低水平存在。在可重复条件下在大批非转基因种子中成功地检测到最低水平0.4%(w/w)的转基因种子。因此检测限被确定为0.4%(w/w)。
在该靶PCR反应中使用下列引物：
靶向T-DNA序列的正向引物：
SHA1305'-CTA.TAT.TCT.ggT.TCC.AAT.TTA.TC-3'(SEDIDNo.12)
靶向3'侧翼序列的反向引物：
SMP1785'-TgA.ggC.ACg.TAT.TgA.TgA.CC-3'(SEQIDNo.13)
PCR反应中来自这些引物的预期的扩增片段为115bp。
对约200ng的按照改良的GentraPuregeneDNA纯化提取试剂盒(Qiagen)从磨碎的大批种子制备的模板DNA进行靶PCR反应。当使用利用其它方法制备的DNA时，应当使用具有已知相对水平的EE-GM3的样品进行测试运行。
理想地应当在单独的PCR运行中进行靶向内源序列的验证参照系统PCR反应，以验证DNA样品对于PCR分析的适合性，以避免假阴性结果。
对于未知测试样品，PCR实验理想地应当包括适当的阳性和阴性对照样品，即：
-MasterMix对照(DNA阴性对照)。这是其中未向反应物中添加DNA的PCR。当对于靶和参照系统反应都观察到预期的结果(无PCR产物)时，这表明PCR混合物未被靶DNA污染。
-DNA阳性对照(已知包含转基因序列的基因组DNA样品)。该阳性对照的成功扩增表明PCR在允许靶序列扩增的条件下运行。
-还可将野生型DNA对照添加进该PCR中。这是其中提供的模板DNA是从非转基因植物制备的基因组DNA。当观察到预期的结果：无转基因PCR产物扩增但内源PCR产物扩增时，这表明在基因组DNA样品中不存在可检测的转基因背景扩增。
在下列条件下获得最佳结果：
很明显，可使用其它Taq聚合酶，从而条件可与下列提供商的推荐不同。
-用于25μl反应的PCR混合物包含：
200ng模板DNA
5μl5x反应缓冲液
0.25μl20mMdNTP
0.7μlSHA130(10皮摩尔/l)
0.4μlSMP178(10皮摩尔/l)
0.1μlGO-TaqDNA聚合酶(5个单位/l)
加水至25μl
遵照以获取最佳结果的热循环设置如下：
在95℃下进行4分钟
随后：在95℃下进行1分钟
在57℃下进行1分钟
在72℃下进行2分钟
进行5个循环
随后：在92℃进行30秒
在57℃下进行30秒
在72℃下进行1分钟
进行30个循环
随后：在72℃下进行10分钟
为了最佳地显现PCR的结果，已确定应当将10至20μl的PCR产物施加在1.5%的琼脂糖凝胶(Tris-硼酸盐缓冲液)上，使用适当的分子量标准(例如50bp梯带)。
当按照上述PCR法进行时，显示可见量的具有期望大小的靶和参照系统PCR产物的泳道表明基因组模板DNA从其制备的测试样品包含高于靶反应的检测限的EE-GM3原种事件的水平。
未显示可见量的靶PCR产物但显示可见量的参照系统PCR产物的泳道表明基因组模板DNA从其制备的测试样品包含低于靶反应的检测限的EE-GM3原种事件的水平
未显示可见量的内源和转基因PCR产物的泳道表明基因组DNA的质量和/或量不允许PCR产物产生。
不能给这些植物评分。应当重复基因组DNA制备，并且必须利用适当的对照进行新的PCR运行。
3.特定整合片段作为用于检测包含EE-GM3的材料的探针的用途
使用特异性引物SOY028(SEQIDNo.4)和SOY029(SEQIDNo.5)通过PCR获得EE-GM3的特定整合片段，产生具有SEQIDNo8的核苷酸序列的扩增子，或通过化学合成获得EE-GM3的特定整合片段，并且标记所述整合片段。将该整合片段用作用于检测生物样品中的EE-GM3的特异性探针。按照标准方法从样品提取核酸。随后将该核酸在经最优化以允许杂交体形成的杂交条件下与特异性探针接触。随后检测杂交体的形成以表明EE-GM3核酸存在于样品中。任选地，使用特异性引物扩增样品的核酸，然后将所述核酸与特异性探针接触。或者，标记所述核酸，然后将其与特异性探针而非整合片段接触。任选地，将特异性探针连接至固体载体(例如，但不限于滤膜、条带或珠粒)，随后将其与样品接触。
4.用于EE-GM3大豆植物材料的接合性状态的基于PCR的确定的方案。
4.1.引物
以使它们彼此相向并且在其之间具有包含除草剂抗性基因的外源DNA的插入位点的方式设计识别原种事件的插入之前的野生型基因座的核苷酸序列的两个引物。这组引物与与外源DNA序列互补并且针对侧翼DNA的第三引物一起允许同时PCR扩增EE-GM3特异性序列以及野生型序列。
发现下列引物在对EE-GM3DNA的接合性评分PCR中产生特别清晰和可重现的结果：
SMP187:5'-ATA.TCA.ACC.CgT.AgC.TCg.AC-3′(SEQIDNo.:6)
(靶:3'侧翼序列的野生型植物DNA上游)
SOY029：5'-CAA.ggC.CTC.gAg.ATT.ATC-3'(SEQIDNo.:5)
(靶:3'侧翼序列的植物DNA)
STV0195'-ggC.ATT.AAA.TTg.gTg.AAA.ATT.gC-3'(SEQIDNo.:7)
(靶:插入物DNA)
4.2.扩增片段
PCR反应中的预期扩增片段为：
对于引物对SMP187-SOY029:319bp(野生型基因座)
对于引物对STV019-SOY029:706bp(EE-GM3基因座)
4.3.模板DNA
按照Edwards等(NucleicAcidResearch,19,p1349,1991)从叶冲孔制备模板DNA。当使用利用其它方法制备的DNA时，应当利用不同量的模板进行测试运行。通常25或50ng的基因组模板DNA产生最佳结果。
4.4.指定的阳性和阴性对照
为了避免假阳性或阴性，期望应当将下列阳性和阴性对照包括在PCR运行中：
-MasterMix对照(DNA阴性对照)。这是其中未向反应物中添加DNA的PCR。当观察到预期的结果(无PCR产物)时，这表明PCR混合物未被靶DNA污染。
-DNA阳性对照(已知包含转基因序列的基因组DNA样品)。该阳性对照的成功扩增表明PCR在允许靶序列扩增的条件下运行。
-野生型DNA对照。这是其中提供的模板DNA是从非转基因植物制备的基因组DNA。当观察到预期的结果：无转基因PCR产物扩增但内源PCR产物扩增时，这表明在基因组DNA样品中不存在可检测的转基因背景扩增。
4.5.PCR条件
在下列条件下获得最佳结果：
很明显，可使用其它Taq聚合酶例如GO-Taq，从而条件可与下列提供商的推荐不同。
-用于25μl反应的PCR混合物包含：
xμl模板DNA(25ng)
2.5μl10x扩增缓冲液(与Taq聚合酶一起由制造商提供的)
0.5μl10mMdNTP's
0.5μlSMP187(10皮摩尔/μl)
0.5μlSTV019(10皮摩尔/μl)
1μlSOY029(10皮摩尔/μl)
0.1μlTaqDNA聚合酶(5个单位/μl)
加水至25μl
遵照以获取最佳结果的热循环设置如下：
在95℃下进行4分钟
随后：在95℃下进行1分钟
在57℃下进行1分钟
在72℃下进行2分钟
进行5个循环
随后：在92℃下进行30秒
在57℃下进行30秒
在72℃下进行1分钟
进行25个循环
随后：在72℃下进行10分钟
4.6.琼脂糖凝胶分析
为了最佳地显现PCR的结果，已确定应当将10至20μl的PCR样品施加在1.5%的琼脂糖凝胶(Tris-硼酸盐缓冲液)上，使用适当的分子量标准(例如100bp梯带Pharmacia)。
4.7.结果的验证
除非：
阳性对照显示预期的PCR产物(转基因靶扩增)
野生型阳性DNA对照显示预期的结果(野生型靶扩增)
阴性对照对于PCR扩增呈阴性(无片段)，
否则来自单个PCR运行和单个PCRMasterMix内的转基因植物DNA样品的数据应当是不可接受的。
显示可见量的预期大小的转基因PCR产物并且未显示可见量的野生型PCR产物的泳道，表明基因组模板DNA从其制备的相应植物对于转基因基因盒是纯合的。
显示可见量的预期大小的转基因PCR产物和野生型PCR产物的泳道，表明基因组模板DNA从其制备的相应植物对于转基因盒是半合子。未显示可见量的转基因PCR产物和显示可见量的野生型PCR产物的泳道，表明基因组DNA从其制备的相应植物未遗传针对其进行测定的转基因序列，从而对于野生型基因座是纯合的。
未显示可见量的转基因PCR产物和野生型PCR产物的泳道，表明基因组DNA的质量和/或量不允许PCR产物产生。不能给这些植物评分。应当重复基因组DNA制备，并且必须利用适当的对照进行新的PCR运行。
4.8.使用接合性评分方案案鉴定含EE-GM3植物的接合性状态
图3举例说明利用针对EE-GM3的接合性评分PCR对许多大豆植物样品获得的结果。发现泳道2和5中的样品仅包含原种事件EE-GM3的PCR片段(706bp)特征，然而泳道4、6和7的样品仅包含wt基因座存在的片段特征。泳道3、8和9包含两种片段。因此泳道2和5包含以纯合子形式存在的EE-GM3，泳道3、8和9包含以半合子形式存在的EE-GM3，以及泳道4、6和7包含以纯合子形式存在的wt基因座(对于EE-GM3是不成对的)。泳道10表示阴性对照(水)样品，泳道1和11表示分子量标准(100bp梯带)。
5.EE-GM3至优选品种的基因渗入
通过重复回交至商业大豆品种来引入原种事件EE-GM3，所述商业大豆品种是例如但不限于大豆品种7631014(US2009252860)；大豆品种7431014(US2009252859)；大豆品种7925084(US2009252858)；大豆品种7311153(US2009252857)；大豆品种S070159(US2009252856)；大豆品种7535357(US2009246353)；大豆品种S070160(US2009246352)；大豆品种26074414(US2009249508)；大豆品种7509171(US2009249507)；大豆品种S070158(US2009246351)；大豆品种7511119(US2009249506)；大豆品种7113111(US2009238945)；大豆品种S06-02RM018047(US7592518)；大豆品种7013345(US2009232957)；大豆品种7041461(US2009235376)；大豆品种7549450(US2009232956)；大豆品种7317090(US2009232955)；大豆品种2N2V58015(US2009226597)；大豆品种7243182(US2009226596)；大豆品种7143182(US2009226595)；大豆品种7043182(US2009220673)；大豆品种S070157(US2009222950)；大豆品种306924721(US2009220672)；大豆品种7614385(US2009220671)；大豆品种7925118(US2009214750)；大豆品种7821295(US2009214749)；大豆品种7811336(US2009214748)；大豆品种S070150(US2009214747)；大豆品种6214260(US2009214746)；大豆品种S070152(US2009214745)；大豆品种7429331(US2009214751)；大豆品种26034631(US2009208634)；大豆品种S07-03JR108674(US7560621)；大豆品种S07-03KL016279(US7560620)；大豆品种S06-CL959411(US7554017)；大豆品种SG3870NRR(US2009158453)；大豆品种HFPR-G(CA2645702)；大豆品种S06-02JR423016(US7521606)；大豆品种S06-01JR119814(US7518039)；大豆品种S06-01JR119448(US7518038)；大豆品种6540220(US2009055960)；大豆品种S060292(US2009055959)；大豆品种S050228(US2009055958)；大豆品种S06-02JR423003(US7491873)；大豆品种S06-02JR423005(US7491872)；大豆品种S06-02JR409114(US7485782)；大豆品种S06-SJ144056(US7473823)；大豆品种(US7470835)；大豆品种6910450(US2008282369)；大豆品种6223012(US7446246)；大豆品种6549250(US7446245)；大豆品种17731225(US2008271204)；大豆品种6928285(US2008271203)；大豆品种6736054(US2008271169)；大豆品种S060299(US2008271199)；大豆品种S060294(US2008271202)；大豆品种6943322(US2008271201)；大豆品种5343260(US2008263719)；大豆品种6439359(US2008263704)；大豆品种6238359(US2008263703)；大豆品种6547272(US2008263702)；大豆品种6929431(US2008263701)；大豆品种6703392(US2008263700)；大豆品种6044483(US2008263699)；大豆品种S050224(US2008263698)；大豆品种6719022(US2008263697)；大豆品种5826056(US2008263696)；大豆品种6265047(US2008263724)；大豆品种6928331(US2008263695)；大豆品种6719331(US2008263694)：大豆品种6636454(US2008263693)；大豆品种6226454(US2008263718)；大豆品种Q0073801(US2008256657)；大豆品种6326393(US2008256652)；大豆品种6408448(US2008256651)；大豆品种6449315(US2008250524)；大豆品种S060296(US2008250523)；大豆品种6012078(US2008250522)；大豆品种6342078(US2008250521)；大豆品种6419156(US2008250520)；大豆品种5723264(US2008250519)；大豆品种S050225(US2008250518)；大豆品种S060298(US2008244783)；大豆品种6935331(US2008244782)；大豆品种6819456(US2008244787)；大豆品种S060297(US2008244781)；大豆品种6135319(US2008244786)；大豆品种6819331(US2008244780)；大豆品种6137445(US2008244779)；大豆品种6917445(US2008244778)；大豆品种6111333(US2008244777)；大豆品种S050229(US2008244776)；大豆品种6114011(US2008244775)；大豆品种6900358(US2008244784)；大豆品种6345184(US2008244774)；大豆品种6836085(US2008244773)；大豆品种6635047(US2008244772)；大豆品种6139105(US2008244771)；大豆品种6434385(US2008244770)；大豆品种S060295(US2008244769)；大豆品种6035184(US2008244768)；大豆品种S060293(US2008209590)；大豆品种6733322(US2008209594)；大豆品种6421326(US2008209593)；大豆品种S060077(US2008209589)；大豆品种6600375(US2008209592)；大豆品种S06-CL821457(US7420104)；大豆品种S07-02KG294306(US7414178)；大豆品种S06-BA046119(US7414175)；大豆品种S07-02KG294307(US7411114)；大豆品种SG3865N(US2008189802)；大豆品种6701475(US7408097)；大豆品种1335025(US2008178316)；大豆品种1686017(US2008178315)；大豆品种2388028(US2008178314)；大豆品种2387029(US2008178313)；大豆品种S06-WW152330(US7388129)；大豆品种6424090(US7385118)；大豆品种6723322(US7385115)；大豆品种SG4377NRR(US7385114)；大豆品种S06-02JR111334(US7381864)；大豆品种6141287(US7378577)；大豆品种MT110501(US7378576)；大豆品种(US7378575)；大豆品种S06-WW169267(US7375261)；大豆品种6223392(US7371939)；大豆品种S06-CL968413(US7371937)；大豆品种S06-CL951107(US7368636)；大豆品种S06-MT9152077(US7361810)；大豆品种4211676(US2008092253)；大豆品种S06-M059029(US7355101)；大豆品种6548193(US7345228)；大豆品种6440261(US7345227)；大豆品种S060291(US7342151)；大豆品种S06-MT9206166(US7339094)；大豆品种S06-WW013107(US7339093)；大豆品种S06-M03256(US7335820)；大豆品种6134466(US7332656)；大豆品种S06-01JR123373(US7329800)；大豆品种S06-MT9211059(US7326831)；大豆品种26170838(US2008016590)；大豆品种306612412(US2008016588)；大豆品种26660135(US2008016587)；大豆品种306734323(US2008016586)；大豆品种S06-01JR122235(US7317144)；大豆品种5900450(US7314986)；大豆品种S06-MT116260(US7314984)；大豆品种S06-SJ143606(US7312381)；大豆品种S06-98181-G01-35167(US7309819)；大豆品种26082635(US7304219)；大豆品种BA922834(US7304217)；大豆品种01JR123480(US7304216)；大豆品种M061422(US7304215)；大豆品种17082821(US2007277262)；大豆品种17621620(US2007277261)；大豆品种00977706(US2007277260)；大豆品种S060182(US2007277259)；大豆品种26312034(US7301078)；大豆品种26143837(US7301077)；大豆品种435.TCS(US2007271626)；大豆品种495.RC(US2007271625)；大豆品种5306230(US7297845)；大豆品种S06-WW167686(US7291772)；大豆品种6141145(US2007245426)；大豆品种S050232(US2007226829)；大豆品种5333301(US2007226828)；大豆品种S050215(US2007226827)；大豆品种3235020(US2007226826)；大豆品种5720482(US2007226825)；大豆品种S050216(US2007226824)；大豆品种5512112(US2007226823)；大豆品种3233021(US2007226822)；大豆品种1336024(US2007226821)；大豆品种5348287(US2007226820)；大豆品种5204220(US2007226819)；大豆品种6188027(US2007226818)；大豆品种4183026(US2007226817)；大豆品种S06-WW157958(US7271325)；大豆品种5733056(US2007209091)；大豆品种90501911(US2007209090)；大豆品种S050221(US2007204361)；大豆品种5802205(US2007204360)；大豆品种1000642(US2007204359)；大豆品种5420128(US2007204358)；大豆品种S050222(US2007199094)；大豆品种S050217(US2007199093)；大豆品种S050223(US2007199092)；大豆品种S050218(US2007199091)；大豆品种5419227(US2007199089)；大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7370)；大豆品种XB19V06(US2006107369)；大豆品种XB43C06(US2006107368)；大豆品种XB22N06(US2006107367)；大豆品种XB38E06(US2006107366)；大豆品种XB37U06(US2006107365)；大豆品种XB37Q06(US2006107364)；大豆品种XB00D06(US2006107363)；大豆品种XB14N06(US2006107362)；大豆品种XB31H06(US2006107361)；大豆品种XB21Z06(US2006107360)；大豆品种XB005B06(US2006107359)；大豆品种XB15W06(US2006107358)；大豆品种XB33N06(US2006107357)：大豆品种XB18W06(US2006107356)：大豆品种XB32M06(US2006107355)；大豆品种XB19F06(US2006107354)；大豆品种S03-95021-55-138-AB(US7026531)；大豆品种94M41(US7002061)：大豆品种91M50(US6998518)；大豆品种92B13(US6989475)；大豆品种93B68(US6989474)；大豆品种93B09(US6979759)；大豆品种92M00(US6972352)；大豆品种XB08P05(US2005120433)；大豆品种XB26V05(US2005150023)；大豆品种XB21R05(US2005108795)；大豆品种XB28E05(US2005114942)；大豆品种XB58K05(US2005114941)；大豆品种XB27B05(US2005114940)；大豆品种XB21S05(US2005150022)；大豆品种XB26U05(US2005138695)；大豆品种XB35K05(US2005150021)；大豆品种XB18S05(US2005120436)；大豆品种XB25C05(US2005120435)；大豆品种90M01(US2005120434)；大豆品种XB22H05(US2005150020)；大豆品种XB22K05(US2005114939)；大豆品种XB58G05(US2005114938)；大豆品种XB57U05(US2005120432)；大豆品种XB49M05(US2005120431)；大豆品种XB20D05(US2005144683)；大豆品种XB41B05(US2005150019)；大豆品种XB38T05(US2005120430)；大豆品种XB13T05(US2005120429)；大豆品种XB19Y05(US2005120428)；大豆品种XB43D05(US2005120427)；大豆品种XB40E05(US2005120426)；大豆品种XB39N05(US2005120425)；大豆品种93M01(US2005120424)；大豆品种XB31W05(US2005223439)；大豆品种XB32C05(US2005114937)；大豆品种XB40D05(US2005120423)；大豆品种92M61(US2005120422)；大豆品种91M91(US2005114936)；大豆品种XB33Y05(US2005120421)；大豆品种XB34A05(US2005120420)；大豆品种XB13U05(US2005114935)；大豆品种XB12K05(US2005114934)；大豆品种XB30P05(US2005120419)；大豆品种XB57T05(US2005114933)；大豆品种XB17S05(US2005114932)；大豆品种XB25Y05(US2005114930)；大豆品种XB25S05(US2005150017)；大豆品种XB43W04(US2004177420)；大豆品种XB44W04(US2004177419)；大豆品种XB53J04(US2004199960)；大豆品种XB43V04(US2004216192)；大豆品种XB49K04(US2004172668)；大豆品种XB27P04(US2004205864)；大豆品种XB29L04(US2004177418)；大豆品种XB29K04(US2004177417)；大豆品种XB41U04(US2004231017)；大豆品种XB34D04(US2004177416)；大豆品种XB09J04(US2004172711)；大豆品种XB32Y04(US2004194169)；大豆品种XB44D04(US2004172710)；大豆品种XB44C04(US2004172709)；大豆品种XB10L04(US2004172708)；大豆品种XB19U04(US2004172707)；大豆品种XB02F04(US2004172706)；大豆品种XB25X04(US2004172705)；大豆品种XB26L04(US2004172704)；大豆品种XB11F04(US2004172703)；大豆品种XB40Z04(US2004177415)；大豆品种XB40Y04(US2004181833)；大豆品种XB007C04(US2004181832)；大豆品种96M20(US2004172702)；大豆品种XB39J04(US2004172701)；大豆品种XB29A04(US2004172700)；大豆品种XB35P04(US2004172699)；大豆品种XB58Z04(US2004177414)；大豆品种XB43R04(US2004172698)；大豆品种XB35L04(US2004172697)；大豆品种XB06H04(US2004172696)；大豆品种XB59U04(US2004172695)；大豆品种XB64C04(US2004172694)；大豆品种95M80(US2004172693)；大豆品种XB35Q04(US2004177413)；大豆品种XB04D04(US2004177412)；大豆品种XB08L04(US2004177411)；大豆品种XB18Q04(US2004177410)；大豆品种XB16Q04(US2004177409)；大豆品种XB55K04(US2004172692)；大豆品种XB57M04(US2004172691)；大豆品种XB25L04(US2004205863)；大豆品种XB48T04(US2004194168)；大豆品种XB42X04(US2004199959)；大豆品种XB31T04(US2004177408)；大豆品种XB31U04(US2004194167)；大豆品种XB30E04(US2004177407)；大豆品种XB31R04(US2004177406)；大豆品种S03-95341-A98-60618(US6909033)；大豆品种SN97-6946(US2004168227)；大豆品种94M70(US6864408)；大豆品种92M70(US6797866)；大豆品种92M71(US6858782)；大豆品种91M40(US6828490)；大豆品种93M80(US6849789)；大豆品种XB39N03(US6864407)；大豆品种93M90(US6846975)；大豆品种90M90(US6852913)；大豆品种92M72(US6960708)；大豆品种91M90(US6849788)；大豆品种92M50(US6855876)；大豆品种92M30(US6951974)；大豆品种93M60(US6797865)；大豆品种93M40(US6791016)；大豆品种93M41(US6835875)；大豆品种XB15P03(US6797864)；大豆品种XB24H03(US6936752)；大豆品种XB05A03(US6815585)；大豆品种92M80(US6849787)；大豆品种XB33S03(US6855875)；大豆品种XB48P03(US6797863)；大豆品种XB29X03(US6806406)；大豆品种XB02C03(US6800795)；大豆品种XB29W03(US6858781)；大豆品种91M10(US6958437)；大豆品种92M10(US6916975)；大豆品种XB10G03(US6806405)；大豆品种92M31(US6846974)；大豆品种XB38D03(US6806404)；大豆品种XB34N03(US6803508)；大豆品种XB30W03(US6809236)；大豆品种XB37J03(US6844488)；大豆品种SE72581(US2004148665)；大豆品种SE90076(US2004148664)；大豆品种SD82997(US2004148663)；大豆品种0037393(US2004148662)；大豆品种0088414(US2004148661)；大豆品种0149926(US2004148660)；大豆品种0037209(US2004148659)；大豆品种93B36(US6833498)；大豆品种90B74(US6812384)；大豆品种90B51(US6818809)；大豆品种91B03(US6815584)；大豆品种95B43(US6818808)；大豆品种95B42(US6815583)；大豆品种92B47(US6812383)；大豆品种SE90346(US2004055055)；大豆品种0007583(US2004010824)；大豆品种0008079(US2004010823)；大豆品种S02-AP98041-2-333-01(US2003121076)；大豆品种S02-98041-2-251-01(US2003182694)；大豆品种S02-AP98041-2-262-02(US2003196220)；大豆品种S02-95021-55-240-BA(US2003188348)；大豆品种APA94-31572(US2003061641)；大豆品种AP98041-1-203(US2003056251)；大豆品种APA95-15294(US2003061640)；大豆品种AP98041-4-117(US2003056250)；大豆品种91B33(US6580018)；大豆品种93B85(US6605762)；大豆品种92B76(US6610911)；大豆品种92B38(US6605761)；大豆品种94B24(US6613967)；大豆品种94B73(US6605760)；大豆品种93B86(US6610910)；大豆品种91B12(US6583343)；大豆品种95B34(US6605759)；大豆品种94B23(US6605758)；大豆品种90B11(US6583342)；大豆品种91B92(US6586659)；大豆品种95B96(US6605757)；大豆品种93B72(US6566589)；大豆品种95B97(US6613966)；大豆品种92B95(US6608243)；大豆品种93B47(US6583341)；大豆品种97B52(US6605756)；大豆品种93B15(US6617499)；大豆品种94B54(US6613965)；大豆品种93B67(US6573433)；大豆品种93B87(US6727410)；大豆品种96B51(US6613964)；大豆品种92B84(US6730829)；大豆品种92B12(US6605755)；大豆品种90A07(US6320105)；大豆品种93B26(US6342659)；大豆品种96B21(US6369301)；大豆品种92B63(US6326529)；大豆品种93B46(US6323402)；大豆品种92B75(US6362400)；大豆品种93B08(US6323401)；大豆品种97B62(US6323400)；大豆品种92B37(US6323399)；大豆品种92B56(US6339186)；大豆品种93B66(US6307131)；大豆品种92B62(US6346657)；大豆品种92B36(US6369300)；大豆品种90B73(US6316700)；大豆品种95B95(US6323398)；大豆品种93B65(US6229074)：大豆品种92B24(US6284950)；大豆品种94B53(US6235976)；大豆品种94B22(US6140557)；大豆品种93B84(US6143956)；大豆品种95B32(US6229073)；大豆品种95B53(US6147283)；大豆品种93B35(US6153816)；大豆品种93B07(US6143955)；大豆品种92B74(US6124526)；大豆品种92B35(US6166296)；大豆品种94B45(US6162968)；大豆品种96B01(US6143954)；大豆品种93B53(US6335197)。
观察到原种事件至这些品种中的基因渗入未显著影响这些品种的任何期望的表型或农艺学特征(无产量抑制现象)，同时转基因的表达(如通过草甘膦和/或异噁唑草酮抗性测定的)满足商业可接受的水平。这将事件EE-GM3的状态确认为原种事件。
有利地可将原种事件EE-GM3与市场上可获得的一种或多种其它大豆事件进一步组合，所述其它大豆事件包括但不限于其它耐除草剂事件，例如USDA-APHIS申请(petition)中描述的事件：09-349-01p、09-201-01p、09-183-01p、09-082-01p、09-015-01p、06-354-01p、06-271-01p、06-178-01p、98-238-01p、98-014-01p、97-008-01p、96-068-01p、95-335-01p、93-258-01p(参见，例如，www.aphis.usda.gov/brs/notieg.html)或US2006-282915中描述的事件MON89788(草甘膦抗性)、WO2008/054747中描述的事件DP-305423-1(高油酸/ALS抑制剂抗性)、分别在WO2006/108674或WO2006/108675中描述的事件A2704-12和A5547-127(草胺膦抗性)、US2009130071中描述的MON87701、US2009036308中描述的事件3560.4.3.5、US2008312082中描述的事件DP-305423-1或WO2010/080829中描述的事件BPS-CV127-9(事件127)。
如下文权利要求中所用，除非另外明确地指出，否则术语“植物”意欲包括任何成熟阶段的植物组织以及采自或来源于任何此类植物的细胞、组织或器官，包括但不限于任何种子、叶、茎、花、根、单细胞、配子、细胞培养物、组织培养物或原生质体。
将包含原种事件EE-GM3的参照种子于2009年10月12日在NCIMB登录号NCIMB41659下作为32-RRMM-0531保藏在NCIMB(FergusonBuilding,CraibstoneEstate,Bucksburn,AberdeenAB9YA,Scotland)，验证其成活力。EE-GM3的别名为事件FG-072或事件
本发明的上述说明旨在举例说明而不非限定。
所述实施方案的各种改变或变更对本领域技术人员来说是可想到的。在不背离本发明的精神或范围的情况下进行此类改变和变更。
Claims (53)
1.一种用于鉴定生物样品中的原种事件EE-GM3的方法，所述方法包括利用特异性引物对或探针检测EE-GM3特异性区域，所述特异性引物对或探针特异性识别EE-GM3的包含耐除草剂基因的外源DNA的5’侧翼区域以及与其邻接的插入的外源DNA，或者特异性识别EE-GM3的包含耐除草剂基因的外源DNA的3’侧翼区域以及与其邻接的插入的外源DNA，所述5'侧翼区域包含SEQIDNo2的核苷酸1至核苷酸1451的核苷酸序列，所述3'侧翼区域包含SEQIDNo3的核苷酸241至核苷酸1408的核苷酸序列，并且其中，与所述5'或3'侧翼区域邻接的所述插入的外源DNA为SEQIDNo.11的核苷酸位置1452至核苷酸位置16638的核苷酸序列。
2.权利要求1所述的方法，所述方法包括使用利用至少两个引物的聚合酶链式反应，从存在于所述生物样品中的核酸扩增50至1000bp的DNA片段，所述引物之一识别EE-GM3的包含耐除草剂基因的外源DNA的5'侧翼区域或EE-GM3的包含耐除草剂基因的外源DNA的3'侧翼区域，所述5'侧翼区域包含SEQIDNo2的核苷酸1至核苷酸1451的核苷酸序列，所述3'侧翼区域包含SEQIDNo3的核苷酸241至核苷酸1408的互补序列的核苷酸序列，所述引物的另一个引物识别所述插入的外源DNA内的序列，所述外源DNA的核苷酸序列为SEQIDNo.11的核苷酸位置1452至核苷酸位置16638的核苷酸序列或其互补序列。
3.权利要求2所述的方法，其中识别5'侧翼区域的所述引物包含选自SEQIDNo2的核苷酸1至核苷酸1451的核苷酸序列的17至200个连续核苷酸的核苷酸序列，或识别EE-GM3的3'侧翼区域的所述引物包含选自SEQIDNo3的核苷酸241至核苷酸1408的互补序列的核苷酸序列的17至200个连续核苷酸的核苷酸序列，并且识别外源DNA内的序列的所述引物包含SEQIDNo.11的核苷酸位置1452至核苷酸位置16638的核苷酸序列或其互补序列的17至200个连续核苷酸。
4.权利要求2所述的方法，其中识别5'侧翼区域的所述引物在其最3'末端包含选自SEQIDNo2的核苷酸1至核苷酸1451的核苷酸序列的至少17个连续核苷酸的核苷酸序列，或识别EE-GM3的3'侧翼区域的所述引物在其最3'末端包含选自SEQIDNo3的核苷酸241至核苷酸1408的互补序列的核苷酸序列的至少17个连续核苷酸的核苷酸序列，并且识别外源DNA内的序列的所述引物在其3'末端包含SEQIDNo.11的核苷酸位置1452至核苷酸位置16638的核苷酸序列或其互补序列的至少17个连续核苷酸。
5.权利要求4所述的方法，其中所述引物分别包含SEQIDNo.5和SEQIDNo.4的序列，或分别包含SEQIDNo7和SEQIDNo.5的序列。
6.权利要求5所述的方法，所述方法包括使用PCR扩增263或706bp的片段。
7.一种用于鉴定生物样品中的原种事件EE-GM3的试剂盒，所述试剂盒包含：一种识别EE-GM3的包含耐除草剂基因的外源DNA的5'侧翼区域的引物，所述5'侧翼区域包含SEQIDNo2的核苷酸1至核苷酸1451的核苷酸序列，以及一种识别与所述5'侧翼序列邻接的外源DNA内的序列的引物，所述外源DNA由SEQIDNo.11的核苷酸位置1452至核苷酸位置16638的互补序列的核苷酸序列组成；或，一种识别EE-GM3的包含耐除草剂基因的外源DNA的3'侧翼区域的引物，所述3'侧翼区域包含SEQIDNo3的核苷酸241至核苷酸1408的互补序列的核苷酸序列，以及一种识别外源DNA内的序列的引物，所述外源DNA由SEQIDNo.11的核苷酸位置1452至核苷酸位置16638的核苷酸序列组成。
8.权利要求7所述的试剂盒，其中识别5'侧翼区域的所述引物包含选自SEQIDNo2的核苷酸1至核苷酸1451的核苷酸序列的17至200个连续核苷酸的核苷酸序列，或识别EE-GM3的3'侧翼区域的所述引物包含选自SEQIDNo3的核苷酸241至核苷酸1408的互补序列的核苷酸序列的17至200个连续核苷酸的核苷酸序列，并且识别外源DNA内的序列的所述引物包含SEQIDNo.11的核苷酸位置1452至核苷酸位置16638的核苷酸序列或其互补序列的17至200个连续核苷酸。
9.权利要求7所述的试剂盒，其中识别所述5'侧翼区域的所述引物在其最3'末端包含选自SEQIDNo2的核苷酸1至核苷酸1451的核苷酸序列的至少17个连续核苷酸的核苷酸序列或识别EE-GM3的所述3'侧翼区域的所述引物在其最3'末端包含选自SEQIDNo3的核苷酸241至核苷酸1408的互补序列的核苷酸序列的至少17个连续核苷酸的核苷酸序列，并且识别所述外源DNA内的序列的所述引物在其3'末端包含SEQIDNo.11的核苷酸位置1452至核苷酸位置16638的核苷酸序列或其互补序列的至少17个连续核苷酸。
10.权利要求7所述的试剂盒，其包括包含SEQIDNo.4的序列的引物和包含SEQIDNo.5的序列的引物或包括包含SEQIDNo.5的序列的引物和包含SEQIDNo.7的序列的引物。
11.一种适用于EE-GM3特异性检测的引物对，其包含第一引物和第二引物，所述第一引物具有在最优化的检测条件下特异性识别EE-GM3的包含耐除草剂基因的外源DNA的5’或3’侧翼区域内的序列的序列，所述5'侧翼区域包含SEQIDNo2的核苷酸1至核苷酸1451的核苷酸序列，并且所述3'侧翼区域包含SEQIDNo3的核苷酸241至核苷酸的1408的互补序列的核苷酸序列，所述第二引物具有在最优化的检测条件下特异性识别与所述外源DNA的5'或3'侧翼区域邻接的插入的外源DNA内的序列的序列，其中与所述5'或3'侧翼区域邻接的所述插入的外源DNA由SEQIDNo.11的核苷酸位置1452至核苷酸位置16638的核苷酸序列组成。
12.权利要求11所述的引物对，其中所述第一引物包含选自SEQIDNo2的核苷酸1至核苷酸1451的核苷酸序列的17至200个连续核苷酸的核苷酸序列或选自SEQIDNo3的核苷酸241至核苷酸1408的互补序列的核苷酸序列的17至200个连续核苷酸的核苷酸序列。
13.权利要求11所述的引物对，其中所述第一引物在其最3'末端包含选自SEQIDNo2的核苷酸1至核苷酸1451的核苷酸序列的至少17个连续核苷酸的核苷酸序列或选自SEQIDNo3的核苷酸241至核苷酸1408的互补序列的核苷酸序列的至少17个连续核苷酸的核苷酸序列。
14.权利要求11所述的引物对，其包含一种在其最3'末端包含SEQIDNo.5的序列的引物。
15.一种引物对，其包括在其最3'末端包含SEQIDNo.5的序列的第一引物和在其最3'末端包含SEQIDNo.4的序列的第二引物。
16.权利要求1所述的方法，所述方法包括将生物样品的核酸与针对EE-GM3的特异性探针杂交。
17.权利要求16所述的方法，其中所述特异性探针在标准严格条件下与包含EE-GM3的5'侧翼序列或3'侧翼序列的部分和与其邻接的外源DNA的序列的部分的序列杂交，其中所述5'侧翼区域包含SEQIDNo2的核苷酸1至核苷酸1451的核苷酸序列，所述3'侧翼区域包含SEQIDNo3的核苷酸241至核苷酸1408的核苷酸序列，并且与所述5'或3'侧翼区域邻接的所述插入的外源DNA由SEQIDNo.11的核苷酸位置1452至核苷酸位置16638的核苷酸序列组成。
18.权利要求17所述的方法，其中所述特异性探针在标准严格条件下与SEQIDNo.2的核苷酸1431至1472或SEQIDNo.3的核苷酸220至261或所述序列的互补序列杂交。
19.一种用于鉴定生物样品中的原种事件EE-GM3的试剂盒，其包括能够与EE-GM3的特异性区域特异性杂交的特异性探针，所述特异性区域包含EE-GM3的5'侧翼序列或3'侧翼序列的部分和与其邻接的外源DNA的序列的部分，其中所述5'侧翼区域包含SEQIDNo2的核苷酸1至核苷酸1451的核苷酸序列，所述3'侧翼区域包含SEQIDNo3的核苷酸241至核苷酸1408的核苷酸序列，与所述5'或3'侧翼区域邻接的所述插入的外源DNA由SEQIDNo.11的核苷酸位置1452至核苷酸位置16638的核苷酸序列组成。
20.权利要求19所述的试剂盒，其中所述特异性探针在标准严格条件下与包含EE-GM3的包含耐除草剂基因的外源DNA的5'侧翼序列或3'侧翼序列的部分和与其邻接的外源DNA的序列的部分的序列杂交，其中所述5'侧翼区域包含SEQIDNo2的核苷酸1至核苷酸1451的核苷酸序列，所述3'侧翼区域包含SEQIDNo3的核苷酸241至核苷酸1408的核苷酸序列，并且与所述5'或3'侧翼区域邻接的所述插入的外源DNA由SEQIDNo.11的核苷酸位置1452至核苷酸位置16638的核苷酸序列组成。
21.权利要求20所述的试剂盒，其中所述特异性探针在标准严格条件下与SEQIDNo.2的核苷酸1441至1462或SEQIDNo.3的核苷酸230至251或所述序列的互补序列杂交。
22.一种用于鉴定生物样品中的原种事件EE-GM3的特异性探针，其在标准严格条件下与包含EE-GM3的包含耐除草剂基因的外源DNA的5'侧翼序列或3'侧翼序列的部分和与其邻接的外源DNA的序列的部分的DNA或其互补序列杂交，其中所述5'侧翼区域包含SEQIDNo2的核苷酸1至核苷酸1451的核苷酸序列，所述3'侧翼区域包含SEQIDNo3的核苷酸241至核苷酸1408的核苷酸序列，并且与所述5'或3'侧翼区域邻接的所述插入的外源DNA由SEQIDNo.11的核苷酸位置1452至核苷酸位置16638的核苷酸序列组成。
23.权利要求22所述的探针，其在标准严格条件下与DNA杂交，所述DNA包含SEQIDNo.2的核苷酸1441至1462或SEQIDNo.3的核苷酸230至251的序列或所述序列的互补序列。
24.权利要求22所述的探针，其包含SEQIDNo.2的核苷酸1441至1462或SEQIDNo.3的核苷酸230至251的核苷酸序列或所述序列的互补序列。
25.权利要求22所述的探针，其包含SEQIDNo.2或SEQIDNo.3的核苷酸序列。
26.一种用于确认种子样品中的种子纯度的方法，所述方法包括用特异性引物对或探针检测EE-GM3特异性区域，所述特异性引物对或探针特异性识别EE-GM3的包含耐除草剂基因的外源DNA的5’或3’侧翼区域以及与其邻接的外源DNA，所述5'侧翼区域包含SEQIDNo2的核苷酸1至核苷酸1451的核苷酸序列，所述3'侧翼区域包含SEQIDNo3的核苷酸241至核苷酸1408的核苷酸序列，并且与所述5'或3'侧翼区域邻接的所述插入的外源DNA由SEQIDNo.11的核苷酸位置1452至核苷酸位置16638的核苷酸序列组成。
27.一种用于在种子批的样品中筛选种子的EE-GM3的存在的方法，所述方法包括用特异性引物或探针检测EE-GM3特异性区域，所述特异性引物或探针特异性识别EE-GM3的包含耐除草剂基因的外源DNA的5’或3’侧翼区域以及与其邻接的外源DNA，所述5'侧翼区域包含SEQIDNo2的核苷酸1至核苷酸1451的核苷酸序列，所述3'侧翼区域包含SEQIDNo3的核苷酸241至核苷酸1408的核苷酸序列，并且与所述5'或3'侧翼区域邻接的所述插入的外源DNA由SEQIDNo.11的核苷酸位置1452至核苷酸位置16638的核苷酸序列组成。
28.一种通过与互补的标记核酸探针杂交来检测原种事件EE-GM3在生物样品中的存在的方法，其中通过再循环靶核酸序列来放大探针：靶核酸比率，所述方法包括：
a)将所述靶核酸序列与作为SEQIDNO:2或其互补序列一部分、并且包含SEQIDNo2的核苷酸1452至核苷酸1469的核苷酸序列或其互补序列的第一核酸寡核苷酸或作为SEQIDNO:3或其互补序列一部分、并且包含SEQIDNo3的核苷酸223至核苷酸240的核苷酸序列或其互补序列的所述第一核酸寡核苷酸杂交；
b)将所述靶核酸序列与作为SEQIDNO:2或其互补序列一部分、并且包含SEQIDNo2的核苷酸1434至核苷酸1451的核苷酸序列或其互补序列的第二核酸寡核苷酸或作为SEQIDNO:3或其互补序列一部分、并且包含SEQIDNo3的核苷酸241至核苷酸258的核苷酸序列或其互补序列的所述第二核酸寡核苷酸杂交，其中所述第一核酸寡核苷酸和第二核酸寡核苷酸重叠至少一个核苷酸，并且其中将所述第一核酸寡核苷酸或所述第二核酸寡核苷酸标记为所述标记核酸探针；
c)用产生选择性探针切割从而导致双链体离解的酶仅切割探针：靶核酸序列双链体内的标记探针，从而使所述靶序列保持完整；
d)通过重复步骤(a)至(c)再循环所述靶核酸序列；和
e)检测切割的标记探针，从而确定所述靶核酸序列的存在，其中所述事件EE-GM3包含在于保藏号NCIMB41659下保藏在NCIMB的参照种子中。
29.转基因大豆植物或其种子或后代用于控制大豆田中的杂草的用途，其中所述植物、种子或后代各自在其基因组中包含原种事件EE-GM3，所述事件包含在于保藏号NCIMB41659下保藏在NCIMB的参照种子中。
30.权利要求29所述的用途，其中所述植物、种子或后代的基因组DNA，当使用PCR，利用两个其核苷酸序列分别为SEQID4和SEQID5的核苷酸序列的引物进行分析时，产生263bp的DNA片段。
31.于保藏号NCIMB41659下保藏在NCIMB的包含原种事件EE-GM3的种子或来源于其的衍生物用于生长耐受除草剂的植物的用途。
32.包含原种事件EE-GM3的大豆细胞，其中所述细胞不是植物繁殖材料，并且其中所述事件包含在于保藏号NCIMB41659下保藏在NCIMB的参照种子中。
33.在其基因组中包含原种事件EE-GM3的大豆植物或种子用于生长耐受除草剂的植物的用途，其中所述原种事件EE-GM3是具有嵌合HPPDPfW366编码基因和嵌合2mEPSPS编码基因的转基因事件，且包含在于保藏号NCIMB41659下保藏在NCIMB的参照种子中，且其中所述植物或种子能够通过从已于保藏号NCIMB41659下保藏在NCIMB的种子生长而来的大豆植物的繁殖和/或与其繁育来获得。
34.包含原种事件EE-GM3的大豆种子用于获得大豆产物的用途，其中所述事件包含在于保藏号NCIMB41659下保藏在NCIMB的参照种子中。
35.权利要求34所述的用途，其中所述大豆产物是大豆膳食、磨碎的大豆种子、大豆粉末或大豆薄片。
36.一种用于产生包含原种事件EE-GM3的大豆植物或种子的方法，包括将权利要求29至31任一项中或权利要求33中所定义的植物与另一种大豆植物杂交，和种植获自所述杂交的种子。
37.包含原种事件EE-GM3的大豆基因组DNA，其中所述事件包含在于保藏号NCIMB41659下保藏在NCIMB的参照种子中。
38.SEQIDNo.2的核苷酸1441至核苷酸1462或SEQIDNo.3的核苷酸230至251的核苷酸序列或所述序列的互补序列。
39.SEQIDNo.2的核苷酸1431至核苷酸1472或SEQIDNo.3的核苷酸220至261的核苷酸序列或所述序列的互补序列。
40.一种大豆植物细胞，其在其基因组中按顺序包含下列核苷酸序列：
a)SEQIDNo2的核苷酸1至1451的核苷酸序列；
b)SEQID1的核苷酸6760至核苷酸6958的核苷酸序列的互补序列的核苷酸序列；
c)SEQID1的核苷酸6874至核苷酸7298的核苷酸序列；
d)SEQID1的核苷酸7至核苷酸7291的核苷酸序列；
e)SEQID1的核苷酸12至核苷酸7265的核苷酸序列；
f)SEQID3的核苷酸217至核苷酸240的核苷酸序列；和
g)SEQIDNo3的核苷酸241至核苷酸1408的核苷酸序列，
其中，所述细胞不是植物繁殖材料。
41.一种按顺序包含下列核苷酸序列的DNA分子：
a)SEQIDNo2的核苷酸1至1451的核苷酸序列；
b)SEQID1的核苷酸6760至核苷酸6958的核苷酸序列的互补序列的核苷酸序列；
c)SEQID1的核苷酸6874至核苷酸7298的核苷酸序列；
d)SEQID1的核苷酸7至核苷酸7291的核苷酸序列；
e)SEQID1的核苷酸12至核苷酸7265的核苷酸序列；
f)SEQID3的核苷酸217至核苷酸240的核苷酸序列；
g)SEQIDNo3的核苷酸241至核苷酸1408的核苷酸序列。
42.转基因大豆植物或种子用于控制杂草的方法中的用途，其中所述植物或种子在其基因组中包含事件EE-GM3，所述事件EE-GM3特征在于包含SEQIDNo.2的核苷酸1441至核苷酸1462的核苷酸序列的核酸分子，以及包含SEQIDNo.3的核苷酸230至251的核苷酸序列或所述序列的互补序列的核酸分子，其中所述事件包含在于保藏号NCIMB41659下保藏在NCIMB的参照种子中。
43.大豆植物或种子用于控制杂草的方法中的用途，其中所述植物或种子在其基因组中包含原种事件EE-GM3，其中所述原种事件的DNA序列是SEQIDNO.11的序列，其中所述事件包含在于保藏号NCIMB41659下保藏在NCIMB的参照种子中。
44.大豆植物或种子用于生长大豆的方法中的用途，其中所述植物或种子包含原种事件EE-GM3，其中所述原种事件的DNA序列是SEQIDNO.11的序列，其中所述事件包含在于保藏号NCIMB41659下保藏在NCIMB的参照种子中。
45.包含原种事件EE-GM3的大豆种子用于生长耐受除草剂的大豆的方法中的用途，其中所述种子在其基因组中包含SEQIDNo.11的核苷酸序列或其互补序列，其中所述事件包含在于保藏号NCIMB41659下保藏在NCIMB的参照种子中。
46.SEQIDNo.11的核苷酸序列或其互补序列。
47.SEQIDNo.2或SEQIDNo.3的核苷酸序列。
48.包含原种事件EE-GM3的大豆产物，其中所述事件EE-GM3包含在于保藏号NCIMB41659下保藏在NCIMB的参照种子中，并且其中所述大豆产物是大豆膳食、磨碎的大豆种子、大豆粉末或大豆薄片。
49.在其基因组中包含原种事件EE-GM3的大豆植物或种子用于生长耐受除草剂的植物中的用途，其中所述原种事件的DNA序列是SEQIDNO.11的序列，其中所述事件包含在于保藏号NCIMB41659下保藏在NCIMB的参照种子中。
50.包含原种事件EE-GM3的大豆种子用于获得大豆产物的用途，其中所述原种事件的DNA序列为SEQIDNO.11的序列，其中所述事件包含在于保藏号NCIMB41659下保藏在NCIMB的参照种子中。
51.权利要求50所述的用途，其中所述大豆产物是大豆膳食、磨碎的大豆种子、大豆粉末或大豆薄片。
52.一种用于产生包含原种事件EE-GM3的大豆植物或种子的方法，包括将包含原种事件EE-GM3的植物与另一种大豆植物杂交，和种植获自所述杂交的种子，其中所述原种事件的DNA序列是SEQIDNO.11的序列,其中所述事件包含在于保藏号NCIMB41659下保藏在NCIMB的参照种子中。
53.包含原种事件EE-GM3的大豆产物，其中所述大豆产物是大豆膳食、磨碎的大豆种子、大豆粉末或大豆薄片，其中所述原种事件的DNA序列是SEQIDNO.11的序列，并且其中所述事件包含在于保藏号NCIMB41659下保藏在NCIMB的参照种子中。
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| US7612259B2 (en) | 2007-06-12 | 2009-11-03 | Monsanto Technology Llc | Soybean variety D4127789 |
| US7622644B2 (en) | 2007-06-12 | 2009-11-24 | Monsanto Technology Llc | Soybean variety D4201139 |
| US7619139B2 (en) | 2007-06-12 | 2009-11-17 | Monsanto Technology Llc | Soybean variety D4572906 |
| US7642409B2 (en) | 2007-06-12 | 2010-01-05 | Monsanto Technology Llc | Soybean variety D4120384 |
| US20080313757A1 (en) | 2007-06-12 | 2008-12-18 | Monsanto Technology Llc | Soybean variety d4256958 |
| US7566820B2 (en) | 2007-06-12 | 2009-07-28 | Monsanto Technology Llc | Soybean variety D4208814 |
| US7629506B2 (en) | 2007-06-12 | 2009-12-08 | Monsanto Technology Llc | Soybean variety D4422957 |
| US7557275B2 (en) | 2007-06-28 | 2009-07-07 | Monsanto Technology Llc | Soybean variety D4210110 |
| US7554015B2 (en) | 2007-06-28 | 2009-06-30 | Monsanto Technology Llc | Soybean variety D4483789 |
| US7569751B2 (en) | 2007-06-28 | 2009-08-04 | Monsanto Technology Llc | Soybean variety D4523081 |
| US7592516B2 (en) | 2007-06-28 | 2009-09-22 | Monsanto Technology Llc | Soybean variety D4328762 |
| US7626089B2 (en) | 2007-06-28 | 2009-12-01 | Monsanto Technology Llc | Soybean variety D4311702 |
| US7700843B2 (en) | 2007-07-03 | 2010-04-20 | Monsanto Technology Llc | Soybean variety D4021792 |
| US7560617B2 (en) | 2007-07-03 | 2009-07-14 | Monsanto Technology Llc | Soybean variety D4253854 |
| US7569752B2 (en) | 2007-07-03 | 2009-08-04 | Monsanto Technology Llc | Soybean variety D4902530 |
| US7550653B2 (en) | 2007-07-03 | 2009-06-23 | Monsanto Technology Llc | Soybean variety D4367012 |
| US7554016B2 (en) | 2007-07-03 | 2009-06-30 | Monsanto Technology Llc | Soybean variety D4923560 |
| US7626090B2 (en) | 2007-07-10 | 2009-12-01 | Monsanto Technology Llc | Soybean variety D4470236 |
| US7557276B2 (en) | 2007-07-10 | 2009-07-07 | Monsanto Technology Llc | Soybean variety D4556201 |
| US7629507B2 (en) | 2007-07-10 | 2009-12-08 | Monsanto Technology Llc | Soybean variety D4201483 |
| US7622645B2 (en) | 2007-07-10 | 2009-11-24 | Monsanto Technology Llc | Soybean variety D4063284 |
| US7629508B2 (en) | 2007-07-10 | 2009-12-08 | Monsanto Technology Llc | Soybean variety D4012368 |
| US7557277B2 (en) | 2007-07-10 | 2009-07-07 | Monsanto Technology Llc | Soybean variety D4526223 |
| US7642410B2 (en) | 2007-07-10 | 2010-01-05 | Monsanto Technology Llc | Soybean variety D4452019 |
| US7626091B2 (en) | 2007-07-10 | 2009-12-01 | Monsanto Technology Llc | Soybean variety D4159630 |
| US7592517B2 (en) | 2007-07-10 | 2009-09-22 | Monsanto Technology Llc | Soybean variety D4463892 |
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| US7939725B2 (en) | 2008-04-03 | 2011-05-10 | Stine Seed Farm, Inc. | Soybean cultivar 7311153 |
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| RU2403807C2 (ru) * | 2009-01-23 | 2010-11-20 | Государственное научное учреждение Всероссийский научно-исследовательский институт сои Российской академии сельскохозяйственных наук | Способ приготовления белковых продуктов |
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| WO2006108674A2 (en) * | 2005-04-08 | 2006-10-19 | Bayer Bioscience N.V. | Elite event a2704-12 and methods and kits for identifying such event in biological samples |
| WO2006108675A2 (en) * | 2005-04-11 | 2006-10-19 | Bayer Bioscience N.V. | Elite event a5547-127 and methods and kits for identifying such event in biological samples |
| WO2007017186A1 (en) * | 2005-08-08 | 2007-02-15 | Bayer Bioscience N.V. | Herbicide tolerant cotton plants and methods for identifying same |
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