CN107779520A - 大豆转基因事件mon 87708及其使用方法 - Google Patents
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Abstract
本发明提供了转基因大豆事件MON 87708植物和源自事件MON 87708的植物、植物细胞、种子、植物部分和商品。本发明也提供了对于事件MON 87708特异性的聚核苷酸和包含事件MON 87708特异性的多核苷酸的植物、植物细胞、种子、植物部分和商品。本发明还提供了与事件MON 87708相关的方法。
Description
本申请是申请日为2010年8月26日和发明名称为“大豆转基因事件MON 87708及其使用方法”的201080047890.1号发明专利申请的分案申请。
相关申请的交叉引用
本申请要求享有于2009年9月17日提交的美国临时申请61/243,227的优先权，该申请在本发明中通过引用完整地引入。
序列表的并入
包含在名为”55544-0001_seqlisting.txt”，大小为19.5千字节(在Microsoft中测定的大小)，于2010年8月13日生成的文件中的序列表通过电子提交方式与本申请一起递交，在本申请中通过引用并入。
技术领域
本发明涉及转基因大豆事件MON 87708。该事件表现出对麦草畏除草剂的耐受性。本发明也涉及与事件MON 87708相关的植物、植物部分、植物种子、植物细胞、农产品和方法，并提供了对该事件独特的、与转基因DNA插入大豆植物基因组中关联而产生的核苷酸分子。
背景技术
大豆(Glycine max)是世界上许多领域中的重要作物，且生物技术方法已经应用于该作物以产生具有所需性状的大豆。一种这样的所需性状就是除草剂耐受性。植物中除草剂耐受转基因的表达可以赋予该植物除草剂耐受性的所需性状，但是转基因的表达可能受染色体位置和转基因插入的基因组结果的影响。例如，在植物已经观察到，在转基因的染色体插入位置不同但其它方面均相同的单个事件间转基因表达的水平和模式经常存在差异。事件之间也可能存在不希望的和/或希望的表型或农艺学差异。因为这一点，通常需要产生并分析大量的单个植物转化事件以挑选具有所需性状和使之适用于商业目的必需的最佳表型和农艺特性的事件。这种选择常常需要在多年内、在多个地点及在多种条件下对许多事件进行温室及田间试验，以便可以收集大量的农艺、表型和分子数据。然后得到的数据和观察结果必须由科学家和农学家的团队以选择商业适用的事件为目的进行分析。然后，这样的事件(一旦被选择)可以被用来用植物育种方法将所需性状渗入其它遗传背景中，并因此产生包含所需性状并适当地适应于特定的本地生长环境的许多不同作物品种。
发明内容
本发明提供了被命名为事件MON87708的转基因大豆植物，其表现出对麦草畏除草剂应用的商业可接受的耐受性，具有以保藏号PTA-9670保藏于美国典型培养物保藏中心(ATCC)的代表性种子。本发明也提供了与大豆事件MON 87708相关的新的DNA分子以及使用这些分子的方法。本发明也提供了大豆事件MON 87708的种子、后代、植物部分、细胞以及商品。本发明也提供了使用大豆事件MON 87708的方法以及产生麦草畏耐受性大豆的方法。
本发明提供了与大豆事件MON 87708相关的重组DNA分子。这些重组DNA分子可以包含具有代表侧邻转基因插入的基因组DNA的区域和/或转基因插入的区域和/或这些区域中任一个的连续序列(如转基因插入和大豆事件MON 87708的侧翼基因组DNA之间连接的区域)的核苷酸序列的核苷酸分子。本发明也提供了可用作诊断大豆事件MON 87708的引物和探针及诊断大豆事件MON 87708存在的扩增子的DNA分子。本发明也公开了包含这些分子的大豆植物、植物细胞、植物部分、商品、后代和种子。
本发明提供了用于检测源自于大豆事件MON 87708的DNA的存在和/或不存在，并因此检测事件的存在和/或不存在的方法、组合物和试剂盒。本发明提供了通过使包含DNA的样品与引物组接触，该引物组在与来自大豆事件MON87708的基因组DNA一起用于核酸扩增反应中时产生诊断大豆事件MON87708的扩增的DNA；进行核酸扩增反应，从而产生扩增的DNA；和检测所述扩增的DNA的存在和/或不存在来检测MON 87708的方法。本发明也提供了通过使包含DNA的样品与探针接触，该探针在与来自大豆事件MON87708的DNA一起用于杂交反应中时与对大豆事件MON87708特异性的DNA分子杂交；进行杂交反应；和检测探针与DNA分子的杂交来检测MON 87708的方法。也提供了可用于检测源自大豆事件MON 87708的DNA的存在的包括本发明的方法和组合物的试剂盒。
本发明提供了源自于大豆事件MON 87708的植物、植物细胞或种子的大豆植物、种子、植物细胞、后代植物、植物部分或商品。本发明也提供了包含具有选自SEQ ID NO：1-8及其互补序列和片段的核苷酸序列的重组DNA分子的大豆植物、种子、植物细胞、后代植物、植物部分或商品。本发明也提供了源自于大豆事件MON 87708的植物或种子并且包含在DNA扩增方法中产生含SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：7和/或SEQ ID NO：8的扩增DNA分子的重组DNA分子的大豆植物、种子、植物细胞、后代植物、植物部分或商品。
本发明提供了通过种植大豆事件MON 87708，然后应用能够控制杂草而不伤害大豆事件MON 87708植物的有效剂量的麦草畏除草剂而在田间控制杂草的方法。本发明也提供了通过应用有效剂量的麦草畏除草剂而控制田间杂草，然后在田间种植大豆事件MON87708而在田间控制杂草的方法。本发明也提供了通过在田间种植麦草畏耐受性大豆品种MON 87708的种子，将足以杀灭毒性杂草品种的发芽后有效剂量麦草畏除草剂应用于田间，并从田间收获种子而产生基本上没有毒性杂草种的大豆种子的方法。
本发明提供了其通过将包含SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：7和/或SEQID NO：8的大豆事件MON 87708植物与第二种大豆植物有性杂交从而产生种子，使种子生长以产生后代植物，以麦草畏处理后代植物，并选择对麦草畏耐受的后代植物而产生耐受麦草畏除草剂应用的大豆植物和/或种子的方法。该方法也可以包括将选择的后代植物自交以产生多个第二代后代植物以及从其中选择麦草畏耐受的植物。该方法也可以包括将选择的后代植物与另一种大豆植物有性杂交以产生种子，使该种子生长以产生第二代后代植物，以麦草畏处理第二代后代植物以及选择对麦草畏耐受的第二代后代植物。本发明提供了通过将包含SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：7和/或SEQ ID NO：8的麦草畏耐受的大豆事件MON 87708植物自交从而产生种子，使种子生长以产生后代植物，以麦草畏处理后代植物，并选择对麦草畏耐受的后代植物而产生耐受麦草畏除草剂应用的大豆植物和/或种子的方法。
本发明提供了测定大豆事件MON 87708植物或种子的接合性的方法，包括将大豆DNA样品与包含SEQ ID NO：12、SEQ ID NO：13和SEQ ID NO：14的引物组及包含SEQ ID NO：15和SEQ ID NO：16的探针组接触；然后利用该样品、引物组和探针组进行核酸扩增反应；然后在随后的核酸扩增反应中检测诊断事件87708的第一荧光信号和不同于第一荧光信号并诊断对应于事件MON 87708转基因的插入位置的天然大豆基因组DNA的第二荧光信号；并分析核酸扩增反应中第一荧光信号和第二荧光信号的存在和/或不存在，其中两种荧光信号的存在指示样品对于事件MON 87708是杂合的而仅第一荧光信号的存在指示样品对于事件MON 87708是纯合的。
本发明也提供了包含在包含麦草畏耐受性基因的连锁群9上大致图谱位置143.5处的并由单倍体型窗口19743和19767进一步限定的大豆单倍体型区域的大豆植物、种子、植物细胞或植物部分。通过下面的详细描述，本发明的上述和其它方面将变得更加明显。
特别地，本发明涉及以下各项：
1.一种重组DNA分子，其包含含有选自SEQ ID NO：1-8及其互补序列的核苷酸序列的核苷酸分子。
2.第1项的重组DNA分子，其中所述重组DNA分子通过将异源核酸分子插入大豆植物、植物细胞或种子的基因组DNA中而形成。
3.第1项的重组DNA分子，其中所述DNA分子源自转基因大豆事件MON 87708，其代表性样品被保藏为ATCC PTA-9670。
4.第1项的重组DNA分子，其中所述DNA分子处于源自大豆事件MON 87708的大豆植物、植物细胞、种子、植物部分或商品中，大豆事件MON 87708的代表性样品被保藏为ATCCPTA-9670。
5.第1项的重组DNA分子，其中所述DNA分子是诊断源自事件MON 87708的DNA存在的扩增子。
6.一种DNA分子，其包含具有SEQ ID NO：6的足够长度的连续核苷酸序列及其互补序列的核苷酸序列，以作为在严格杂交条件下与包含选自SEQ ID NO：1-8的核苷酸序列的DNA分子杂交并且在严格杂交条件下不与不含选自SEQ ID NO：1-8的核苷酸序列的DNA分子杂交的DNA探针。
7.一对DNA分子，由第一DNA分子以及不同于第一DNA分子的第二DNA分子组成，其中所述第一DNA分子和第二DNA分子各包含具有SEQ ID NO：6的足够长度的连续核苷酸的核苷酸序列或其互补序列的核苷酸分子，以与源自事件MON 87708的DNA一起用于扩增反应中以产生诊断样品中大豆事件MON 87708的扩增子时作为DNA引物。
8.一种检测样品中源自大豆事件MON87708的DNA分子的存在的方法，所述方法包括：
a.将样品与第6项的DNA探针接触；
b.使所述样品和所述DNA探针经受严格杂交条件；和
c.检测所述DNA探针与所述样品中DNA分子的杂交，其中所述DNA探针与所述DNA分子的杂交指示了所述样品中源自大豆事件MON 87708的DNA分子的存在。
9.一种检测样品中源自大豆事件MON87708的DNA分子的存在的方法，所述方法包括：
a.将样品与第7项的DNA分子对接触；
b.进行扩增反应，其足以产生包含选自SEQ ID NO：1-8及其互补序列的序列的DNA扩增子；和
c.检测所述反应中所述DNA扩增子的存在，其中所述DNA扩增子在所述反应中的存在指示了所述样品中源自大豆事件MON 87708的DNA分子的存在。
10.一种DNA检测试剂盒，其包括至少一种包含SEQ ID NO：6的足够长度的连续核苷酸的核苷酸序列及其互补序列的DNA分子，该DNA分子作为对于检测源自大豆事件MON87708的DNA的存在特异性的DNA引物或探针，其中所述DNA的检测诊断样品中所述事件MON87708 DNA的存在。
11.一种重组大豆植物、其种子、细胞或植物部分，其包含具有选自SEQ ID NO：1-8及其互补序列的核苷酸序列的核苷酸分子。
12.第11项的重组大豆植物、其种子、细胞或植物部分，其中所述植物、其种子、细胞或植物部分耐受麦草畏除草剂处理。
13.第11项的重组大豆植物、其种子、细胞或植物部分，当在DNA扩增方法中测试时，其基因组产生包含选自SEQ ID NO：1-8及其互补序列的DNA分子的扩增子。
14.一种大豆植物或种子，其中所述大豆植物或种子源自大豆事件MON 87708，其代表性样品以ATCC保藏号No.PTA-9670保藏。
15.第14项的大豆植物或种子，其中所述大豆植物或种子是具有至少一个源自大豆事件MON 87708的亲本的杂种。
16.一种无生命植物材料，其包含选自SEQ ID NO：1-8及其互补序列的重组DNA分子。
17.一种微生物，其包含具有选自SEQ ID NO：1-8及其互补序列的核苷酸序列的核苷酸分子。
18.第17项的微生物，其中所述微生物是植物细胞。
19.一种商品，其包含具有选自SEQ ID NO：1-8及其互补序列的核苷酸序列的核苷酸分子。
20.第19项的商品，其中所述商品选自完整或加工的种子、动物饲料、油、粗粉、面粉、薄片、麸皮、乳、奶酪、纸、奶油、酒、生物质和燃料产品。
21.一种在田间控制杂草的方法，包括在田间种植大豆事件MON 87708植物并在所述田间应用有效剂量的麦草畏除草剂以控制杂草而不伤害所述大豆事件MON 87708植物。
22.第21项的方法，其中所述有效剂量的麦草畏除草剂是每英亩约0.005磅至约8磅。
23.一种用于在田间控制杂草的方法，包括应用有效剂量的麦草畏除草剂以控制田间杂草，然后在所述田间种植大豆事件MON 87708。
24.第23项的方法，其中所述有效剂量的麦草畏除草剂是每英亩约0.005磅至约8磅，且所述种植大豆事件MON 87708是在所述应用有效剂量的麦草畏除草剂的14天内。
25.一种用于产生基本上没有毒性杂草种子的大豆种子的方法，所述方法包括：
a.在田间种植大豆事件MON 87708的种子；
b.将有效剂量的麦草畏除草剂应用于所述田间以杀灭所述田间的毒性杂草而不伤害所述大豆事件MON 87708植物；和
c.从所述田间收获大豆种子。
26.第25项的方法，其中所述毒性杂草种子选自群心菜属(Cardaria spp)、天芥菜属(Heliotropium spp)、矢车菊属(Centaurea spp.)、千里光属(Senecio spp.)、猪屎豆属(Crotalaria spp.)、茄属(Solanum spp.)、苍耳属(Xanthium spp.)、琴颈草属(Amsinckiaspp.)、山扁豆属(Cassia spp.)、田菁属(Sesbania spp.)、曼陀罗属(Datura spp.)、蓖麻属(Ricinus spp.)、蓟罂粟属(Argemone spp.)、黄麻属(Corchorus spp.)、牵牛花属(Impomoea spp.)和蓝蓟属(Echium spp.)。
27.一种产生耐受麦草畏除草剂应用的大豆植物的方法，包括：
a.将包含具有选自SEQ ID NO：1-8及其互补序列的核苷酸序列的核苷酸分子的转基因大豆事件MON 87708植物与第二种大豆植物有性杂交；
b.收集由所述杂交产生的种子；
c.使所述种子生长以产生多个后代植物；
d.以麦草畏处理所述多个后代植物；和
e.选择对麦草畏耐受的后代植物。
28.一种产生耐受麦草畏除草剂应用的大豆植物的方法，包括：
a.使包含具有选自SEQ ID NO：1-8及其互补序列的核苷酸序列的核苷酸分子的转基因大豆事件MON 87708植物自交；
b.收集由所述自交产生的种子；
c.使所述种子生长以产生多个后代植物；
d.以麦草畏处理所述多个后代植物；和
e.选择对麦草畏耐受的后代植物。
29.一种确定大豆事件MON87708植物或种子的接合性的方法，包括：
a.将包含大豆DNA的样品与包含SEQ ID NO：12、SEQ ID NO：13和SEQ ID NO：14的引物组以及包含SEQ ID NO：15和SEQ ID NO：16的探针组接触；
b.用所述样品、引物组和探针组进行核酸扩增反应；
c.在所述核酸扩增反应中检测诊断事件MON 87708的第一荧光信号和与所述第一荧光信号不同且诊断对应于事件MON 87708转基因的插入位置的天然大豆基因组DNA的第二荧光信号；和
d.分析所述核酸扩增反应中所述第一荧光信号和所述第二荧光信号的存在和/或缺乏，其中两种荧光信号的存在指示所述样品对于事件MON 87708是杂合的，而仅所述第一荧光信号的存在指示所述样品对于事件MON 87708是纯合的。
30.包含麦草畏耐受基因的在连锁群9上大致图谱位置143.5处的大豆单倍体型区域，且进一步由单倍体型窗口19743和19767限定。
附图说明
图1说明了大豆事件MON 87708的基因组中转基因插入片段的组织：[A]对应于为大豆基因组DNA和转基因插入DNA的5’部分之间的接头的60个核苷酸的SEQ ID NO：1的相对位置；[A‘]对应于为大豆基因组DNA和转基因插入DNA的5’部分之间的接头的100个核苷酸的SEQ ID NO：7的相对位置；[B]对应于为大豆基因组DNA和转基因插入DNA的3’部分之间的接头的60个核苷酸的SEQ ID NO：2的相对位置；[B’]对应于为大豆基因组DNA和转基因插入DNA的3’部分之间的接头的100个核苷酸的SEQ ID NO：8的相对位置；[C]对应于SEQ ID NO：3的相对位置，SEQ ID NO：3是位于事件MON 87708中整合到基因组中的表达盒的任意分配/指定的5′末端侧翼的大豆基因组序列；[D]对应于SEQ ID NO：4的相对位置，SEQ ID NO：4是位于事件MON 87708中整合到基因组中的表达盒的任意分配/指定的3′末端侧翼的大豆基因组序列；[E]代表包含SEQ ID NO：5的各种元件，并且是插入到事件MON 87708的基因组中的表达盒的序列；和[F]代表包含如图中从左至右示出的SEQ ID NO：3、SEQ ID NO：5和SEQID NO：4的连续序列(如SEQ ID NO：6所示)，其中包括了SEQ ID NO：1、SEQ ID NO：2、SEQ IDNO：7和SEQ ID NO：8，因为这些序列存在于事件MON 87708的基因组中。
序列简要说明
SEQ ID NO：1是代表大豆基因组DNA与整合的转基因表达盒之间的5’接头的60个核苷酸的序列。SEQ ID NO：1位于SEQ ID NO：6中第1097-1156位核苷酸处。
SEQ ID NO：2是代表大豆基因组DNA与整合的转基因表达盒之间的3’接头的60个核苷酸的序列。SEQ ID NO：2位于SEQ ID NO：6中第4100-4159位核苷酸处。
SEQ ID NO：3是位于大豆事件MON 87708的插入DNA侧翼直到并包括转基因DNA插入区域的5’序列。
SEQ ID NO：4是位于大豆事件MON 87708的插入DNA侧翼直到并包括转基因DNA插入的3’序列。
SEQ ID NO：5是整合的转基因表达盒的序列。
SEQ ID NO：6是代表位于大豆事件MON 87708的插入DNA侧翼的5’序列(SEQ IDNO：3)、插入DNA的序列(SEQ ID NO：5)和位于大豆事件MON 87708的插入DNA侧翼的3’序列(SEQ ID NO：4)的叠连群的核苷酸序列，并包括SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：7和SEQ ID NO：8。
SEQ ID NO：7是代表大豆基因组DNA与整合的转基因表达盒之间的5’接头的100个核苷酸的序列。
SEQ ID NO：8是代表大豆基因组DNA与整合的转基因表达盒之间的3’接头的100个核苷酸的序列。
SEQ ID NO：9是称为Primer SQ13570的引物的序列，且被用于鉴定大豆事件MON87708。它与靠近3’转基因插入边界的区域处的插入表达盒互补。由使用引物SQ13570和SQ13571(SEQ ID NO：10)的组合的(PE Applied Biosystems，Foster City，CA)试验产生的PCR扩增子对于事件MON87708的存在是阳性结果。
SEQ ID NO：10是称为Primer SQ13571的引物的序列，且被用于鉴定大豆事件MON87708。它与位于插入表达盒侧翼并靠近转基因DNA插入边界的3’区域互补。由使用引物SQ13570(SEQ ID NO：9)和SQ13571的组合的(PE Applied Biosystems，Foster City，CA)试验产生的PCR扩增子对于事件MON 87708的存在是阳性结果。
SEQ ID NO：11是称为Probe PB4655的探针的序列，且用于鉴定大豆事件MON87708。它与跨越插入表达盒和基因组DNA的3’接头的区域互补。该探针是6-FAMTM-标记的合成寡核苷酸。试验中，在使用引物SQ13570和SQ13571与6-FAMTM-标记的探针PB4655结合的扩增反应中荧光信号的释放是事件MON 87708诊断性的。
SEQ ID NO：12是称为Primer SQ20632的引物的序列，且被用于鉴定MON 87708事件接合性。
SEQ ID NO：13是称为Primer SQ20636的引物的序列，且被用于鉴定大豆野生型和MON 87708事件接合性。
SEQ ID NO：14是称为Primer SQ20637的引物的序列，且被用于鉴定大豆野生型接合性。
SEQ ID NO：15是称为Probe PB10130的探针的序列，且用于MON 87708事件接合性分析。
SEQ ID NO：16是称为Probe PB10131的探针序列，且用于大豆野生型接合性分析。
具体实施方式
提供下面的定义和方法以更好地定义本发明并指导本领域普通技术人员实施本发明。除非另有说明，可以根据相关领域的普通技术人员的常规用法来理解术语。
本发明提供了表现出商业可接受的对麦草畏应用的耐受性的转基因大豆事件MON87708。该事件包括插入大豆种质的染色体/基因组中的转基因DNA单一插入。“事件””通过：(i)以包括感兴趣的转基因的核酸构建体转化植物细胞，(ii)再生由转基因插入植物基因组而获得的植物群体，和(iii)选择特征为转基因插入植物基因组中特定位置的特定植物而产生术语”事件”是指包括插入植物基因组中特定位置的转基因的原始转化体。术语”事件”也指包括插入植物基因组中特定位置的转基因的转化体的后代。也可以通过转化体或其后代与另一植物之间的有性异交而产生这种后代。这种另外的植物可以是包含相同或不同转基因的转基因植物和/或非转基因植物，如来自不同品种的植物。甚至在与轮回亲本反复回交后，来自转化的亲本的插入DNA和侧翼DNA存在于杂交后代中同一基因组位置处。
本文所用的术语”大豆”是指大豆(Glycine max)，且包括可用大豆繁育的所有植物品种，包括野生大豆种以及那些属于允许在种之间繁育的大豆属的植物。
术语”事件”也指来自包括插入DNA和与插入DNA的任一侧紧邻的侧翼大豆基因组DNA的原始转化体的DNA分子。通过将转基因DNA插入大豆植物基因组的行为，即通过转化的活动而产生这种DNA分子。因此，这种DNA分子包括对事件特异性的和转基因DNA插入其中的大豆植物的基因组特有的核苷酸序列，因为这种核苷酸序列包含大豆基因组DNA的特定区域和转基因DNA插入片段的序列。因此，插入的DNA在大豆事件MON 87708中相关于周围大豆植物基因组DNA的排列对于大豆事件MON 87708是特异性的且独特的。这种DNA分子也是事件MON 87708的大豆染色体的整合部分，且由此在植物中是静态的并可以传递至植物的后代。
事件MON 87708包含赋予大豆植物对麦草畏除草剂应用的耐受性的转基因。“麦草畏”是指3，6-二氯-2-甲氧基苯甲酸。麦草畏是用于控制阔叶杂草的合成的植物生长素除草剂。大豆植物以麦草畏单加氧酶(从土壤根围中常见的嗜麦芽寡养单胞菌(Stenotrophomonas maltophilia)克隆的酶)转化。麦草畏单加氧酶是通过O-去甲基反应催化麦草畏灭活为非除草剂化合物3，5-二氯水杨酸的酶。在世界上一些地区，毒性杂草种可能污染收获的大豆种子，这可以影响以污染的大豆商品饲养的动物的健康和营养。通过麦草畏除草剂处理可以从大豆田间将这些植物清除。该组毒性杂草的成员包括群心菜属(Cardaria spp)、天芥菜属(Heliotropium spp)、矢车菊属(Centaurea spp.)、千里光属(Senecio spp.)、猪屎豆属(Crotalaria spp.)、茄属(Solanum spp.)、苍耳属(Xanthiumspp.)、琴颈草属(Amsinckia spp.)、山扁豆属(Cassia spp.)、田菁属(Sesbania spp.)、曼陀罗属(Datura spp.)、蓖麻属(Ricinus spp.)、蓟罂粟属(Argemone spp.)、黄麻属(Corchorus spp.)、牵牛花属(Impomoea spp.)和蓝蓟属(Echium spp.)。
本文所用的术语”重组”是指通常不能在自然界中发现并且因此通过人类干预产生的DNA和/或蛋白和/或生物体的形式。这种人类干预可产生重组DNA分子和/或重组植物。此处所用的”重组DNA分子”是包含非天然地存在在一起而是由人类干预产生的DNA分子的组合的DNA分子，即由至少两种彼此异源的DNA分子的组合组成的DNA分子，和/或人工合成并包含源自通常自然界中存在的聚核苷酸序列的DNA分子，和/或包含人工引入宿主细胞基因组DNA中的转基因和宿主细胞基因组的相关侧翼DNA的DNA分子。重组DNA分子的例子是此处所述的由转基因插入大豆基因组DNA中而产生的DNA分子，这种插入可能最终导致重组RNA和/或蛋白分子在该生物体中的表达。此处所用的”重组植物”是在自然界通常不存在，是人类干预的结果并且包含并入其基因组中的转基因和/或异源DNA分子的植物。作为这种基因组改变的结果，重组植物与相关的野生型植物明显不同。重组植物的一个例子是此处描述为事件MON 87708的大豆植物。
此处所用的术语”转基因”是指人工引入宿主细胞基因组中的核苷酸分子。这种转基因可以是对于宿主细胞异源的。术语”转基因植物”是指包含这种转基因的植物。
此处所用的术语”异源的”是指自然界中第一分子通常不被发现与第二分子组合。例如，分子可以源自第一物种并插入第二物种的基因组中。因此这种分子对于宿主是异源的并被人工引入宿主细胞的基因组中。
此处所用的术语”嵌合的”是指通过将第一DNA分子与第二DNA分子融合而产生的单一DNA分子，其中无论第一DNA分子还是第二DNA分子通常都不会以这种构型(即与另一种融合)存在。因此，嵌合DNA分子是通常在自然界中不会另外发现的新的DNA分子。
本发明提供了DNA分子及其对应的核苷酸序列。此处所用的术语”DNA”、”DNA分子”、”核苷酸分子”是指基因组源或合成来源的DNA分子，即脱氧核糖核苷酸碱基的聚合物或聚核苷酸分子，从5’端(上游)至3’端(下游)阅读。此处所用的术语”DNA序列”、”核苷酸序列”或”聚核苷酸序列”指的是DNA分子的核苷酸序列。此处所用的命名法是美国联邦法规§1.822的第37条所要求的，并记述在WIPO标准ST.25(1998)附录2的表1和3的表格中。根据惯例，作为SEQ ID NO：1-8及其片段提供的本发明核苷酸序列仅根据两条互补核苷酸序列链中的一条链公开。通过隐含公开，互补序列(即互补链的序列)(在本领域中也称为反向互补序列)在本发明的范围之内并明确地意图包括在要求保护的主题的范围之内。
对应于插入的转基因DNA和与插入的转基因DNA任一端侧邻的大豆基因组DNA的实质片段的完整核苷酸序列的核苷酸序列此处作为SEQ ID NO：6提供。该序列的一个分段是作为SEQ ID NO：5提供的插入转基因DNA。通过磷酸二酯键与插入的转基因DNA的5’端物理连接并因此在其侧翼的大豆基因组DNA的核苷酸序列如SEQ ID NO：3所示。通过磷酸二酯键与插入的转基因DNA的3’端物理连接并因此在其侧翼的大豆基因组DNA的核苷酸序列如SEQID NO：4所示。
大豆事件MON 87708进一步包括两个区域，一个横跨其中转基因DNA被插入基因组DNA的5’位置和一个横跨3’位置，在此处分别被称为5’和3’接头。“连接序列”或“接头”是指横跨插入的转基因DNA和相邻的侧翼基因组DNA的DNA序列和/或相应的DNA分子。连接序列可以任意地由如SEQ ID NO：1和SEQ ID NO：2提供的两个60个核苷酸的序列代表，其各显示与插入DNA的30个核苷酸相邻并连续的侧翼基因组DNA的30个核苷酸。或者，连接序列可以任意地由如SEQ ID NO：7和SEQ ID NO：8提供的两个100个核苷酸的序列代表，其各显示与插入DNA的50个核苷酸相邻并连续的侧翼基因组DNA的50个核苷酸。这些核苷酸通过磷酸二酯键相连，并在大豆事件MON 87708中作为基因组的部分存在。在大豆中，在来自大豆植物、种子或植物部分的样品中鉴定出SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：7和SEQ ID NO：8中的一个或多个可确定该DNA从大豆事件MON 87708中获得，且可诊断样品中来自大豆事件MON 87708的DNA的存在。本发明因此提供了包含至少在SEQ ID NO：1、SEQ ID NO：2、SEQ IDNO：7和/或SEQ ID NO：8中所示的核苷酸序列的DNA分子。足以包括SEQ ID NO：1、SEQ IDNO：2、SEQ ID NO：7和/或SEQ ID NO：8的源自转基因大豆事件MON 87708的任何DNA片段都在本发明的范围内。另外，包含与本段所述的任何序列互补的序列的任何聚核苷酸都在本发明的范围内。图1显示了按5’至3’方向排列的SEQ ID NO：1-5和7-8相对于SEQ ID NO：6的物理排列。
本发明提供了可用作用于诊断样品中源自大豆植物事件MON 87708的DNA的存在的引物或探针的示例性DNA分子。这种引物或探针对于目标核酸序列是特异性的，且因此可用于通过此处所述的本发明的方法鉴定大豆事件MON 87708核酸序列。
“引物”通常是被设计用于涉及热扩增的特异性退火或杂交方法中的高度纯化的、分离的聚核苷酸。在热扩增、如聚合酶链式反应(PCR)中可以将一对引物用于模板DNA如大豆基因组DNA的样品以产生扩增子，其中由这种反应产生的扩增子具有对应于位于引物与模板杂交的两个位点之间的模板DNA序列的DNA序列。此处所用的“扩增子””是用扩增技术合成的DNA的小段或片段。本发明的扩增子包含至少SEQ ID NO：1、SEQ ID NO：2、SEQ IDNO：7和/或SEQ ID NO：8。引物通常设计为与互补的靶DNA链杂交以形成引物和靶DNA链之间的杂合链，且引物的存在是聚合酶的识别点以利用靶DNA链为模板开始引物的延伸(即将额外的核苷酸聚合到延长的核苷酸分子中)。本发明所用的引物对意图指以由引物对的单个成员靶向结合的位置之间线性扩增聚核苷酸片段为目的使用两条结合双链核苷酸片段的相反链的两个引物，通常在热扩增反应或其它常规核酸扩增方法中。可用作引物的示例性DNA分子如SEQ ID NO：9-10所示。如SEQ ID NO：9和SEQ ID NO：10所示的引物对可用作第一DNA分子和不同于第一DNA分子的第二DNA分子，且二者各为SEQ ID NO：4、SEQ ID NO：5或SEQ ID NO：6中任一个的足够长度的连续核苷酸，以在与源自大豆事件MON 87708的模板DNA一起用于热扩增反应中时产生包含SEQ ID NO：2的扩增子。
“探针”是与靶核酸的链互补的分离的核酸。本发明的探针不仅包括脱氧核糖核酸或核糖核酸，而且包括特异性地结合靶DNA序列的聚酰胺和其它探针材料，并且这种结合的检测可用于诊断、区分、测定或验证具体样品中该靶DNA序列的存在。探针可以结合于常规的可检测标记或报告分子，例如放射性同位素、配体、化学发光剂或酶。可用作探针的示例性DNA分子如SEQ ID NO：11所示。
本发明的探针和引物可以具有与靶序列的完全序列同一性，尽管可以通过常规方法设计不同于靶序列而保留与靶序列优先杂交的能力的探针。为了使核酸分子用作引物或探针，它只需要在序列上充分互补以在所采用的特定的溶剂和盐浓度下形成稳定的双链结构。任何常规的核酸杂交或扩增方法可以用于鉴定样品中来自大豆事件MON 87708的转基因DNA的存在。探针和引物的长度通常至少约11个核苷酸、至少约18个核苷酸、至少约24个核苷酸或至少约30个核苷酸或更长。这样的探针和引物在严格杂交条件下特异性地与靶DNA序列杂交。Sambrook等人，1989和Haymes等人，在Nucleic Acid Hybridization，APractical Approach，IRL Press，Washington，DC(1985)描述了常规的严格性条件。如本文所用，如果两个核酸分子能够形成反平行双链核酸结构，那么这两个分子能够特异性地彼此杂交。如果两个核酸分子表现出完全的互补性，则一个核酸分子对于另一核酸分子是“互补的”。如本文所用，当一个分子的每个核苷酸都与另一分子的核苷酸互补时，它们显示出”完全的互补性”。如果两个分子可以以足够的稳定性互相杂交从而允许它们在至少常规的”低严格性”条件下保持互相退火，那么这两个分子为“最低互补的”。类似地，如果分子可以以足够的稳定性互相杂交从而允许它们在常规的“高严格性”条件下保持互相退火，那么它们是”互补的”。因此偏离完全互补性是可允许的，只要这种偏离没有完全消除该分子形成双链结构的能力。
此处所用的术语“分离的”是指至少部分地将分子与在自然或天然状态下通常与其伴随的其它分子分离。在一个实施方式中，术语”分离的”是指至少部分地与在自然或天然状态下通常在DNA分子侧翼的核酸分离的该DNA分子。因此，与通常与其无关的调控或编码序列融合的DNA分子(例如作为重组技术的结果)在此处被认为是分离的。甚至当被整合到宿主细胞的染色体中或与其它DNA分子一起存在于核酸溶液中时，这种分子被认为是分离的。
本领域技术人员熟知的多种方法可用来分离和操作本发明公开的DNA分子或其片段。例如，PCR(聚合酶链式反应)技术可以被用来扩增特定初始DNA分子和/或产生初始分子的变体。DNA分子或其片段也可以通过其他技术获得，如通过化学方法直接合成该片段，正如通常用自动寡核苷酸合成仪所实施的。
因此，本文所提供的DNA分子及相应的核苷酸序列可用于特别是鉴定大豆事件MON87708，选择包含大豆事件MON 87708的植物品种或杂种，检测样品中源自转基因大豆事件MON 87708的DNA的存在以及监测样品的大豆事件MON 87708存在和/或不存在或者源自大豆事件MON 87708的植物部分。
本发明提供了大豆植物、后代、种子、植物细胞、植物部分(如花粉、胚珠、荚、花组织、根组织、茎组织和叶组织)和商品。这些植物、后代、种子、植物细胞、植物部分和商品包含可检测量的本发明的聚核苷酸，即，如具有至少如SEQ ID NO：1-8所示的序列之一的聚核苷酸。本发明的植物、后代、种子、植物细胞和植物部分也包含一种或多种另外的转基因。这种转基因可以是编码蛋白质或RNA分子的任何核苷酸序列，该蛋白质或RNA分子赋予期望的性状，包括但不限于增强的昆虫抗性、增强的水利用效率、增强的产量性能、增强的耐旱性、提高的种子质量、改进的营养质量和/或增强的除草剂耐受性，其中该期望的性状是相对于缺乏该另外的转基因的大豆植物而测量的。
本发明提供了源自于转基因大豆植物事件MON 87708的大豆植物、后代、种子、植物细胞和植物部分，如花粉、胚珠、荚、花、根或茎组织和叶。为了使本发明能够实施，大豆事件MON 87708的种子已经根据布达佩斯条约进行了保藏。选择用于接受该保藏的保藏中心是美国典型培养物保藏中心(ATCC)，其地址为10801University Boulevard，Manassas，Virginia USA，邮编20110。该ATCC保藏中心将登录号No.PTA-9670分配给该事件MON87708的种子。
本发明提供了其基因组中包含SEQ ID NO：1和SEQ ID NO：2的DNA分子的微生物。这种微生物的一个例子是转基因植物细胞。微生物，如本发明的植物细胞，可用于许多工业应用中，包括但不限于：(i)用作科学调查或工业研究的研究工具；(ii)在培养中用于产生可能用于随后的科学研究或用作工业产品的内源的或重组的碳水化合物、脂质、核酸或蛋白质产品或小分子；以及(iii)用于现代植物组织培养技术以产生随后可用于农业研究或生产的转基因植物或植物组织培养物。微生物如转基因植物细胞的生产和使用利用现代微生物技术和人类干预以产生人造的独特的微生物。在该过程中，重组DNA被插入植物细胞的基因组中以产生与天然存在的植物细胞分离且独特的转基因植物细胞。然后该转基因植物细胞可以用现代微生物技术非常类似于细菌和酵母一样进行培养，且可以以未分化的单细胞状态存在。新的植物细胞的遗传组成和表型是由异源DNA整合进细胞基因组而产生的技术效果。本发明的另一方面是使用本发明的微生物的方法。本发明的使用微生物如转基因植物细胞的方法包括(i)通过将重组DNA整合进细胞的基因组和然后用该细胞衍生具有同样的异源DNA的额外细胞的方法；(ii)用现代微生物技术培养包含重组DNA的细胞的方法；(iii)产生并纯化来自培养细胞的内源或重组的碳水化合物、脂质、核酸或蛋白质产品的方法；以及(iv)使用现代植物组织培养技术利用转基因植物细胞产生转基因植物或转基因植物组织培养物的方法。
本发明的植物可以将事件DNA(包括转基因)传递至后代。此处所用的“后代”包括包含源自祖先植物的事件DNA和/或具有至少如SEQ ID NO：1和SEQ ID NO：2所示序列中之一的聚核苷酸的任何植物、种子、植物细胞和/或可再生植物部分。植物、后代和种子对于转基因而言可以是纯合的或杂合的。后代可以由大豆事件MON 87708植物产生的种子和/或用来自大豆事件MON 87708植物的花粉受精的植物产生的种子生长而成。
后代植物可以自花授粉(也称为“自交”)以产生植物的纯育品系(true breedingline)，即对转基因纯合的植物。适当后代的自交可以产生对于两种增加的外源基因而言为纯合的植物。
另外，后代植物可以进行异交，例如与另一种无关植物育种以产生变种或者杂交种子或植物。另外的无关植物可以是转基因或非转基因的。因此，本发明的变种或者杂交种子或植物可以通过将缺乏大豆事件MON 87708的特异和独特的DNA的第一亲本与包含大豆事件MON 87708的第二亲本杂交而获得，从而产生包含大豆事件MON 87708的特异和独特的DNA的杂种。各亲本可以是杂种或近交系/变种，只要杂交或育种产生本发明的植物或种子，即具有至少一个包含大豆事件MON 87708特异和独特的DNA和/或SEQ ID NO：1和SEQ IDNO：2的等位基因的种子。因此，可以将两种不同的转基因植物交配以产生包含两种独立地隔离的、添加的、外源基因的杂种后代。例如，MON 87708麦草畏耐受性大豆可以与其它转基因大豆植物杂交以产生具两种转基因亲本的特征的植物。它的一个例子是MON 87708麦草畏耐受性大豆与具有一种或多种额外性状如除草剂耐受性(例如大豆事件40-3-2或大豆事件MON 89788(美国专利申请公开号No.20060282915))、昆虫控制(例如大豆事件MON 87701(美国专利申请公开号No.20090130071))和/或其它期望的性状(例如增强的油组成，如大豆事件MON 87769(PCT专利申请WO2009102873))的植物杂交，从而产生对麦草畏耐受并具有一种或多种额外性状的后代植物或种子。已经证明其转基因植物耐受性以及本发明的方法可以应用的除草剂包括但不限于：草甘膦、草丁膦、磺酰脲类、咪唑啉酮类、溴草腈、茅草枯(delapon)、环己二酮、原卟啉原(protoporphyrionogen)氧化酶抑制剂和异噁唑草酮(isoxasflutole)除草剂。编码涉及除草剂耐受性的蛋白质的核苷酸分子是本领域已知的，且包括但不限于编码如下物质的核苷酸分子：草甘膦耐受的5-烯醇丙酮莽草酸-3-磷酸合酶(EPSPS)(参见例如美国专利号5,627,061；5,633,435；6,040,497；5,094,945；5,804,425；6,248,876；7,183,110；RE39,247)；草甘膦氧化还原酶(GOX)(参见例如美国专利号5,776,760)；草甘膦-n-乙酰转移酶(GAT)；用于磺酰脲类、咪唑啉酮类、三唑并嘧啶(triazolopyrimidine)类、嘧啶氧苯甲酸酯(pyrimidinyl oxybenzoate)类、磺酰氨基羰基三唑啉酮类和/或杂芳基醚耐受性的除草剂耐受乙酰乳酸合成酶(ALS，也称为乙酰羟酸合成酶(AHAS))；用于芳氧基苯氧基丙酸酯(AOPP)(如吡氟氯禾灵、喹禾灵、dichlorofop和禾草灵)耐受性的除草剂耐受乙酰辅酶A羧化酶(ACCase)或R-2，4-二氯苯氧基丙酸双加氧酶(rdpA)；用于合成植物生长素除草剂耐受性的解毒蛋白如2，4-D双加氧酶(tfdA)、R-2，4-二氯苯氧基丙酸双加氧酶(rdpA)、芳氧基链烷酸双加氧酶(AAD)和/或S-2，4-滴丙酸双加氧酶(sdpA)；用于溴草腈耐受性的溴草腈腈水解酶(Bxn)(参见例如美国专利号4,810,648)；用于达草灭耐受性的八氢番茄红素去饱和酶(crtI)；用于草丁膦和双丙氨膦耐受性的双丙氨膦抗性(bar)或草胺磷乙酰转移酶(PAT)蛋白(参见例如美国专利号5,646,024和5,276,268)；以及用于三酮(甲基磺草酮(mezotrione)、tembotrione、苯唑草酮(topromezone)、异噁唑)除草剂耐受性的蛋白质如耐受的4-羟基苯基丙酮酸双加氧酶(HPPD)、解毒的细胞色素P450或HPPD通路旁路如球形节杆菌(Artbrobacter globiformis)HPP氧化酶(HPPO)和食酸假单胞菌(Pseudomonas acidovorans)4-HPA 1-羟化酶(HPAH)和NADH氧化还原酶(HPAC)。
也考虑与亲本植物的回交以及与非转基因植物和异交，以及无性繁殖。通常用于不同性状和作物的其它育种方法的描述可以在许多参考文献之一中找到，例如Fehr，在Breeding Methods for Cultivar Development，Wilcox J.等，American Society ofAgronomy，Madison WI(1987)中。
本发明提供了源自大豆事件MON 87708的植物部分。此处所用的“植物部分”是指由源自大豆事件MON 87708植物的材料组成的植物的任何部分。植物部分包括但不限于：花粉、胚珠、荚、花、根或茎组织、纤维和叶。植物部分可以是有存活力的、非存活的、可再生的和/或不可再生的。
本发明提供了源自大豆事件MON 87708的商品。此处所用的“商品”是指由源自大豆事件MON 87708植物、种子、植物细胞或植物部分的材料组成的任何组合物或产物。商品可以卖给消费者且可以是存活的或非存活的。非存活的商品包括但不限于非存活的种子和谷粒；加工的种子、种子部分和植物部分；脱水的植物组织、冷冻的植物组织和加工的植物组织；被加工用于陆地的和/或水生动物消耗的动物饲料的种子和植物部分、油、粗粉、面粉、薄片、麸皮、纤维、乳、奶酪、纸、奶油、酒和任何其它供人类消耗的食物；以及生物质和燃料产品。存活的商品包括但不限于种子和植物细胞。因此大豆事件MON 87708可用于生产通常是由大豆获得的任何商品。源自大豆事件MON 87708的任何这种商品可以包含至少可检测量的对应于大豆事件MON 87708的特异和独特的DNA，且特别地可以包含可检测量的含SEQ ID NO：1或SEQ ID NO：2的至少15个连续核苷酸的聚核苷酸。可能使用任何检测核苷酸分子的标准方法，包括此处公开的检测方法。如果商品中有任何可检测量的SEQ ID NO：1或SEQ ID NO：2，那么商品在本发明的范围之内。
因此本发明的植物、后代、种子、植物细胞、植物部分(如花粉、胚珠、荚、花、根或茎组织和叶)和商品可用于特别是为了农业目的产生大豆事件MON 87708的种子和/或植物部分而种植植物，为了植物育种和研究目的而产生大豆事件MON 87708的后代，为了工业和研究应用而用于微生物技术和销售给消费者。
本发明提供了用麦草畏除草剂和大豆事件MON 87708控制杂草的方法和生产植物的方法。提供了在田间控制杂草的方法，其包括将大豆事件MON 87708变种或杂交植物种植于田间，和为了控制田间杂草而不伤害MON 87708植物的目的将除草有效剂量的麦草畏应用于田间。这种麦草畏除草剂的应用可以是在发芽前，即在MON 87708种子种植后和MON87708植物萌发前的任何时间，或在发芽后，即在MON 87708植物萌发后的任何时间。也提供了另一种在田间控制杂草的方法，其包括应用有效剂量的麦草畏除草剂以控制田间杂草，和然后在田间种植大豆事件MON 87708。这种麦草畏除草剂的应用可以是在种植前，即在MON 87708种子种植前，且可以在种植前的任何时间进行，包括但不限于种植前约14天至种植前约1天。本发明也提供了用于产生基本上没有毒性杂草种种子的大豆种子的方法，该方法通过在田间种植麦草畏耐受品种MON 87708的种子，应用足以杀灭毒性杂草种的发芽后有效剂量的麦草畏除草剂于田间和从田间收获种子而进行。用于田间的除草有效剂量的麦草畏在一个生长季应在每英亩约0.005磅至每英亩8磅的范围内。麦草畏的多次应用可用于一个生长季，例如，两次应用(如种植前应用和发芽后应用或发芽前应用和发芽后应用)或三次应用(如种植前应用、发芽前应用和发芽后应用)。
提供了用于产生包含对本发明的转基因事件MON 87708特异和独特的DNA序列的除草剂耐受性大豆植物的方法。这些方法中所用的转基因植物对于该转基因而言可以是纯合的或杂合的。这些方法产生的后代植物可以是变种或杂交植物；可以由大豆事件MON87708产生的种子和/或用大豆事件MON 87708植物的花粉受精的植物产生的种子生长而成；且对于该转基因而言可以是纯合的或杂合的。随后，后代植物可以自花传粉以产生植物的纯育品系，即对转基因纯合的植物，或者可以异交，例如与另一种无关植物配种以产生变种或杂交种子或植物。
可以通过将包含含有SEQ ID NO：1和SEQ ID NO：2的序列的核苷酸分子的事件MON87708植物与另一种大豆植物有性杂交而因而产生种子(其随后生长为后代植物)而产生耐受麦草畏除草剂应用的大豆植物。然后用麦草畏除草剂处理这些后代植物以选择对麦草畏除草剂耐受的后代植物。或者，可以用诊断方法分析这些后代植物以选择包含事件MON87708 DNA的后代植物。在杂交中使用的另外的可以植物对麦草畏除草剂耐受或不耐受，且可以是或不是转基因的。产生的后代植物和/或种子可以是变种或杂交种子。在实施该方法时，可以通过人类干预而完成或帮助一种植物与另一种植物有性杂交的步骤，即异花授粉，例如：通过手工收集一种植物的花粉并将该花粉与第二种植物的花柱或柱头接触；通过人手和/或行为去除、破坏或覆盖植物的雄蕊或花药(例如通过去雄或通过应用化学杀配子剂)以防止自然的自花传粉，且为使受精发生，异花授粉可以通过人为将传粉昆虫放置到”定向授粉”的位置(例如，通过在果园或田间设置蜂房或通过将植物和传粉昆虫笼养)；通过人为开放或去除花的部分以允许外来花粉在花柱或柱头上安置或接触(例如，在天然具有阻碍或防止异花授粉的花的大豆中，使其在没有人为干预的情况下使它们天然的强制自花传粉)；通过植物的选择性安置(例如故意将植物种植于传粉邻近区)；和/或应通过用化学物质以促使开花或促进(柱头对花粉)的感受性而发生。
可以通过将包含含有SEQ ID NO：1和SEQ ID NO：2的序列的核苷酸分子的事件MON87708植物自交而因此产生种子(其随后生长为后代植物)而产生耐受麦草畏除草剂应用的大豆植物。然后可以用麦草畏除草剂处理这些后代植物以选择对麦草畏除草剂耐受的后代植物。或者，可以用诊断方法分析这些后代植物以选择包含事件MON 87708 DNA的后代植物。在实施该方法时，可以通过人类干预完成或帮助一个植物与其自身有性杂交的步骤，即自花传粉或自交，例如：通过手工收集植物的花粉并将该花粉与同一植物的花柱或柱头接触，且然后任选地防止植物的进一步受精；通过人手和/或行为去除、破坏或覆盖其它相近植物的雄蕊或花药(例如通过去雄或通过应用化学杀配子剂)以防止自然的自花传粉且为使受精发生，自花传粉可以通过人为将传粉昆虫安置到“定向授粉”的位置(例如，通过将植物和传粉昆虫笼养)；通过人为操作花或其部分以允许自花传粉；通过植物的选择性安置(例如故意将植物种植于传粉邻近区)；和/或通过应用化学物质以促使开花或促进(柱头对花粉的)的感受性而发生。
这些方法涵盖的以及通过使用这些方法产生的后代大豆植物与其他大豆植物明显不同，例如因为后代大豆植物和种子：是重组的而因此由人为干预产生；是麦草畏除草剂耐受的；包含至少一种由本发明的转基因DNA组成的等位基因；和/或包含可检测量的选自SEQ ID NO：1和SEQ ID NO：2的聚核苷酸序列。种子可以选自单个后代植物，且只要该种子包含SEQ ID NO：1和SEQ ID NO：2，它就在本发明的范围之内。
在实施本发明时，可以将两种不同的转基因植物杂交以产生包含两种独立隔离的异源基因的杂种后代。适宜后代的自交可以产生对于两种基因为纯合的植物。也考虑与亲本植物的回交以及与非转基因植物的异交，以及无性繁殖。常用于不同性状和作物的其它方法的描述可以在许多参考文献之一中找到，例如Fehr，在Breeding Methods forCultivar Development，Wilcox J.等，American Society of Agronomy，Madison WI(1987)中。
此处公开的方法中使用的植物和种子也可以包含一种或多种额外的转基因。这类转基因可以是编码蛋白质或RNA分子的任何核苷酸序列，该蛋白质或RNA分子赋予期望的性状，包括但不限于增强的昆虫抗性、增强的水利用效率、增强的产量性能、增强的耐旱性、提高的种子质量、改进的营养质量和/或增强的除草剂耐受性，其中该期望的性状是相对于缺乏这种额外转基因的大豆植物而测量的。
因此本发明的方法可用于特别是当为了农业或研究目的产生大豆事件MON 87708的种子和/或植物部分而种植植物的同时控制田间杂草，为了植物育种或研究的目的选择大豆事件MON 87708的后代，以及产生大豆事件MON 87708的后代植物和种子。
本发明的植物、后代、种子、植物细胞、植物部分(如花粉、胚珠、荚、花、根或茎组织和叶)和商品可以对于DNA组成、基因表达和/或蛋白表达进行评估。这种评估可以通过使用任何标准方法如PCR、northern印记、southern分析、western印记、免疫沉淀和ELISA或通过使用此处提供的检测方法和/或检测试剂盒而进行。
提供了检测样品中源自大豆事件MON 87708的大豆细胞、组织、种子或植物的DNA的存在的方法。一种方法包括(i)从至少一个大豆细胞、组织、种子或植物中提取DNA样品，(ii)将DNA样品与能够在适于DNA扩增的条件下从事件MON 87708 DNA产生扩增子的引物对接触，(iii)进行DNA扩增反应，和然后(iv)检测扩增子分子和/或确认扩增子的核苷酸序列包含事件MON 87708特异性的核苷酸序列，如选自SEQ ID NO：1-8的序列。该扩增子应当是事件MON87708特异性的，如包含SEQ ID NO：1或SEQ ID NO：2的扩增子。扩增子中对于事件MON 87708特异性的核苷酸序列的检测可以确定和/或诊断样品中大豆事件MON 87708特异性DNA的存在。能够在适于DNA扩增的条件下从事件MON 87708 DNA产生扩增子的引物对的例子如SEQ ID NO：10-11所示。本领域技术人员可以轻易地设计其它引物对，它们包含SEQID NO：6的至少一个片段。检测样品中源自大豆事件MON 87708的大豆细胞、组织、种子或植物的DNA的存在的另一种方法包括(i)从至少一个大豆细胞、组织、种子或植物中提取DNA样品，(ii)将DNA样品与对事件MON 87708特异性的DNA探针接触，(iii)在严格杂交条件下使探针和DNA样品进行杂交，和然后(iv)检测探针和靶DNA样品之间的杂交。对于事件MON87708 DNA特异性的DNA探针的序列的例子如SEQ ID NO：11所示。本领域技术人员可以轻易地设计其它探针，它们包含SEQ ID NO：6的至少一个片段。探针与DNA样品的杂交的检测可诊断样品中大豆事件MON 87708特异性DNA的存在。或者，不存在杂交可诊断样品中大豆事件MON 87708特异性DNA的不存在。
提供了可用于鉴定样品中的大豆事件MON87708 DNA的DNA检测试剂盒，而且也可以应用于培育含有适宜的事件DNA的大豆植物的方法。这种试剂盒包括包含SEQ ID NO：1-8的片段的DNA引物和/或探针。这种试剂盒的一个例子包含SEQ ID NO：6的足够长度的连续核苷酸的至少一种DNA分子，以用作可用于检测样品中源自转基因大豆事件MON 87708的DNA的存在和/或不存在的DNA探针。源自转基因大豆事件MON 87708的DNA包含SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：7和/或SEQ ID NO：8。提供了足以用作DNA探针的DNA分子，其用于确定、检测或诊断样品中大豆事件MON 87708DNA的存在和/或不存在，如SEQ ID NO：11所示。本领域技术人员可以轻易地设计其它探针，它们包含SEQ ID NO：6的至少15个连续核苷酸且对于大豆事件MON 87708 DNA足够独特以鉴定源自该事件的DNA。另一种类型的试剂盒包含可用于产生用于检测样品中源自于转基因大豆事件MON 87708的DNA的存在和/或不存在的扩增子的引物对。这种试剂盒采用的方法包括将目标DNA样品与此处所述的引物对接触，然后进行足以产生包含SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：7和/或SEQ ID NO：8的扩增子的核酸扩增反应，和然后检测扩增子的存在和/或不存在。这种方法也可以包括对扩增子或其片段进行测序，这可以确定，即诊断目标DNA样品中大豆事件MON 87708特异性DNA的存在。本领域技术人员可以轻易地设计其它引物对，它们包含SEQ ID NO：6的至少15个连续核苷酸且对于大豆事件MON 87708 DNA足够独特以鉴定源自该事件的DNA。
可以通过本领域已知的各种核酸扩增方法中的任何一种，包括热扩增方法，完成核酸扩增。许多用于对这些方法产生的扩增子进行检测、定量和/或测序的技术是本领域已知的。用于实施本发明的一种示例性技术是(PE Applied Biosystems，Foster City，CA)。
本发明的试剂盒和检测方法可用于特别是鉴定大豆事件MON 87708，选择包含大豆事件MON 87708的植物品种或杂种，检测样品中源自转基因大豆事件MON 87708的DNA的存在以及监测样品的大豆事件MON 87708的存在和/或不存在或源自大豆事件MON 87708的植物部分。
来自大豆事件MON 87708(典型种子样品作为ATCC PTA-9670保藏)的异源DNA插入序列、连接序列或侧翼序列可以通过使用源自本发明提供的序列的引物扩增来自所述事件的这种序列，接着对扩增子或克隆的DNA进行标准DNA测序来验证(如果必要，进行校正)。
此处所述的术语”包含”是指”包括，但不限于”。
包括的下面的实施例证明本发明的某些优选实施方式的实施例。本领域的技术人员应该理解，下面的实施例中公开的技术代表了本发明人发现在实施本发明中运用良好的技术，因此可以被认为是构成实施本发明的优选方式的实施例。然而，本领域的技术人员根据本公开内容应该理解，在不背离本发明的精神和范围的情况下，可以对公开的具体实施方式进行许多改变而仍然获得相同或类似的结果。
实施例
实施例1：大豆A3525的转化及MON 87708事件选择
通过大豆的农杆菌介导转化产生大豆植物MON 87708。将大豆细胞进行转化并再生成完整的大豆植物，且从表现出植物表达盒的完整性和对麦草畏的耐受性的植物群体中选择单个植物。从该群体中选择和鉴定大豆植物事件MON 87708。
利用转化载体PV-GMHT4355通过大豆分生组织的农杆菌介导转化产生转基因麦草畏耐受的大豆植物MON 87708。该方法如美国专利号6,384,301(在此处通过引用包含)所述，其允许在不利用愈伤组织的情况下产生转化的植物。简言之，从萌发的A3525大豆种子(Asgrow，St Louis，MO)的胚中切取分生组织。在与携带该载体的农杆菌共培养后，将分生组织置于含草甘膦(Monsanto，St Louis，MO)、氨苄青霉素二钠盐、头孢噻肟钠盐和替卡西林二钠盐/克拉维酸钾混合物的选择培养基上以抑制未转化的植物细胞和过量农杆菌的生长。然后将分生组织置于有利于发芽和根发育的培养基中。选择具有正常表型特征的生根的植物并转移至土壤中进行生长及进一步评价。
将通过上述转化产生的R0植物转移至土壤进行生长，然后自交以产生R1种子。在随后的R0植物自交以产生R1代的过程中，未连锁的T-DNA I(dmo表达盒)和T-DNA II(cp4epsps表达盒)插入发生分离。将非致死剂量的草甘膦应用于R1植物。选择具有小损伤的植物用于进一步分析，而未显示损伤，即含T-DNA II(cp4 epsps表达盒)的植物从后续发展中去除。随后，鉴定仅含单一T-DNA I插入片段(即dmo基因盒)的R0植物。T-DNA I表达盒包含具有双重增强子区域(P-PClSV.FLt-enh)的花生褪绿条纹病毒(Peanut Chlorotic StreakVirus)(PClSV)启动子；可操作地与源自烟草蚀纹病毒(L-TEV)RNA转录本的DNA前导序列连接；可操作地与编码来自豌豆(Pisum sativum)的1，5-二磷酸核酮糖羧化酶小亚基(SSU)的N末端叶绿体转运肽(TS-RbcS-3C)的DNA分子连接；可操作地与豌豆的1，5-二磷酸核酮糖羧化酶小亚基(SSU)的成熟蛋白质的部分(CR-RbcS-3C)连接；可操作地与编码来自嗜麦芽寡养单胞菌(Stenotrophomonas maltophilia)的麦草畏单加氧酶(DMO)的DNA分子连接(嗜麦芽假单胞菌(Pseudomonas maltophilia)是DMO基因来源的原始名称，随后该来源生物体先被重新分类为嗜麦芽黄单胞菌(Xanthomonas maltophilia)，然后重新分类为嗜麦芽寡养单胞菌(Stenotrophomonas maltophilia))；可操作地与源自豌豆的1，5-二磷酸核酮糖羧化酶小亚基的3’UTR DNA分子(T-Ps.RbcS2-E9)连接。通过分析技术(包括TaqMan、PCR分析和喷洒除草剂)的组合选择植物。基于其优势表型特征、综合分子谱分析及其期望的单倍体型关联从约2400个单个转基因事件中选择MON 87708事件。然后将事件MON 87708与事件MON 89788(草甘膦耐受性的)进行杂交。该杂交的后代用麦草畏(BASF，Research Triangle Park，NC)、草甘膦(RoundupMonsanto Co.，StLouis，MO)或麦草畏和草甘膦的组合进行处理。该处理在种植前、种植后的营养3生长期(V3)和种植后的繁殖1期(R1)进行。处理的植物对于种植前除草剂处理在处理后14天(DAT)、对于萌发后VE期的处理在3DAT以及对于萌发后R1期的处理在3DAT对生长抑制百分比进行评分。如表1所示以各种不同的每英亩比率应用除草剂。抑制百分比测量值代表该重复的平均值。
表1：利用MON89788x MON 87708的麦草畏和/或Roundup的耐受性测试
麦草畏耐受转基因在大豆事件MON 87708中被映射到连锁群9大致图谱位置143.5处。相关的单倍体型窗口19743和19767对产量、成熟、高度或倒伏无影响。单倍体型相关信息如表2所示，其中GM_A92205表示事件MON 87708。
表2：单倍体型相关LG9，Pos 143.5
实施例2：MON 87708 DNA序列的鉴定
通过详细的分子分析鉴定插入大豆植物MON 87708基因组中的DNA和侧翼序列。这些分析包括：插入序列、插入数(在大豆基因组中的整合位点数)、拷贝数(一个基因座中转基因DNA的拷贝数)、插入基因盒的完整性、侧翼序列和插入与大豆基因组的单倍体型区域的关联。
使用了包括植物表达盒的完整编码区域及其各自的调控元件、启动子、内含子和聚腺苷酸化序列的分子DNA探针。分析显示MON 87708包含具有一个拷贝的表达盒的单转基因DNA插入。进行反向PCR和DNA序列分析以确定5’和3’插入-植物基因组接头，确认插入片段内元件的组织(图1)和确定大豆植物MON 87708中插入片段的完整DNA序列(此处如SEQID NO：5所示)。本发明的一个方面是在其基因组中包含如图1所示的连锁的转基因遗传元件和对麦草畏耐受的大豆植物。
使用Ochman等人，1990(PCR Protocols：A guide to Methods andApplications，Academic Press，Inc.)所描述的反向PCR和/或基因组步移技术确定MON87708中侧邻转基因DNA插入的序列。从A3525和来自标准温室条件下生长的组织的转基因大豆株系分离植物基因组DNA。将约1克嫩叶组织与液氮混合并用研钵和研杵研磨成细粉。根据厂商的方案用NucleonTM PhytoPureTM基因组DNA提取试剂盒(RPN8511，Amersham，Piscataway，NJ)提取DNA。在最终的沉淀步骤之后，将DNA重悬于0.5ml TE(10mM Tris-HClpH 8.0，1mM EDTA)中。本领域技术人员可以改进该方法以从大豆的任何组织(包括但不限于种子)中提取DNA。另外，用基于转基因DNA的限制分析选择的限制性内切酶消化DNA的等份试样。经过限制性片段的自身连接后，使用由转基因DNA序列设计的引物进行PCR，其将扩增由转基因DNA的5’和3’端延伸的序列。通过琼脂糖凝胶电泳分离PCR产物，并使用QIAGEN凝胶纯化试剂盒(Qiagen，Valencia，CA)纯化。直接使用标准DNA测序方案对随后的DNA产物测序。延伸至表达盒转基因DNA的右边界序列中的5’侧翼序列如SEQ ID NO：3所示([C]，见图1)。延伸至表达盒转基因DNA的左边界序列中的3’侧翼序列如SEQ ID NO：4所示([D]，见图1)。完全整合入A3525基因组DNA中的表达盒DNA的部分如SEQ ID NO：5所示([E]，见图1)。
将分离的DNA分子序列与转基因DNA序列进行比较以鉴定侧翼序列和共分离的转基因DNA片段。通过使用基于推导的侧翼序列数据和已知的转基因DNA序列设计的引物进行PCR来确认表达盒的存在。使用由MON87705中的侧翼序列设计的引物分离对应于转基因DNA整合到转化株系中的同一区域的野生型序列。使用扩增系统(Invitrogen，Carlsbad，CA)进行PCR反应。相对于多个核苷酸和蛋白质数据库分析MON87708中的侧翼DNA序列和A3525野生型序列。该信息用于检验转基因与植物基因组的关系，并观察插入位点的完整性。利用侧翼序列和野生型序列设计用于用来鉴定该事件的TaqMan终点分析中的引物。使用该信息开发了接合性分析。
实施例3：事件特异性的终点分析
本实施例描述了开发用于鉴定样品中事件MON 87708的事件特异性终点热扩增方法。用于事件MON 87708特异性终点方法的条件的实例如下：步骤1：将18兆欧水调节至10μl的最终体积。步骤2：将5.0μl的2X通用主混合物(dNTPs，酶，缓冲液)调节至1X最终浓度。步骤3：将0.5μl事件引物-1(SQ13570)和事件引物-2(SQ13571)混合物(重悬于18兆欧水中至各引物的浓度为20uM)调节至1.0μM最终浓度(例如在微量离心管中，加入以下物质以20uM最终浓度下达到500μl体积：100μM浓度下100μl的引物SQ13570(SEQ ID NO：9)；100μM浓度下100μl的引物SQ13571(SEQ ID NO：10)；300μl的18兆欧的水)。步骤4：将0.2μl事件6-FAMTM MGB探针PB4655(重悬浮于18兆欧的水中至10μM浓度)(SEQ ID NO：11)调节至0.2μM最终浓度。步骤5：将0.5μl内部对照引物-1和内部对照引物-2的混合物(重悬于18兆欧的水中至各引物的浓度为20μM)调节至1.0μM最终浓度。步骤6：将0.2μl内部对照VIC TM探针调节至0.2μM最终浓度(重悬于18兆欧的水中至10μM浓度)。步骤7：3.0μl提取的DNA(模板)用于以下各种的各样品：1.待分析的叶样品；2.阴性对照(非转基因DNA)；3.阴性水对照(无模板)；4.阳性对照MON 87708 DNA。步骤8：热循环条件如下：50℃ 2分钟，1个循环；95℃ 10分钟，1个循环；95℃ 15秒然后64℃ 1分钟，10个循环，-1℃/循环；95℃ 15秒然后54℃ 1分钟，30个循环；10℃的最终循环。
用于该终点分析的DNA引物是引物SQ13570(SEQ ID NO：9)、SQ13571(SEQ ID NO：10)和6-FAMTM标记的探针PB4655(SEQ ID NO：11)。6-FAMTM是连接到DNA探针上的AppliedBiosystems(Foster City，CA)的荧光染料产品。对于MGBTM探针，Taq DNA聚合酶的5’核酸外切酶活性从5’-末端在荧光团和猝灭剂之间切割探针。当与靶DNA链杂交时，猝灭剂和荧光团充分分离从而产生荧光信号，因此释放荧光。当在这些反应方法中采用PB4655(SEQ ID NO：11)时，SQ13570(SEQ ID NO：9)和SQ13571(SEQ ID NO：10)产生诊断事件MON87708 DNA的DNA扩增子。用于此分析的对照应包括来自包含事件MON87708 DNA的大豆的阳性对照、来自非转基因大豆的阴性对照和不包含模板DNA的阴性对照。此外，PCR反应的对照包括对大豆基因组中单拷贝基因特异的内部对照引物和内部对照探针。本领域技术人员知道如何设计对大豆基因组中单拷贝基因特异的引物。优化这些分析方法以用于Applied BiosystemsPCR System 9700(以最高速运行)或MJ Research DNAEngine PTC-225热循环仪。本领域技术人员已知产生鉴定事件MON87708 DNA的扩增子的其它方法和仪器在本领域的技能范围内。
使得显示出表达盒存在的R0植物发育成完全成熟的植物。使用基于麦草畏耐受转基因盒的序列设计的探针进行Southern印记分析以确定连锁。也用Southern分析和终点的组合评价R0植物的表达盒的拷贝数。
接合性分析可用于确定包含事件的植物对于事件DNA是否是纯合的，即在染色体对的各染色体上的相同位置包含外源DNA；或者对于事件DNA是否是杂合的，即在染色体对的仅一条染色体上包含外源DNA；或者对于事件DNA为空，即是野生型的。也使用终点热扩增方法进行事件MON 87708的接合性分析。本实施例描述了开发用于确定样品中事件MON 87708的接合性的事件特异性终点热扩增方法。对于该分析，采用三引物分析方法，其中引物SQ20632(SEQ ID NO：12)从插入的外源DNA和基因组DNA的3’接头处杂交并特异性延伸，引物SQ20636(SEQ ID NO：13)从侧邻插入的外源DNA的3′端的DNA杂交并特异性延伸，引物SQ20637(SEQ ID NO：14)从其中整合插入的外源DNA的基因组DNA杂交并特异性延伸。这三个引物可诊断事件。在本实施例中，引物SQ20636(SEQ IDNO：13)和引物SQ20632(SEQ ID NO：12)以及6-FAMTM-标记的寡核苷酸探针PB10130(SEQ IDNO：15)在存在插入的外源DNA的拷贝时进行诊断。在本实施例中，SQ20636(SEQ ID NO：13)和引物SQ20637(SEQ ID NO：14)以及VICTM-标记的寡核苷酸探针PB10131(SEQ ID NO：16)在基因组DNA中不存在插入的外源DNA的拷贝时(即野生型)进行诊断。当这三个引物与两个探针在PCR反应中与从事件MON 87708纯合的植物中提取的DNA一起混合时，具有仅仅来自6-FAMTM-标记的寡核苷酸探针PB10130(SEQ ID NO：15)的荧光信号，这指示和诊断事件对于MON 87708纯合的植物。当这三个引物与两个探针在PCR反应中与从事件MON 87708杂合的植物中提取的DNA一起混合时，存在来自6-FAMTM-标记的寡核苷酸探针PB10130(SEQ ID NO：15)和VICTM-标记的寡核苷酸探针PB10131(SEQ ID NO：16)的荧光信号，这指示和诊断对于事件MON 87708为杂合的植物。当这三个引物与两个探针在PCR反应中与从对于事件MON87708为空的植物(即野生型)中提取的DNA一起混合时，仅存在来自VICTM-标记的寡核苷酸探针PB10131(SEQ ID NO：16)的荧光信号，这指示和诊断对于事件MON 87708为空的植物。可用于该方法的条件的例子如下。步骤1：将18兆欧的水调节至10μl的最终体积。步骤2：将5.0μl的2X通用主混合物(Applied Biosystems cat#4304437；dNTPs，酶，缓冲液)调节至1X最终浓度。步骤3：将0.5μl接合性引物SQ20632、SQ20636、SQ20637(重悬浮于18兆欧的水中至各引物的浓度为20μM)调节至1.0μM最终浓度。步骤4：将0.2μl 6-FAMTM探针PB10130(SEQID NO：15)(重悬浮于18兆欧的水中至10μM浓度)调节至0.2μM最终浓度。步骤5：将0.2μlVICTM探针PB10131(SEQ ID NO：16)(重悬浮于18兆欧的水中至10μM浓度)调节至至0.2μM最终浓度。步骤6：3.0μl提取的DNA(模板)用于以下各种的各样品：1.待分析的叶样品(在水中稀释的4-80ng基因组DNA)；2.阴性对照(非转基因大豆DNA；4ng，稀释于水中)；3.阴性水对照(无模板；DNA重悬于其中的溶液)；4.来自已知杂合事件的阳性对照MON 87708基因组DNA(4ng，稀释于水中)；5.来自已知纯合事件的阳性对照MON 87708基因组DNA(4ng，稀释于水中)。步骤7：温和混合。步骤8：使用Applied BiosystemsPCR System 9700(以最高速运行)或MJ Research DNA Engine PTC-225热循环仪的热循环仪条件如下：50℃ 2分钟，1个循环；95℃ 10分钟，1个循环；95℃ 15秒然后64℃ 1分钟(-1℃/循环)，10个循环；95℃ 15秒然后54℃ 1分钟，30个循环；任选地，在终点分析过程中，另外10-20个循环的95℃ 15秒然后64℃ 1分钟(-1℃/循环)可以提供更明显的群体分离；保持在10℃一个循环。
实施例4：在任何MON 87708育种活动中鉴定事件MON 87708
下面的实施例描述如何用大豆事件MON 87708在任何育种活动的后代中鉴定MON87705事件。
使用DNA事件引物对产生诊断大豆事件MON87708的扩增子。诊断MON87708的扩增子包含至少一个连接序列，如SEQ ID NO：1或SEQ ID NO：2或SEQ ID NO：7或SEQ ID NO：8所示。产生MON87705的诊断扩增子的事件引物对包括基于侧翼序列和插入的表达盒的引物对。为了获得其中存在SEQ ID NO：1的诊断扩增子，设计了基于SEQ ID NO：3的碱基1至1126的正向引物分子和基于插入表达盒DNA序列(SEQ ID NO：5的1至3303位)的反向引物分子，其中该引物分子是长度足以与SEQ ID NO：3和SEQ ID NO：5特异性杂交的连续核苷酸。为了获得其中存在SEQ ID NO：2的诊断扩增子，设计了基于插入表达盒DNA序列(SEQ ID NO：5的1至3303位)的正向引物分子和基于3’侧翼序列(SEQ ID NO：4的碱基131至1947)的反向引物分子，其中该引物分子是长度足以与SEQ ID NO：4和SEQ ID NO：5特异性杂交的连续核苷酸。从实际的目的来看，应设计产生具有限定的大小范围，例如100至1000个碱基的扩增子的引物。较小(较短的聚核苷酸长度)的大小调节的扩增子通常在PCR反应中更可靠地产生，允许更短的循环时间，并且可以容易地在琼脂糖凝胶上分离和显现或适用于终点样分析中。可以产生并使用本领域已知的DNA扩增子检测方法检测更小的扩增子。此外，使用引物对产生的扩增子可以被克隆到载体中，增殖，分离并测序，或者可以用本领域确立的方法直接测序。可用于产生诊断MON87708或其后代的扩增子的DNA扩增方法的源自SEQ ID NO：3和SEQ ID NO：5的组合或SEQ ID NO：4和SEQ ID NO：5的组合的以任何引物对是本发明的方面。可用于产生诊断MON87708或其后代的扩增子的DNA扩增方法中的包含SEQ ID NO：3的至少11个连续核苷酸或其互补序列的任何单个分离的DNA多核苷酸引物分子是本发明的方面。可用于产生诊断MON 87708或其后代的扩增子的DNA扩增方法中的包含SEQ ID NO：4的至少11个连续核苷酸或其互补序列的任何单个分离的DNA多核苷酸引物分子是本发明的方面。可用于产生诊断MON 87708或其后代的扩增子的DNA扩增方法中的包含SEQ ID NO：5的至少11个连续核苷酸或其互补序列的任何单个分离的DNA多核苷酸引物分子是本发明的方面。
实施例3中显示了用于这种分析的扩增条件的实例。然而，对于这些方法的任何改变或使用与SEQ ID NO：3或SEQ ID NO：4或MON87708转基因插入片段(SEQ ID NO：5)中包含的遗传元件的DNA序列同源或互补的DNA引物产生诊断MON 87708的扩增子都属于本领域的范围。诊断扩增子包含与至少一个转基因/基因组连接DNA(SEQ ID NO：1或SEQ ID NO：2或SEQ ID NO：7或SEQ ID NO：8)或其实质部分同源或互补的DNA分子。
对事件MON87708植物组织样品的分析应包括来自事件MON 87708的阳性组织对照、来自非事件MON87708的大豆植物(例如但不限于A3525)的阴性对照和不包含大豆基因组DNA的阴性对照。扩增内源性大豆DNA分子的引物对用作DNA扩增条件的内部对照。另外的引物序列可以由DNA扩增方法领域中的技术人员从SEQ ID NO：3、SEQ ID NO：4或SEQ IDNO：5中选择，且选择由实施例3所示的方法产生扩增子的条件可能会有所不同，但产生诊断事件MON87708 DNA的扩增子。对实施例3的方法进行改良的这些DNA引物序列的应用在本发明的范围内。由源自SEQ ID NO：3、SEQ ID NO：4或SEQ ID NO：5的至少一个DNA引物序列产生的诊断MON 87708的扩增子是本发明的方面。
包含至少一个源自SEQ ID NO：3、SEQ ID NO：4或SEQ ID NO：5的足够长度的连续核苷酸的DNA引物的DNA检测试剂盒是本发明的方面，该DNA引物在DNA扩增方法中使用时产生对于MON 87708或其后代的诊断扩增子。在DNA扩增法中测试时产生诊断MON 87708的扩增子的MON 87708大豆植物、植物部分、植物细胞、种子或商品是本发明的方面。可以通过使用Applied BiosystemsPCR System 9700(以最高速运行)或MJ ResearchDNA Engine PTC-225热循环仪或任何其它如实施例3所示能够用来产生诊断MON 87708的扩增子的其它扩增系统对MON 87708扩增子进行分析。
以上公开的并且在权利要求书中所述的大豆事件MON 87708种子的代表性样品已经按照布达佩斯条约的规定在美国典型培养物保藏中心(ATCC)，10801UniversityBoulevard，Manassas，VA.20110进行了保藏。该保藏的ATCC保藏号为PTA-9670。该保藏将在该保藏机构保存30年的时间或最后请求后的5年或专利的有效期间，以较长者为准，并且在此期间必要时将进行更换。
上面已经说明并描述了本发明的原理，本领域技术人员应当明白，可以在不背离这种原理的情况下在配置和细节方面对本发明进行修改。我们要求保护在所附的权利要求的精神和范围内的所有修改。
Claims (21)
1.一种重组DNA分子，其包含含有选自SEQ ID NO：1-4和6-8及其互补序列的核苷酸序列的核苷酸分子。
2.权利要求1的重组DNA分子，其中所述重组DNA分子通过将异源核酸分子插入大豆植物、植物细胞或种子的基因组DNA中而形成。
3.权利要求1的重组DNA分子，其中所述DNA分子源自转基因大豆事件MON 87708，其代表性种子样品被保藏为ATCC PTA-9670。
4.权利要求1的重组DNA分子，其中所述DNA分子处于源自大豆事件MON 87708的大豆植物、植物细胞、植物部分或商品中，大豆事件MON 87708的代表性种子样品被保藏为ATCCPTA-9670。
5.权利要求1的重组DNA分子，其中所述DNA分子处于源自大豆事件MON 87708的种子中，大豆事件MON 87708的代表性种子样品被保藏为ATCC PTA-9670。
6.权利要求1的重组DNA分子，其中所述DNA分子是诊断源自事件MON 87708的DNA存在的扩增子，大豆事件MON 87708的代表性种子样品被保藏为ATCC PTA-9670。
7.一种DNA分子，其包含具有SEQ ID NO：6的足够长度的连续核苷酸序列及其互补序列的核苷酸序列，以作为在严格杂交条件下与包含选自SEQ ID NO：1-4和6-8的核苷酸序列的DNA分子杂交并且在严格杂交条件下不与不含选自SEQ ID NO：1-4和6-8的核苷酸序列的DNA分子杂交的DNA探针。
8.一对DNA分子，由第一DNA分子以及不同于第一DNA分子的第二DNA分子组成，其中所述第一DNA分子和第二DNA分子各包含具有SEQ ID NO：6的足够长度的连续核苷酸的核苷酸序列或其互补序列的核苷酸分子，以与源自事件MON 87708的DNA一起用于扩增反应中以产生诊断样品中大豆事件MON 87708的扩增子时作为DNA引物，大豆事件MON 87708的代表性种子样品被保藏为ATCC PTA-9670。
9.一种检测样品中源自大豆事件MON 87708的DNA分子的存在的方法，所述方法包括：
a.将样品与权利要求7的DNA探针接触；
b.使所述样品和所述DNA探针经受严格杂交条件；和
c.检测所述DNA探针与所述样品中DNA分子的杂交，其中所述DNA探针与所述DNA分子的杂交指示了所述样品中源自大豆事件MON 87708的DNA分子的存在，大豆事件MON 87708的代表性种子样品被保藏为ATCC PTA-9670。
10.一种检测样品中源自大豆事件MON 87708的DNA分子的存在的方法，所述方法包括：
a.将样品与权利要求8的DNA分子对接触；
b.进行扩增反应，其足以产生包含选自SEQ ID NO：1-4和6-8及其互补序列的序列的DNA扩增子；和
c.检测所述反应中所述DNA扩增子的存在，其中所述DNA扩增子在所述反应中的存在指示了所述样品中源自大豆事件MON87708的DNA分子的存在，大豆事件MON 87708的代表性种子样品被保藏为ATCC PTA-9670。
11.一种DNA检测试剂盒，其包括一对DNA引物或至少一种DNA探针，其中a)所述DNA引物对包括至少一个包含SEQ ID NO：6的足够长度的连续核苷酸的核苷酸序列或其互补序列的DNA分子，该DNA引物对能够产生诊断事件MON 87708的扩增子，且其中所述扩增子包含SEQID NO：1或SEQ ID NO：2；b)所述至少一种DNA探针诊断事件MON 87708并包含SEQ ID NO：1或SEQ ID NO：2，其中大豆事件MON 87708的代表性种子样品被保藏为ATCC PTA-9670。
12.一种在田间控制杂草的方法，包括在田间种植大豆事件MON87708植物并在所述田间应用有效剂量的麦草畏除草剂以控制杂草而不伤害所述大豆事件MON 87708植物，大豆事件MON 87708的代表性种子样品被保藏为ATCC PTA-9670。
13.权利要求12的方法，其中所述有效剂量的麦草畏除草剂是每英亩0.005磅至8磅。
14.一种用于在田间控制杂草的方法，包括应用有效剂量的麦草畏除草剂以控制田间杂草，然后在所述田间种植大豆事件MON 87708，大豆事件MON 87708的代表性种子样品被保藏为ATCC PTA-9670。
15.权利要求14的方法，其中所述有效剂量的麦草畏除草剂是每英亩0.005磅至8磅，且所述种植大豆事件MON 87708是在所述应用有效剂量的麦草畏除草剂的14天内。
16.一种用于产生基本上没有毒性杂草种子的大豆种子的方法，所述方法包括：
a.在田间种植大豆事件MON 87708的种子，大豆事件MON 87708的代表性种子样品被保藏为ATCC PTA-9670；
b.将有效剂量的麦草畏除草剂应用于所述田间以杀灭所述田间的毒性杂草而不伤害所述大豆事件MON 87708植物；和
c.从所述田间收获大豆种子。
17.权利要求16的方法，其中所述毒性杂草种子选自群心菜属(Cardaria spp)、天芥菜属(Heliotropium spp)、矢车菊属(Centaurea spp.)、千里光属(Senecio spp.)、猪屎豆属(Crotalaria spp.)、茄属(Solanum spp.)、苍耳属(Xanthium spp.)、琴颈草属(Amsinckiaspp.)、山扁豆属(Cassia spp.)、田菁属(Sesbania spp.)、曼陀罗属(Datura spp.)、蓖麻属(Ricinus spp.)、蓟罂粟属(Argemone spp.)、黄麻属(Corchorus spp.)、牵牛花属(Impomoea spp.)和蓝蓟属(Echium spp.)。
18.一种产生耐受麦草畏除草剂应用的大豆植物的方法，包括：
a.将包含具有选自SEQ ID NO：1-4和6-8及其互补序列的核苷酸序列的核苷酸分子的转基因大豆事件MON 87708植物与第二种大豆植物有性杂交，大豆事件MON 87708的代表性种子样品被保藏为ATCC PTA-9670；
b.收集由所述杂交产生的种子；
c.使所述种子生长以产生多个后代植物；
d.以麦草畏处理所述多个后代植物；和
e.选择对麦草畏耐受的后代植物。
19.一种产生耐受麦草畏除草剂应用的大豆植物的方法，包括：
a.使包含具有选自SEQ ID NO：1-4和6-8及其互补序列的核苷酸序列的核苷酸分子的转基因大豆事件MON 87708植物自交，大豆事件MON 87708的代表性种子样品被保藏为ATCCPTA-9670；
b.收集由所述自交产生的种子；
c.使所述种子生长以产生多个后代植物；
d.以麦草畏处理所述多个后代植物；和
e.选择对麦草畏耐受的后代植物。
20.一种确定大豆事件MON 87708植物或种子的接合性的方法，大豆事件MON 87708的代表性种子样品被保藏为ATCC PTA-9670，包括：
a.将包含大豆DNA的样品与包含SEQ ID NO：12、SEQ ID NO：13和SEQ ID NO：14的引物组以及包含SEQ ID NO：15和SEQ ID NO：16的探针组接触；
b.用所述样品、引物组和探针组进行核酸扩增反应；
c.在所述核酸扩增反应中检测诊断事件MON 87708的第一荧光信号和与所述第一荧光信号不同且诊断对应于事件MON 87708转基因的插入位置的天然大豆基因组DNA的第二荧光信号；和
d.分析所述核酸扩增反应中所述第一荧光信号和所述第二荧光信号的存在和/或缺乏，其中两种荧光信号的存在指示所述样品对于事件MON 87708是杂合的，而仅所述第一荧光信号的存在指示所述样品对于事件MON 87708是纯合的。
21.包含麦草畏耐受基因的在连锁群9上大致图谱位置143.5处的大豆单倍体型区域，且进一步由单倍体型窗口19743和19767限定。
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| US9468180B1 (en) | 2015-05-22 | 2016-10-18 | Monsanto Technology Llc | Soybean variety 01051028 |
| US9585338B2 (en) | 2015-05-22 | 2017-03-07 | Monsanto Technology Llc | Soybean variety 01051129 |
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| US9497920B1 (en) | 2015-05-22 | 2016-11-22 | Monsanto Technology Llc | Soybean variety 01051652 |
| US9497922B1 (en) | 2015-05-22 | 2016-11-22 | Monsanto Technology Llc | Soybean variety 01051809 |
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| US9521824B1 (en) | 2015-06-03 | 2016-12-20 | Monsanto Technology Llc | Soybean variety 01050628 |
| US9532544B2 (en) | 2015-06-03 | 2017-01-03 | Monsanto Technology Llc | Soybean variety 01050668 |
| US9510551B1 (en) | 2015-06-03 | 2016-12-06 | Monsanto Technology Llc | Soybean variety 01050909 |
| US9675030B2 (en) | 2015-06-03 | 2017-06-13 | Monsanto Technology Llc | Soybean variety 01050762 |
| US9585342B2 (en) | 2015-06-03 | 2017-03-07 | Monsanto Technology Llc | Soybean variety 01050585 |
| US9585343B2 (en) | 2015-06-03 | 2017-03-07 | Monsanto Technology Llc | Soybean variety 01050761 |
| US9609829B2 (en) | 2015-06-03 | 2017-04-04 | Monsanto Technology Llc | Soybean variety 01050652 |
| US9510549B1 (en) | 2015-06-03 | 2016-12-06 | Monsanto Technology Llc | Soybean variety 01050689 |
| US9675029B2 (en) | 2015-06-03 | 2017-06-13 | Monsanto Technology Llc | Soybean variety 01050834 |
| US9510550B1 (en) | 2015-06-03 | 2016-12-06 | Monsanto Technology Llc | Soybean variety 01050509 |
| US9743631B2 (en) | 2015-06-25 | 2017-08-29 | Monsanto Technology Llc | Soybean variety 01058733 |
| US9781897B2 (en) | 2015-06-25 | 2017-10-10 | Monsanto Technology Llc | Soybean variety 01058741 |
| US9986704B2 (en) | 2015-06-25 | 2018-06-05 | Monsanto Technology Llc | Soybean variety 01057162 |
| US10104848B2 (en) | 2015-06-25 | 2018-10-23 | Monsanto Technology Llc | Soybean variety 01051773 |
| US9775320B2 (en) | 2015-06-25 | 2017-10-03 | Monsanto Technology Llc | Soybean variety 01058744 |
| US9832964B2 (en) | 2015-06-25 | 2017-12-05 | Monsanto Technology Llc | Soybean variety 01051705 |
| US9974268B2 (en) | 2015-06-25 | 2018-05-22 | Monsanto Technology Llc | Soybean variety 01051074 |
| US9907262B2 (en) | 2015-06-25 | 2018-03-06 | Monsanto Technology Llc | Soybean variety 01051117 |
| US9814206B2 (en) | 2015-06-25 | 2017-11-14 | Monsanto Technology Llc | Soybean variety 01051877 |
| US9832963B2 (en) | 2015-06-25 | 2017-12-05 | Monsanto Technology Llc | Soybean variety 01058403 |
| US9788516B2 (en) | 2015-06-25 | 2017-10-17 | Monsanto Technology Llc | Soybean variety 01058627 |
| US9894860B2 (en) | 2015-06-25 | 2018-02-20 | Monsanto Technology Llc | Soybean variety 01058780 |
| US9743630B2 (en) | 2015-06-25 | 2017-08-29 | Monsanto Technology Llc | Soybean variety 01058664 |
| US9807961B2 (en) | 2015-06-25 | 2017-11-07 | Monsanto Technology Llc | Soybean variety 01058472 |
| US9807962B2 (en) | 2015-06-25 | 2017-11-07 | Monsanto Technology Llc | Soybean variety 01058613 |
| US9814205B2 (en) | 2015-06-25 | 2017-11-14 | Monsanto Technology Llc | Soybean variety 01058730 |
| US9826701B2 (en) | 2015-06-25 | 2017-11-28 | Monsanto Technology Llc | Soybean variety 01050769 |
| US10076093B2 (en) | 2015-06-25 | 2018-09-18 | Monsanto Technology Llc | Soybean variety 01058524 |
| US9781898B2 (en) | 2015-06-25 | 2017-10-10 | Monsanto Technology Llc | Soybean variety 01058771 |
| US10051807B2 (en) | 2015-06-28 | 2018-08-21 | Monsanto Technology Llc | Soybean variety 01058772 |
| US9924676B2 (en) | 2015-06-28 | 2018-03-27 | Monsanto Technology Llc | Soybean variety 01057057 |
| US9807963B2 (en) | 2015-06-28 | 2017-11-07 | Monsanto Technology Llc | Soybean variety 01052094 |
| US9907266B2 (en) | 2015-06-28 | 2018-03-06 | Monsanto Technology Llc | Soybean variety 01057255 |
| US9974269B2 (en) | 2015-06-28 | 2018-05-22 | Monsanto Technology Llc | Soybean variety 01058766 |
| US9820458B2 (en) | 2015-06-28 | 2017-11-21 | Monsanto Technology Llc | Soybean variety 01052110 |
| US9844200B2 (en) | 2015-06-28 | 2017-12-19 | Monsanto Technology Llc | Soybean variety 01058749 |
| US9924677B2 (en) | 2015-06-28 | 2018-03-27 | Monsanto Technology Llc | Soybean variety 01052136 |
| US9844199B2 (en) | 2015-06-28 | 2017-12-19 | Monsanto Technology Llc | Soybean variety 01058475 |
| US9907265B2 (en) | 2015-06-28 | 2018-03-06 | Monsanto Technology Llc | Soybean variety 01058380 |
| US9826703B2 (en) | 2015-06-28 | 2017-11-28 | Monsanto Technology Llc | Soybean variety 01058683 |
| US9848569B2 (en) | 2015-06-28 | 2017-12-26 | Monsanto Technology Llc | Soybean variety 01057209 |
| US9924679B2 (en) | 2015-06-29 | 2018-03-27 | Monsanto Technology Llc | Soybean variety 01057010 |
| US9968057B2 (en) | 2015-06-29 | 2018-05-15 | Monsanto Technology Llc | Soybean variety 01058336 |
| US9844201B2 (en) | 2015-06-29 | 2017-12-19 | Monsanto Technology Llc | Soybean variety 01050745 |
| US9907270B2 (en) | 2015-06-29 | 2018-03-06 | Monsanto Technology Llc | Soybean variety 01057497 |
| US9907268B2 (en) | 2015-06-29 | 2018-03-06 | Monsanto Technology Llc | Soybean variety 01057333 |
| US9907271B2 (en) | 2015-06-29 | 2018-03-06 | Monsanto Technology Llc | Soybean variety 01056960 |
| US9924678B2 (en) | 2015-06-29 | 2018-03-27 | Monsanto Technology Llc | Soybean variety 01057325 |
| US9901057B2 (en) | 2015-06-29 | 2018-02-27 | Monsanto Technology Llc | Soybean variety 01050566 |
| US9907269B2 (en) | 2015-06-29 | 2018-03-06 | Monsanto Technology Llc | Soybean variety 01050536 |
| US9907267B2 (en) | 2015-06-29 | 2018-03-06 | Monsanto Technology Llc | Soybean variety 01057383 |
| US9901056B2 (en) | 2015-06-29 | 2018-02-27 | Monsanto Technology Llc | Soybean variety 01057377 |
| US10034445B2 (en) | 2015-06-29 | 2018-07-31 | Monsanto Technology Llc | Soybean variety 01050611 |
| US9901055B2 (en) | 2015-06-30 | 2018-02-27 | Monsanto Technology Llc | Soybean variety 01050596 |
| US10004197B2 (en) | 2015-06-30 | 2018-06-26 | Monsanto Technology Llc | Soybean variety 01050595 |
| US9826702B2 (en) | 2015-06-30 | 2017-11-28 | Monsanto Technology Llc | Soybean variety 01051835 |
| US9907263B2 (en) | 2015-06-30 | 2018-03-06 | Monsanto Technology Llc | Soybean variety 01050612 |
| US9832965B2 (en) | 2015-06-30 | 2017-12-05 | Monsanto Technology Llc | Soybean variety 01051604 |
| US10070606B2 (en) | 2015-06-30 | 2018-09-11 | Monsanto Technology Llc | Soybean variety 01058789 |
| US9814207B2 (en) | 2015-06-30 | 2017-11-14 | Monsanto Technology Llc | Soybean variety 01058565 |
| US9820459B2 (en) | 2015-06-30 | 2017-11-21 | Monsanto Technology Llc | Soybean variety 01058808 |
| US9907264B2 (en) | 2015-06-30 | 2018-03-06 | Monsanto Technology Llc | Soybean variety 01052085 |
| US10034444B2 (en) | 2015-06-30 | 2018-07-31 | Monsanto Technology Llc | Soybean variety 01050609 |
| US9258973B1 (en) | 2015-08-05 | 2016-02-16 | Monsanto Technology Llc | Soybean cultivar 46092117 |
| US9351471B1 (en) | 2015-08-05 | 2016-05-31 | Monsanto Technology Llc | Soybean cultivar 46434117 |
| US9351470B1 (en) | 2015-08-05 | 2016-05-31 | Monsanto Technology Llc | Soybean cultivar 46043017 |
| CN105002291B (zh) * | 2015-08-10 | 2018-01-12 | 吉林省农业科学院 | 转基因大豆mon87708的lamp检测引物组、试剂盒及检测方法 |
| WO2017042259A1 (en) | 2015-09-11 | 2017-03-16 | Bayer Cropscience Aktiengesellschaft | Hppd variants and methods of use |
| US9901059B2 (en) | 2015-09-24 | 2018-02-27 | Monsanto Technology Llc | Soybean variety 01051956 |
| US9894862B2 (en) | 2015-09-24 | 2018-02-20 | Monsanto Technology Llc | Soybean variety 01058666 |
| US9901060B2 (en) | 2015-09-24 | 2018-02-27 | Monsanto Technology Llc | Soybean variety 01051946 |
| US9907274B2 (en) | 2015-09-24 | 2018-03-06 | Monsanto Technology Llc | Soybean variety 01052030 |
| US9907273B2 (en) | 2015-09-24 | 2018-03-06 | Monsanto Technology Llc | Soybean variety 01051571 |
| US9901062B2 (en) | 2015-09-24 | 2018-02-27 | Monsanto Technology Llc | Soybean variety 01052029 |
| US9901061B2 (en) | 2015-09-24 | 2018-02-27 | Monsanto Technology Llc | Soybean variety 01052008 |
| US9629325B1 (en) | 2015-12-01 | 2017-04-25 | Monsanto Technology Llc | Soybean variety 01057518 |
| CN105483239A (zh) * | 2015-12-26 | 2016-04-13 | 吉林省农业科学院 | 转基因植物中dmo基因的LAMP检测引物组、试剂盒及检测方法 |
| US9861053B1 (en) | 2016-03-21 | 2018-01-09 | Monsanto Technology Llc | Soybean variety XR36AC15X |
| US9596819B1 (en) | 2016-03-21 | 2017-03-21 | Monsanto Technology Llc | Soybean variety XR33AU15X |
| US10010040B1 (en) | 2016-03-21 | 2018-07-03 | Monsanto Technology Llc | Soybean variety XR50E15X |
| US9699999B1 (en) | 2016-03-21 | 2017-07-11 | Monsanto Technology Llc | Soybean variety XR22K15X |
| US9861055B1 (en) | 2016-03-21 | 2018-01-09 | Monsanto Technology Llc | Soybean variety XR39U15X |
| US9700000B1 (en) | 2016-03-21 | 2017-07-11 | Monsanto Technology Llc | Soybean variety XR38W15X |
| US9560829B1 (en) | 2016-03-21 | 2017-02-07 | Monsanto Technology Llc | Soybean variety XR47AG15X |
| US9788508B1 (en) | 2016-03-21 | 2017-10-17 | Monsanto Technology Llc | Soybean variety XR20AH15X |
| US9560827B1 (en) | 2016-03-21 | 2017-02-07 | Monsanto Technology Llc | Soybean variety XR45C15X |
| US9603331B1 (en) | 2016-03-21 | 2017-03-28 | Monsanto Technology Llc | Soybean variety XR39V15X |
| US9861056B1 (en) | 2016-03-21 | 2018-01-09 | Monsanto Technology Llc | Soybean variety XR56J15X |
| US9565821B1 (en) | 2016-03-21 | 2017-02-14 | Monsanto Technology Llc | Soybean variety XR47AF15X |
| US9585355B1 (en) | 2016-03-21 | 2017-03-07 | Monsanto Technology Llc | Soybean variety XR40AA15X |
| US9763412B1 (en) | 2016-03-21 | 2017-09-19 | Monsanto Technology Llc | Soybean variety XR09AP15X |
| US9763414B1 (en) | 2016-03-21 | 2017-09-19 | Monsanto Technology Llc | Soybean variety XR25AL15X |
| US9763413B1 (en) | 2016-03-21 | 2017-09-19 | Monsanto Technology Llc | Soybean variety XR23AQ15X |
| US9596818B1 (en) | 2016-03-21 | 2017-03-21 | Monsanto Technology Llc | Soybean variety XR30AX15X |
| US9565822B1 (en) | 2016-03-21 | 2017-02-14 | Monsanto Technology Llc | Soybean variety XR37AE15X |
| US9560828B1 (en) | 2016-03-21 | 2017-02-07 | Monsanto Technology Llc | Soybean variety XR46Z15X |
| US9763411B1 (en) | 2016-03-21 | 2017-09-19 | Monsanto Technology Llc | Soybean variety XR09AL15X |
| US9585354B1 (en) | 2016-03-21 | 2017-03-07 | Monsanto Technology Llc | Soybean variety XR48C15X |
| US9861054B1 (en) | 2016-03-21 | 2018-01-09 | Monsanto Technology Llc | Soybean variety XR36Z15X |
| US9730415B1 (en) | 2016-03-21 | 2017-08-15 | Monsanto Technology Llc | Soybean variety XR08A15X |
| US9648836B1 (en) * | 2016-03-22 | 2017-05-16 | Monsanto Technology Llc | Soybean cultivar 50262411 |
| US9648835B1 (en) * | 2016-03-22 | 2017-05-16 | Monsanto Technology Llc | Soybean cultivar 53030521 |
| US9655336B1 (en) * | 2016-03-22 | 2017-05-23 | Monsanto Technology Llc | Soybean cultivar 58150703 |
| US9907247B2 (en) | 2016-05-31 | 2018-03-06 | Monsanto Technology Llc | Soybean variety 01056929 |
| US9888647B2 (en) | 2016-05-31 | 2018-02-13 | Monsanto Technology Llc | Soybean variety 01057072 |
| US9872469B2 (en) | 2016-05-31 | 2018-01-23 | Monsanto Technology Llc | Soybean variety 01056991 |
| US10051806B2 (en) | 2016-05-31 | 2018-08-21 | Monsanto Technology Llc | Soybean variety 01057079 |
| US9877450B2 (en) | 2016-05-31 | 2018-01-30 | Monsanto Technology Llc | Soybean variety 01056979 |
| US9888648B2 (en) | 2016-05-31 | 2018-02-13 | Monsanto Technology Llc | Soybean variety 01057115 |
| US9756823B1 (en) | 2016-05-31 | 2017-09-12 | Monsanto Technology Llc | Soybean variety 01056961 |
| US9867351B2 (en) | 2016-05-31 | 2018-01-16 | Monsanto Technology Llc | Soybean variety 01057076 |
| US9907246B2 (en) | 2016-05-31 | 2018-03-06 | Monsanto Technology Llc | Soybean variety 01056904 |
| US9867352B2 (en) | 2016-05-31 | 2018-01-16 | Monsanto Technology Llc | Soybean variety 01057081 |
| US9743624B1 (en) | 2016-05-31 | 2017-08-29 | Monsanto Technology Llc | Soybean variety 01056994 |
| US9901051B2 (en) | 2016-05-31 | 2018-02-27 | Monsanto Technology Llc | Soybean variety 01057075 |
| US9743623B1 (en) | 2016-05-31 | 2017-08-29 | Monsanto Technology Llc | Soybean variety 01056977 |
| US9907245B2 (en) | 2016-05-31 | 2018-03-06 | Monsanto Technology Llc | Soybean variety 01056902 |
| US9924672B2 (en) | 2016-05-31 | 2018-03-27 | Monsanto Technology Llc | Soybean variety 01056984 |
| US9750218B1 (en) | 2016-05-31 | 2017-09-05 | Monsanto Technology Llc | Soybean variety 01056968 |
| US9901052B2 (en) | 2016-05-31 | 2018-02-27 | Monsanto Technology Llc | Soybean variety 01057176 |
| US9888646B2 (en) | 2016-05-31 | 2018-02-13 | Monsanto Technology Llc | Soybean variety 01057021 |
| US9713311B1 (en) | 2016-05-31 | 2017-07-25 | Monsanto Technology Llc | Soybean variety 01057181 |
| US10226001B2 (en) | 2016-05-31 | 2019-03-12 | Monsanto Technology Llc | Soybean variety 01056938 |
| US9907251B2 (en) | 2016-06-01 | 2018-03-06 | Monsanto Technology Llc | Soybean variety 01058313 |
| US10104847B2 (en) | 2016-06-01 | 2018-10-23 | Monsanto Technology Llc | Soybean variety 01057454 |
| US9848562B1 (en) | 2016-06-01 | 2017-12-26 | Monsanto Technology Llc | Soybean variety 01058349 |
| US9706739B1 (en) | 2016-06-01 | 2017-07-18 | Monsanto Technology Llc | Soybean variety 01057290 |
| US9907252B2 (en) | 2016-06-01 | 2018-03-06 | Monsanto Technology Llc | Soybean variety 01058320 |
| US9854772B2 (en) | 2016-06-01 | 2018-01-02 | Monsanto Technology Llc | Soybean variety 01058360 |
| US9706738B1 (en) | 2016-06-01 | 2017-07-18 | Monsanto Technology Llc | Soybean variety 01057282 |
| US9907250B2 (en) | 2016-06-01 | 2018-03-06 | Monsanto Technology Llc | Soybean variety 01057493 |
| US10231398B2 (en) | 2016-06-01 | 2019-03-19 | Monsanto Technology Llc | Soybean variety 01057400 |
| US9907249B2 (en) | 2016-06-01 | 2018-03-06 | Monsanto Technology Llc | Soybean variety 01057459 |
| US9788515B1 (en) | 2016-06-01 | 2017-10-17 | Monsanto Technology Llc | Soybean variety 01057347 |
| US9907248B2 (en) | 2016-06-01 | 2018-03-06 | Monsanto Technology Llc | Soybean variety 01057390 |
| US9788514B1 (en) | 2016-06-01 | 2017-10-17 | Monsanto Technology Llc | Soybean variety 01057345 |
| US9832953B1 (en) | 2016-06-01 | 2017-12-05 | Monsanto Technology Llc | Soybean variety 01057348 |
| US10226002B2 (en) | 2016-06-01 | 2019-03-12 | Monsanto Technology Llc | Soybean variety 01057408 |
| US9974264B2 (en) | 2016-06-01 | 2018-05-22 | Monsanto Technology Llc | Soybean variety 01057212 |
| US9661823B1 (en) | 2016-06-01 | 2017-05-30 | Monsanto Technology Llc | Soybean variety 01057424 |
| US9968054B2 (en) | 2016-06-01 | 2018-05-15 | Monsanto Technology Llc | Soybean variety 01057207 |
| US9848563B1 (en) | 2016-06-01 | 2017-12-26 | Monsanto Technology Llc | Soybean variety 01058353 |
| US9700008B1 (en) | 2016-06-01 | 2017-07-11 | Monsanto Technology Llc | Soybean variety 01057341 |
| US9867354B2 (en) | 2016-06-02 | 2018-01-16 | Monsanto Technology Llc | Soybean variety 01058529 |
| US9907253B2 (en) | 2016-06-02 | 2018-03-06 | Monsanto Technology Llc | Soybean variety 01058513 |
| US9867353B2 (en) | 2016-06-02 | 2018-01-16 | Monsanto Technology Llc | Soybean variety 01058528 |
| US9907256B2 (en) | 2016-06-02 | 2018-03-06 | Monsanto Technology Llc | Soybean variety 01058822 |
| US9832955B1 (en) | 2016-06-02 | 2017-12-05 | Monsanto Technology Llc | Soybean variety 01058718 |
| US9872470B2 (en) | 2016-06-02 | 2018-01-23 | Monsanto Technology Llc | Soybean variety 01058469 |
| US9907255B2 (en) | 2016-06-02 | 2018-03-06 | Monsanto Technology Llc | Soybean variety 01058767 |
| US9848564B1 (en) | 2016-06-02 | 2017-12-26 | Monsanto Technology Llc | Soybean variety 01058864 |
| US9832954B1 (en) | 2016-06-02 | 2017-12-05 | Monsanto Technology Llc | Soybean variety 01058711 |
| US9907254B2 (en) | 2016-06-02 | 2018-03-06 | Monsanto Technology Llc | Soybean variety 01058698 |
| US9807960B1 (en) | 2016-06-02 | 2017-11-07 | Monsanto Technology Llc | Soybean variety 01063888 |
| US9832957B1 (en) | 2016-06-02 | 2017-12-05 | Monsanto Technology Llc | Soybean variety 01058863 |
| US9901053B2 (en) | 2016-06-02 | 2018-02-27 | Monsanto Technology Llc | Soybean variety 01058606 |
| US9693530B1 (en) | 2016-06-02 | 2017-07-04 | Monsanto Technology Llc | Soybean variety 01063884 |
| US9700009B1 (en) | 2016-06-02 | 2017-07-11 | Monsanto Technology Llc | Soybean variety 01058861 |
| US9854773B2 (en) | 2016-06-02 | 2018-01-02 | Monsanto Technology Llc | Soybean variety 01058444 |
| US9693529B1 (en) | 2016-06-02 | 2017-07-04 | Monsanto Technology Llc | Soybean variety 01063878 |
| US9832956B1 (en) | 2016-06-02 | 2017-12-05 | Monsanto Technology Llc | Soybean variety 01063886 |
| US9700010B1 (en) | 2016-06-02 | 2017-07-11 | Monsanto Technology Llc | Soybean variety 01063871 |
| US9907259B2 (en) | 2016-06-03 | 2018-03-06 | Monsanto Technology Llc | Soybean variety 01063916 |
| US10058047B2 (en) | 2016-06-03 | 2018-08-28 | Monsanto Technology Llc | Soybean variety 01063918 |
| US9832958B1 (en) | 2016-06-03 | 2017-12-05 | Monsanto Technology Llc | Soybean variety 01064122 |
| US9750221B1 (en) | 2016-06-03 | 2017-09-05 | Monsanto Technology Llc | Soybean variety 01063935 |
| US9907261B2 (en) | 2016-06-03 | 2018-03-06 | Monsanto Technology Llc | Soybean variety 01063928 |
| US9907257B2 (en) | 2016-06-03 | 2018-03-06 | Monsanto Technology Llc | Soybean variety 01063892 |
| US9743625B1 (en) | 2016-06-03 | 2017-08-29 | Monsanto Technology Llc | Soybean variety 01063903 |
| US9730419B1 (en) | 2016-06-03 | 2017-08-15 | Monsanto Technology Llc | Soybean variety 01064108 |
| US9901054B2 (en) | 2016-06-03 | 2018-02-27 | Monsanto Technology Llc | Soybean variety 01063900 |
| US9907258B2 (en) | 2016-06-03 | 2018-03-06 | Monsanto Technology Llc | Soybean variety 01063898 |
| US9717213B1 (en) | 2016-06-03 | 2017-08-01 | Monsanto Technology Llc | Soybean variety 01064120 |
| US9848565B1 (en) | 2016-06-03 | 2017-12-26 | Monsanto Technology Llc | Soybean variety 01063939 |
| US10219462B2 (en) | 2016-06-03 | 2019-03-05 | Monsanto Technology Llc | Soybean variety 01063907 |
| US9717211B1 (en) | 2016-06-03 | 2017-08-01 | Monsanto Technology Llc | Soybean variety 01063940 |
| US9730418B1 (en) | 2016-06-03 | 2017-08-15 | Monsanto Technology Llc | Soybean variety 01063920 |
| US9717212B1 (en) | 2016-06-03 | 2017-08-01 | Monsanto Technology Llc | Soybean variety 01064113 |
| US9737037B1 (en) | 2016-06-03 | 2017-08-22 | Monsanto Technology Llc | Soybean variety 01063921 |
| US9907260B2 (en) | 2016-06-03 | 2018-03-06 | Monsanto Technology Llc | Soybean variety 01063906 |
| US9955645B2 (en) | 2016-06-03 | 2018-05-01 | Monsanto Technology Llc | Soybean variety 01063902 |
| US9867356B2 (en) | 2016-06-06 | 2018-01-16 | Monsanto Technology Llc | Soybean variety 01064143 |
| US9924673B2 (en) | 2016-06-06 | 2018-03-27 | Monsanto Technology Llc | Soybean variety 01064169 |
| US9832962B1 (en) | 2016-06-06 | 2017-12-05 | Monsanto Technology Llc | Soybean variety 01064172 |
| US9839187B1 (en) | 2016-06-06 | 2017-12-12 | Monsanto Technology Llc | Soybean variety 01064165 |
| US9832959B1 (en) | 2016-06-06 | 2017-12-05 | Monsanto Technology Llc | Soybean variety 01064131 |
| US9867355B2 (en) | 2016-06-06 | 2018-01-16 | Monsanto Technology Llc | Soybean variety 01064176 |
| US9743626B1 (en) | 2016-06-06 | 2017-08-29 | Monsanto Technology Llc | Soybean variety 01064148 |
| US9743627B1 (en) | 2016-06-06 | 2017-08-29 | Monsanto Technology Llc | Soybean variety 01064182 |
| US9730420B1 (en) | 2016-06-06 | 2017-08-15 | Monsanto Technology Llc | Soybean variety 01064123 |
| US9795106B1 (en) | 2016-06-06 | 2017-10-24 | Monsanto Technology Llc | Soybean variety 01064150 |
| US9872471B2 (en) | 2016-06-06 | 2018-01-23 | Monsanto Technology Llc | Soybean variety 01064177 |
| US9832960B1 (en) | 2016-06-06 | 2017-12-05 | Monsanto Technology Llc | Soybean variety 01064152 |
| US10064356B2 (en) | 2016-06-06 | 2018-09-04 | Monsanto Technology Llc | Soybean variety 01064137 |
| US9844197B1 (en) | 2016-06-06 | 2017-12-19 | Monsanto Technology Llc | Soybean variety 01064151 |
| US9848566B1 (en) | 2016-06-06 | 2017-12-26 | Monsanto Technology Llc | Soybean variety 01064124 |
| US9832961B1 (en) | 2016-06-06 | 2017-12-05 | Monsanto Technology Llc | Soybean variety 01064167 |
| US9848567B1 (en) | 2016-06-06 | 2017-12-26 | Monsanto Technology Llc | Soybean variety 01064142 |
| US9968055B2 (en) | 2016-06-07 | 2018-05-15 | Monsanto Technology Llc | Soybean variety 01062064 |
| US9924674B2 (en) | 2016-06-07 | 2018-03-27 | Monsanto Technology Llc | Soybean variety 01057531 |
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| US10561105B1 (en) | 2018-08-24 | 2020-02-18 | Monsanto Technology Llc | Soybean variety 01072235 |
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| US10631501B2 (en) | 2018-08-24 | 2020-04-28 | Monsanto Technology Llc | Soybean variety 01067793 |
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| US10631507B2 (en) | 2018-08-24 | 2020-04-28 | Monsanto Technology Llc | Soybean variety 01068485 |
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