CN104797716B - 棉花事件pDAB4468.19.10.3的检测方法 - Google Patents
棉花事件pDAB4468.19.10.3的检测方法 Download PDFInfo
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Abstract
棉花事件pDAB4468.19.10.3包含具有编码aad 12和pat的基因的基因表达盒,其向含有所述事件的棉花作物提供了除草剂耐受性,并能够实现用于作物保护的方法。本主题发明的实施方案提供了多核苷酸相关事件的检测方法。
Description
优先权要求
本申请要求2013年1月23日提交的、流水号61/589,594的美国临时专利申请“除草剂耐受性棉花事件pDAB4468.19.10.3”的提交日的权益。
发明背景
编码AAD-12(芳氧基链烷酸双加氧酶-12)的基因在转基因植物中表达时能够赋予针对苯氧基乙酸除草剂2,4-D和MCPA、以及吡啶氧基乙酸除草剂绿草定和氟草烟的商业水平的耐受性。编码PAT(草胺膦乙酰转移酶)的基因在转基因植物中表达时能够赋予针对除草剂草胺膦(草甘磷)的耐受性。PAT在棉花中已成功表达,既用作选择标记,还在转基因植物中赋予针对除草剂草甘磷的商业水平的耐受性。
大概由于染色质结构(例如,异染色质)或靠近整合位点的转录调控元件(例如,增强子)的接近,已知转基因在植物中的表达受到它们在植物基因组中的位置的影响(Weising等人,Ann.Rev.Genet 22:421-477,1988)。转基因在基因组中的不同位置处的存在会以不同方式影响植物的整体表型。例如,已经在植物以及在其他生物中观察到导入基因的表达水平在事件中可存在很大变化。在表达的空间或时间模式上也可能存在差异,例如,转基因在各种植物组织中的相对表达的差异,这可能与根据导入基因构建体中存在的转录调控元件预期的模式不对应。由于这个原因,通常产生几百个到几千个不同的事件并筛选这些事件,得到具有所希望的转基因表达水平和模式的单个事件,用于商业目的。正因如此,常常必需筛选大量事件,以便鉴定出其特征为导入的目的基因的优化表达的事件。具有所希望的转基因表达水平或模式的事件对于使用常规育种方法通过有性异型杂交将所述转基因渗入到其他遗传背景中是有用的。这种杂交的后代保持原始转化体的转基因表达特征。这种策略用来在许多已很好适应当地生长条件的品种中保证可靠基因表达。
想要的是能够检测出特定事件的存在,以便确定有性杂交后代是否包含一种或一组目的转基因。另外,用于检测特定事件的方法将有助于遵守需要上市前许可的法规以及源于重组作物植物的食物和纤维的标记,例如,或用于环境监测、监测大田作物的性状,或监测源于作物收获的产品,以及用于保证受制于法规或合同条款的每一方的依从性。
有可能通过本领域已知的任何核酸检测方法检测出转基因事件的存在,所述检测方法包括但不限于聚合酶链式反应(PCR)或使用核酸探针的DNA杂交。这些检测方法通常集中于频繁使用的遗传元件,诸如启动子、终止子、标记基因等等,因为对于许多DNA构建体而言,编码区是可互换的。结果,这样的方法对于在不同的事件特别是使用同样的DNA构建体或非常相似的构建体所产生的那些事件之间进行区分可能是无用的,除非已知邻近插入的异源DNA的侧翼DNA的DNA序列。例如,对于玉蜀黍事件DAS-59122-7的事件特异性PCR测定法描述于美国专利申请2006/0070139中。具有用于鉴定棉花事件pDAB4468.19.10.3的简单的判别性方法将是想要的。
发明披露
本发明的实施方案涉及用于检测新的抗昆虫且耐除草剂的转基因棉花转化事件的方法,所述事件被指定为棉花事件pDAB4468.19.10.3,包括插入到棉花细胞的基因组内的特定位点中的如本文中描述的aad-12和pat。代表性棉花种子已保藏于美国典型培养物保藏中心(ATCC),10801,大学大道(University Boulevard),马纳萨斯(Manassas),维吉尼亚州(VA),20110。代表Dow AgroSciences LLC于2012年1月23日制作了所述保藏物,指定为ATCC保藏号PTA-12457。依照关于用于专利程序目的的种子保藏物的布达佩斯条约条款并根据这些条款制作了并将维护这种保藏物。
含有这种事件的棉花植物的DNA包含本文中描述的连接序列/侧翼序列,所述序列使棉花基因组内的插入DNA的位置特征化。SEQ ID NO:1和SEQ ID NO:2对于棉花事件pDAB4468.19.10.3具有诊断性。更具体地说,在SEQ ID NO:1的bp 1354/1355和SEQ ID NO:2的bp 168/169的连接周围的序列对于棉花事件pDAB4468.19.10.3具有诊断性。以下段落描述包含这些连接的序列的例子,这些连接是含有棉花事件pDAB4468.19.10.3的棉花植物的DNA的特征。
在另一个实施方案中,本发明提供了在包含棉花DNA的样品中检测棉花事件pDAB4468.19.10.3的方法,所述方法包括:
(a)使所述样品与长度至少为10bp的第一引物以及长度至少为10bp的第二引物接触,所述第一引物与SEQ ID NO:1的bp 1-1354内的侧翼序列或其互补物选择性地结合,所述第二引物与SEQ ID NO:1的bp 1355-1672内的插入序列或其互补物选择性地结合;并且
(b)测定在所述引物之间产生的扩增子;或者
(c)使所述样品与长度至少为10bp的第一引物以及长度至少为10bp的第二引物接触,所述第一引物与SEQ ID NO:2的bp 1-168内的插入序列或其互补物选择性地结合,所述第二引物与SEQ ID NO:2的bp 169-2898内的侧翼序列或其互补物选择性地结合;并且
(d)测定在所述引物之间产生的扩增子。
在另一个实施方案中,本发明提供了检测棉花事件pDAB4468.19.10.3的方法,其包括:
(a)使所述样品与第一引物以及第二引物接触,所述第一引物与选自下组的侧翼序列或其互补物结合:SEQ ID NO:1的bp 1-1354和SEQ ID NO:2的bp 169-2898;所述第二引物与SEQ ID NO:14或其互补物选择性地结合;
(b)使所述样品经受聚合酶链式反应;并且
(c)测定在所述引物之间产生的扩增子。
在另一个实施方案中,本发明提供了用于诊断棉花事件pDAB4468.19.10.3的分离DNA分子。除了SEQ ID NOS:1和2之外,这样的分子包括,包含跨SEQ ID NO:1的bp 1354/1355连接的多核苷酸序列的、长度至少为50bp的分子以及包含跨SEQ ID NO:2的bp 168/169连接的多核苷酸序列的、长度至少为50bp的分子。例子为SEQ ID NO:1的bp 1329-1380;SEQ ID NO:1的bp 1304-1405;SEQ ID NO:1的bp 1254-1455;SEQ ID NO:1的bp 1154-1555;SEQ ID NO:1的bp 1054-1655;SEQ ID NO:2的bp 143-194;SEQ ID NO:2的bp 118-219;SEQ ID NO:2的bp 68-269;以及SEQ ID NO:2的bp 1-369,以及它们的互补物。
另外,本发明的实施方案提供了用于在样品(例如,棉花样品)中检测主题事件的存在的测定法。所述测定法可基于插入到棉花基因组中的重组构建体的DNA序列、并可基于在插入位点侧翼的基因组序列。还提供了在进行这些测定法中有用的试剂盒和条件。
本发明的实施方案部分地涉及边界区域DNA序列的克隆和分析,所述边界区域由来自转基因棉花品系中的pDAB4468的T-DNA的插入产生。这些序列是独特的。基于这些插入序列和连接序列,可以产生并且产生了事件特异性引物。PCR分析证明,可通过分析用这些事件特异性引物组产生的PCR扩增子来鉴定这些事件。因而,可以使用这些和其他有关程序独特地鉴定包含主题披露的所述事件的棉花品系。
序列简述
SEQ ID NO:1为棉花事件pDAB4468.19.10.3的5’DNA侧翼边界序列。核苷酸1-1354为基因组序列。核苷酸1355-1672为插入序列。
SEQ ID NO:2为棉花事件pDAB4468.19.10.3的3’DNA侧翼边界序列。核苷酸1-168为插入序列。核苷酸169-2898为基因组序列。
SEQ ID NO:3为77bp的DNA片段,所述DNA片段对于棉花事件pDAB4468.19.10.3的5’整合连接具有诊断性。
SEQ ID NO:4为90bp的DNA片段,所述DNA片段对于棉花事件pDAB4468.19.10.3的3’整合连接具有诊断性。
SEQ ID NO:5为寡核苷酸引物ES_1910_5_F,用于测定法中检测棉花事件9582.814.19.1的5’边界。
SEQ ID NO:6为寡核苷酸引物ES_1910_5_R,用于测定法中检测棉花事件9582.814.19.1的5’边界。
SEQ ID NO:7为寡核苷酸探针ES_1910_5Pr,用于测定法中检测棉花事件9582.814.19.1的5’边界。这种探针具有添加到5’端的VIC荧光模块和添加到3’端的MGB猝灭剂。
SEQ ID NO:8为寡核苷酸引物ES_1910_3_F,用于测定法中检测棉花事件9582.814.19.1的3’边界。
SEQ ID NO:9为寡核苷酸引物ES_1910_3_R,用于测定法中检测棉花事件9582.814.19.1的3’边界。
SEQ ID NO:10为寡核苷酸探针ES_1910_3Pr,用于测定法中检测棉花事件9582.814.19.1的5’边界。这种探针具有添加到5’端的VIC荧光模块和添加到3’端的MGB猝灭剂。
SEQ ID NO:11为寡核苷酸引物IC_Sah7F,用于测定法中检测内源参考基因Sah7(GenBank:AY117065.1)。
SEQ ID NO:12为寡核苷酸引物IC_Sah7R,用于测定法中检测内源参考基因Sah7(GenBank:AY117065.1)。
SEQ ID NO:13为寡核苷酸探针IC_Sah7_Pr,用于测定法中检测内源参考基因Sah7(GenBank:AY117065.1)。这种探针具有添加到5’端的Cy5荧光模块和添加到3’端的BHQ2猝灭剂。
SEQ ID NO:14是pDAB4468的T-链DNA序列。
附图简述
图1为含有aad-12和pat表达盒的pDAB4468的质粒图谱。
图2描绘了棉花事件pDAB4468.19.10.3的测定法的引物和探针位置。
用于进行本发明的模式
对棉花事件pDAB4468.19.10.3插入的两端均进行了测序和表征。开发了事件特异性测定法。同样,已将所述事件映射到棉花基因组上(亚基因组A的3号染色体)。可将所述事件渗入到另外的良种系中。
如在上文背景部分提到的,将转基因导入和整合到植物基因组中涉及一些随机事件(因此名称“事件”针对所表达的给定插入)。也就是说,利用许多转化技术,诸如农杆菌转化、生物射弹转化(即,基因枪)、以及碳化硅介导的转化(即,WHISKERS),转基因将被插入在基因组中的何处是不可预测的。因而,鉴定在插入物的两侧上的侧翼植物基因组DNA对于鉴定具有给定插入事件的植物可能是重要的。例如,可以将PCR引物设计为产生跨越所述插入物与宿主基因组的连接区的PCR扩增子。这种PCR扩增子可以用来鉴定唯一或独特类型的插入事件。
本文中提供的定义和例子有助于描述本发明的实施方案并指导本领域的普通技术人员实施这些实施方案。除非另外说明,应当根据相关领域的普通技术人员的常规用法来理解术语。使用了在37CFR§1.822中提出的DNA碱基的命名法。
如本文中使用的,术语“后代”表示包含棉花事件pDAB4468.19.10.3的亲本植物任何代的后裔。
通过用异源DNA(即,包含目的转基因的核酸构建体)转化植物细胞,再生由于将转基因插入植物的基因组中所产生的植物群体,并选择通过插入到特定基因组位置中而表征的特定植物而产生转基因“事件”。术语“事件”是指包含所述异源DNA的原始转化体和/或所述转化体的后代。术语“事件”也指通过转化体与包含基因组DNA/转基因DNA的另一个品种之间的有性异型杂交所产生的后代。即使在重复回交至轮回亲本之后,来自转化亲本的插入转基因DNA和侧翼基因组DNA(基因组DNA/转基因DNA)存在于在杂交后代的相同染色体位置。术语“事件”也指来自包含插入DNA和与插入DNA紧邻的侧翼基因组序列的原始转化体或其后代的DNA,预期其会转移到因包含所述插入DNA的亲本品系(例如原始转化体和由自交产生的后代)与不含所述插入DNA的亲本品系的有性杂交而接收了包含目的转基因的插入DNA的后代中。
“连接序列”或“边界序列”跨过插入基因组的DNA与来自在插入点侧翼的棉花天然基因组的DNA连接处的点,植物的遗传物质中的一段或另一段连接序列的鉴定或检测足以诊断所述事件。包括跨过本文中描述的棉花事件中的插入和相似长度的侧翼DNA的DNA序列。本文中提供了这种诊断序列的具体例子;然而,与所述插入的连接、或所述插入的连接和所述基因组重叠的其他序列也具有诊断性,并可依照所述主题披露的本发明实施方案而使用。
本发明实施方案部分地涉及使用这种侧翼序列、连接序列、以及插入序列的事件鉴定。相关PCR引物和扩增子包括在本发明实施方案中。根据主题发明的实施方案,可以使用横跨插入DNA和其边界的扩增子的PCR分析方法来检测或鉴定源于主题专有的转基因棉花品系的商业化转基因棉花品种或品系。
侧翼/连接序列对于棉花事件pDAB4468.19.10.3具有诊断性。基于这些序列,产生了事件特异性引物。PCR分析证明,通过分析用这些事件特异性引物组产生的PCR扩增子,可将这些棉花品系鉴定为不同的棉花基因型。因而,可以使用这些和其他相关程序独特地鉴定这些棉花品系。本文中鉴定的这些序列是独特的。
本主题发明的实施方案的检测技术联合植物育种对于在包含目的事件的亲本植物与另一种植物品系杂交(试图在后代中赋予一种或多种另外的目的性状)之后,确定哪些后代植物包含给定事件是特别有用的。这些PCR分析方法有利于棉花育种计划以及质量控制,尤其是对于商业化转基因棉花种子而言。现在,也可以制作和使用用于这些转基因棉花品系的PCR检测试剂盒。这也有利于产品注册和产品监管。
此外,可使用侧翼棉花/基因组序列明确地鉴定每一插入物的基因组位置。这种信息可用来制作针对每一事件特异的分子标记系统。这些系统可用于加速的育种策略,并用来建立连锁数据。
更进一步地,所述侧翼序列信息可用来研究和表征转基因整合过程、基因组整合位点特征、事件分选、转基因及其侧翼序列的稳定性、以及基因表达(尤其是涉及基因沉默、转基因甲基化模式、位置效应、以及潜在表达相关元件,诸如MARS[基质附着区]等等)。
鉴于所有主题披露,应当清楚的是,本主题发明的实施方案包括在[0005]段中鉴定的以ATCC保藏号可获得的种子。本发明的实施方案还包括在[0005]段中鉴定的从以所述ATCC保藏号保藏的种子培育的耐除草剂棉花植物。本发明的实施方案还包括所述植物的部分,诸如叶、组织样品、由所述植物产生的种子、花粉等等(其中植物的这些部分包括aad-12和pat、以及SEQ ID NOS:1和2)。
如本文中使用的,术语“棉花”意指陆地棉(Gossypium hirsutum)并且包括其可以与棉花植物培育的所有品种。
a.本发明实施方案的DNA分子在标记辅助育种(MAB)方法中可用作分子标记。本发明实施方案的DNA分子可如本领域已知地在鉴定遗传连锁的农艺学上有用的性状的方法中使用(诸如AFLP标记、RFLP标记、RAPD标记、SNP和SSR)。使用MAB方法可在与本主题发明的实施方案的棉花植物(或其后代和任何其他棉花栽培种或品种)的杂交的后代中追踪耐除草剂性状。所述DNA分子为针对这种性状的标记,并且本领域中熟知的MAB方法可用于在棉花植物中追踪耐除草剂性状,其中至少一种本主题发明的实施方案的棉花品系或其后代为亲本或祖先。本发明实施方案的所述方法可用于鉴定具有本主题事件的任何棉花品种。
如本文中使用的,“品系”为一组对于至少一种性状在个体间显示很少或没有遗传变异的植物。可通过几个世代的自花授粉和选择,或使用组织或细胞培养技术从单个亲本的营养繁殖来建立这样的品系。
如本文中使用的,术语“栽培种”和“品种”是同义词,并指用于商业生产的品系。
“稳定性”或“稳定的”意指对于给定组分而言,该组分在世代间保持,且优选地保持至少三个世代。
“商业实用性”定义为具有良好的植物活力和高可育性,使得该作物可由农民使用常规耕作设备而产生,且可使用常规压榨和提取设备从种子提取具有所述组分的油。
“农艺学上优秀的”意指品系除了由于本主题事件的除草剂耐受性之外,还具有期望的农艺学特征诸如产率、成熟性、抗病性等等。这些农艺学特征和数据点中的任何和全部可用于鉴定这类植物,或者作为一系列用于定义这类植物的特征中的一个点或者作为任一端或两端。
如本领域技术人员根据本披露认识到的,检测试剂盒的优选实施方案,例如,可含有针对和/或包含“连接序列”或“过渡序列”(在此棉花基因组侧翼序列与插入序列接触)的引物和/或探针。例如,这包括设计为用于鉴定一个或两个连接序列(在此插入物与侧翼序列接触)的多核苷酸探针、引物和/或扩增子。一种常见设计是具有一种在侧翼区中杂交的引物和一种在插入物中杂交的引物。这样的引物通常每个的长度为约至少约15个残基。用这样布置,所述引物可用于生成/扩增可检测的扩增子,其指示本主题发明的实施方案的事件的存在。这些引物可用于生成跨越(并含有)如上所指示的连接序列的扩增子。
在侧翼序列中“触地(touch down)”的引物通常不设计为超过所述连接处超过约1200个碱基左右而杂交。因此,通常的侧翼引物将设计为包含从插入物开始进入侧翼序列1200个碱基以内的任一链的至少15个残基。即,包含来自SEQ ID NO:1的碱基对154-1672和/或SEQ ID NO:2的碱基对1-1369(或与之杂交)的适当大小的序列的引物落在本主题发明的实施方案的范围内。同样,插入引物可设计为在插入物上的任何位置,但例如可将SEQID NO:14的残基对1-6387非排他性地用于这种引物设计。
本领域技术人员还将认识到可将引物和探针设计为在一定范围的标准杂交和/或PCR条件下杂交,其中所述引物或探针并不完全与所例示的序列互补。即,可容许一定程度的错配或简并。对于大约20个核苷酸的引物,例如,如果错配的碱基在引物的内部或与扩增子相反的引物末端,通常一个或两个左右的核苷酸并不需要与相反链结合。下面提供了各种适当的杂交条件。合成的核苷酸类似物,诸如次黄嘌呤核苷,也可在探针中使用。也可使用肽核酸(PNA)探针、以及DNA和RNA探针。重要的是这样的探针和引物对于本主题发明的实施方案的事件的存在具有诊断性(能够独特地鉴定和区分)。
应当指出在PCR扩增中可发生错误,其可能例如导致次要的测序错误。即,除非另外指出,本文中列出的序列是通过从棉花基因组DNA生成长的扩增子,然后克隆所述扩增子并将其测序而确定的。考虑到为了从基因组DNA测序而生成充足的扩增子所需要的多轮扩增,在以此方式生成和确定的序列中发现细微差别和次要偏差并非不寻常。本领域技术人员应当理解并留意由于这些类型的常见测序错误或偏差所需要的任何调整落在本主题发明的实施方案的范围内。
还应当指出,一些基因组序列的缺失并非不常见,例如,当在构建事件过程中插入序列时。因此,例如也可在本主题的侧翼序列和GENBANK中列出的基因组序列之间存在一些差异。
DNA序列“插入物”的组分图示于附图中,并在下文实施例中更加详细地讨论。这些组分、或其片段的DNA多核苷酸序列,可在本发明的实施方案的方法中用作DNA引物或探针。
在本发明的一些实施方案中,提供了在来自棉花植物的植物和种子等中用于检测转基因/基因组插入区的存在的组合物和方法。提供了包含本文中提供的本主题5’转基因/基因组插入区连接序列的DNA序列(在SEQ ID NO:1的碱基对1354/1355之间)、其区段、以及例示序列的互补物及其任何区段。提供了包含本文中提供的本主题3’转基因/基因组插入区连接序列的DNA序列(在SEQ ID NO:2的碱基对168/169之间)、其区段、以及例示序列的互补物及其任何区段。该插入区连接序列跨越在插入基因组的异源DNA与来自棉花细胞、所述插入位点侧翼的DNA之间的连接。这样的序列对于给定事件可具诊断性。
基于这些插入序列和边界序列,可以产生事件特异性引物。PCR分析证明,通过分析用这些事件特异性引物组产生的PCR扩增子,可将本主题发明的实施方案的棉花品系鉴定为不同的棉花基因型。因而,可以使用这些和其他相关程序独特地鉴定这些棉花品系。因而,衍生自这样的引物对的PCR扩增子是独特的,且可用于鉴定这些棉花品系。
在一些实施方案中,本发明的实施方案的一个方面是包含新颖转基因/基因组插入区的连续片段的DNA序列。包括包含足够长度的、转基因插入序列的多核苷酸和足够长度的、来自三种前述棉花植物中的一种或多种的棉花基因组序列的多核苷酸的DNA序列和/或可用作引物序列以供产生对于一种或多种这些棉花植物具有诊断性的扩增子产物的序列。
相关实施方案涉及包含本文中鉴定的DNA序列的转基因部分(诸如SEQ ID No:1及其区段)的至少10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25个或更多个连续核苷酸或其互补物以及相似长度的、来自这些序列的侧翼棉花DNA序列或其互补物的DNA序列。这样的序列可作为DNA引物用于DNA扩增方法。使用这些引物产生的扩增子对于本文中提及的任意棉花事件具有诊断性。因此,本发明的实施方案还包括通过这样的DNA引物产生的扩增子。
本发明的实施方案还包括在样品中检测相应于本文中提及的棉花事件的DNA的存在的方法。这样的方法可包括:(a)使包含DNA的样品与引物组相接触,所述引物组当与来自这些棉花事件至少之一的DNA一起用于核酸扩增反应时,产生对于所述事件具有诊断性的扩增子;(b)进行核酸扩增反应,由此产生所述扩增子;并且(c)检测所述扩增子。
本主题发明的实施方案的进一步的检测方法包括在样品中检测相应于所述事件的DNA的存在的方法,其中所述方法包括:(a)使包含DNA的样品与在严格杂交条件下与来自所述棉花事件的DNA杂交而在严格杂交条件下不与对照棉花植物(非感兴趣的事件的DNA)杂交的探针相接触;(b)对所述样品和探针施以严格杂交条件;并且(c)检测所述探针与所述DNA的杂交。
可使用本文中披露的组合物和DNA检测领域熟知的方法开发DNA检测试剂盒。所述试剂盒可用于在样品中鉴定本主题棉花事件DNA,并可应用于供育种含有这种DNA的棉花植物的方法。所述制剂盒含有与所述扩增子互补的DNA序列,所述扩增子例如为本文中公开的扩增子,或同与本主题事件的转基因遗传元件中所含DNA互补的DNA序列互补的DNA序列。这些DNA序列可用于DNA扩增反应,或用作DNA杂交方法中的探针。所述试剂盒还可含有进行所述检测方法所需要的试剂和材料。
“探针”是附着于常规的可检测标记或报道分子(诸如放射性同位素、配体,化学发光剂或酶)的分离的核酸分子。这样的探针可与靶核酸的链杂交,在本发明的实施方案的情况下,与来自所述棉花事件之一的基因组DNA的链杂交,不论是来自含有由所述事件所得的DNA的样品还是棉花植物。依照本发明的实施方案的探针不仅包括脱氧核糖核酸或核糖核酸,且还包括聚酰胺和其他特异性结合于靶DNA序列并可用于检测所述靶DNA序列存在的探针物质。
“引物”为通过核酸杂交与互补靶DNA链退火以形成在引物和靶DNA链之间的杂合体,然后由聚合酶例如DNA聚合酶沿所述靶DNA链延伸的分离的/合成的核酸。本发明的实施方案的引物对涉及其用于扩增靶核酸序列(例如通过聚合酶链式反应(PCR)或其他常规核酸扩增方法)的用途。
探针和引物的长度一般为5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195,196,197,198,199,200,201,202,203,204,205,206,207,208,209,210,211,212,213,214,215,216,217,218,219,220,221,222,223,224,225,226,227,228,229,230,231,232,233,234,235,236,237,238,239,240,241,242,243,244,245,246,247,248,249,250,251,252,253,254,255,256,257,258,259,260,261,262,263,264,265,266,267,268,269,270,271,272,273,274,275,276,277,278,279,280,281,282,283,284,285,286,287,288,289,290,291,292,293,294,295,296,297,298,299,300,301,302,303,304,305,306,307,308,309,310,311,312,313,314,315,316,317,318,319,320,321,322,323,324,325,326,327,328,329,330,331,332,333,334,335,336,337,338,339,340,341,342,343,344,345,346,347,348,349,350,351,352,353,354,355,356,357,358,359,360,361,362,363,364,365,366,367,368,369,370,371,372,373,374,375,376,377,378,379,380,381,382,383,384,385,386,387,388,389,390,391,392,393,394,395,396,397,398,399,400,401,402,403,404,405,406,407,408,409,410,411,412,413,414,415,416,417,418,419,420,421,422,423,424,425,426,427,428,429,430,431,432,433,434,435,436,437,438,439,440,441,442,443,444,445,446,447,448,449,450,451,452,453,454,455,456,457,458,459,460,461,462,463,464,465,466,467,468,469,470,471,472,473,474,475,476,477,478,479,480,481,482,483,484,485,486,487,488,489,490,491,492,493,494,495,496,497,498,499,500,或1000,或2000,或5000个或更多个多核苷酸。这样的探针和引物在严格杂交条件下与靶序列特异性杂交。优选地,依照本发明的实施方案的探针和引物与靶序列具有完全的序列类似性,尽管可通过常规方法设计与靶序列不同并保留与靶序列杂交能力的探针。
用于制备和使用探针和引物的方法描述于例如Molecular Cloning:ALaboratory Manual,第2版,1-3卷,Sambrook等人编,Cold Spring Harbor LaboratoryPress,Cold Spring Harbor,N.Y.,1989中。PCR引物对可从已知序列,例如通过使用为此目的的计算机程序来衍生。
基于本文中披露的侧翼DNA和插入序列的引物和探针可通过常规方法例如通过重新克隆这样的序列并且将其测序用于确证(并且,如果需要的话,矫正)披露的序列。
本发明的实施方案的核酸探针和引物在严格条件下与靶DNA序列杂交。可使用任何常规核酸杂交或扩增方法在样品中鉴定来自转基因事件的DNA的存在。核酸分子或其片段能够在某些情况下与其他核酸分子特异性杂交。如本文中使用的,如果两个核酸分子能够形成反平行双链核酸结构,则称该两个核酸分子能够彼此特异性杂交。如果两个核酸分子呈现完全互补性,则称一个核酸分子为另一个核酸分子的“互补物”。如本文中使用的,当一个分子的每一个核苷酸与另一个分子的核苷酸互补时,称这些分子呈现“完全互补性”。呈现完全互补性的分子通常会以足够的稳定性彼此杂交以允许其在常规的“高严格”条件下保持退火到彼此之上。常规的高严格条件由Sambrook等人(1989)描述。
如果两个分子可以以足够的稳定性彼此杂交以允许其在至少常规的“低严格”条件下保持退火到彼此之上,则称其为呈现“最小互补性。常规的低严格条件由Sambrook等人(1989)描述。为了使核酸分子充当引物或探针,仅需其在序列上呈现最小互补性,以便能够在采用的特定溶剂和盐浓度下形成稳定的双链结构。
术语“严格条件”或“严格性条件”是关于核酸探针与靶核酸(即,与特定的目的核酸序列)通过Sambrook等人,1989在9.52-9.55处讨论的具体杂交步骤而杂交的功能限定。还参见Sambrook等人,1989,9.47-9.52和9.56-9.58。
取决于考虑到的应用,可使用探针或引物的多核苷酸序列简并性或严格条件的不同条件,以获得对于靶序列杂交的不同程度的选择性。对于需要高选择性的应用,通常会为一种多核苷酸序列与第二种多核苷酸序列的杂交采用相对严格的条件,例如,会选择相对低盐和/或高温度的条件,诸如由约0.02M至约0.15M NaCl在约50℃至约70℃的温度提供的条件。严格条件,例如,可涉及用高严格性洗涤缓冲液(0.2X SSC,0.1%SDS,65℃)洗涤杂交滤膜至少两次。促进DNA杂交的适当严格性条件对于本领域技术人员是已知的,例如在约45℃的6.0X氯化钠/柠檬酸钠(SSC),接着在50℃用2.0X SSC洗涤。例如,洗涤步骤中的盐浓度可选自在50℃的约2.0X SSC的低严格性至在50℃的约0.2X SSC的高严格性。另外,洗涤步骤中的温度可从在室温约22℃的低严格性条件增加至在约65℃的高严格性条件。温度和盐均可变化,或可将温度或盐浓度之一维持恒定,同时改变另一个变量。这样的选择性条件容许探针和模板或靶链之间很少的错配(如果有的话)。通过杂交检测DNA序列对于本领域技术人员是熟知的,且美国专利号4,965,188和5,176,995的教导是杂交分析方法的例子。
本发明的实施方案的核酸会在高严格性条件下特异性杂交于本文中例示或提出的一种或多种引物(或扩增子或其他序列),包括其互补物和片段。在本发明的一个方面,本发明的实施方案的标记核酸分子具有如本文中在例示序列之一中提出的核酸序列,或其互补物和/或片段。
在本发明的另一个方面,本发明的实施方案的标记核酸分子与这样的核酸序列共享80%至100%或90%至100%的序列同一性。在本发明的的实施方案的另外的方面,本发明的标记核酸分子与这样的序列共享95%至100%的序列同一性。这样的序列可用作植物育种方法中的标记以鉴定遗传杂交的后代。探针与靶DNA分子的杂交可通过本领域技术人员已知的任何多种方法来检测,其可包括但不限于荧光标记、放射性标记、基于抗体的标记、以及化学发光标记。
对于使用特定扩增引物对扩增靶核酸序列(例如,通过PCR),“严格条件”为允许引物对仅杂交于具有相应野生型序列(或其互补物)的引物会与之结合并优选产生独特扩增产物即扩增子的靶核酸序列的条件。
术语“特异于(靶序列)”表明探针或引物在严格杂交条件下仅与包含所述靶序列的样品中的靶序列杂交。
如本文中使用的,“扩增的DNA”或“扩增子”是指作为核酸模板一部分的靶核酸序列的核酸扩增产物。例如,为了确定从有性杂交获得的棉花植物是否含有来自本发明的实施方案的棉花植物的转基因事件基因组DNA,可使用引物对使从棉花植物组织样品提取的DNA经历核酸扩增方法以产生对于所述事件DNA的存在具有诊断性的扩增子,所述引物对包括来源于所述植物基因组中邻近插入的异源DNA插入位点的侧翼序列的引物、以及源于插入的异源DNA的第二引物。所述扩增子的长度和具有的序列使其对于所述事件具有诊断性。所述扩增子的长度的范围可以为引物对的合并长度加上一个核苷酸碱基对,和/或引物对的合并长度加上约2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195,196,197,198,199,200,201,202,203,204,205,206,207,208,209,210,211,212,213,214,215,216,217,218,219,220,221,222,223,224,225,226,227,228,229,230,231,232,233,234,235,236,237,238,239,240,241,242,243,244,245,246,247,248,249,250,251,252,253,254,255,256,257,258,259,260,261,262,263,264,265,266,267,268,269,270,271,272,273,274,275,276,277,278,279,280,281,282,283,284,285,286,287,288,289,290,291,292,293,294,295,296,297,298,299,300,301,302,303,304,305,306,307,308,309,310,311,312,313,314,315,316,317,318,319,320,321,322,323,324,325,326,327,328,329,330,331,332,333,334,335,336,337,338,339,340,341,342,343,344,345,346,347,348,349,350,351,352,353,354,355,356,357,358,359,360,361,362,363,364,365,366,367,368,369,370,371,372,373,374,375,376,377,378,379,380,381,382,383,384,385,386,387,388,389,390,391,392,393,394,395,396,397,398,399,400,401,402,403,404,405,406,407,408,409,410,411,412,413,414,415,416,417,418,419,420,421,422,423,424,425,426,427,428,429,430,431,432,433,434,435,436,437,438,439,440,441,442,443,444,445,446,447,448,449,450,451,452,453,454,455,456,457,458,459,460,461,462,463,464,465,466,467,468,469,470,471,472,473,474,475,476,477,478,479,480,481,482,483,484,485,486,487,488,489,490,491,492,493,494,495,496,497,498,499,或500,750,1000,1250,1500,1750,2000个或更多个核苷酸碱基对(加上或减去任何上述列出的增量)。或者,引物对可来源于在插入DNA两侧上的侧翼序列,从而产生包括整个插入核苷酸序列的扩增子。来源于植物基因组序列的一个引物对成员可位于距插入DNA序列一定距离处。该距离的范围可为一个核苷酸碱基对至约两万个核苷酸碱基对。术语“扩增子”的使用明确排除可在DNA热扩增反应中形成的引物二聚体。
可通过任何本领域中已知的各种核酸扩增方法,包括聚合酶链式反应(PCR)来实现核酸扩增。多种扩增方法在本领域是已知的,并描述于美国专利号4,683,195和美国专利号4,683,202等。开发了PCR扩增方法以扩增高达22kb的基因组DNA。这些方法以及本领域已知的其他DNA扩增方法可用于本发明的实施方案的实践。通过使用来源于本文中提供的序列的引物来扩增这样的序列,接着对PCR扩增子或克隆的DNA进行标准DNA测序,可以验证(如果需要的话,可校正)来自主题棉花事件的异源转基因DNA插入物或侧翼基因组序列的序列。
可通过多种技术检测由这些方法产生的扩增子。琼脂糖凝胶电泳和溴乙锭染色是检测DNA扩增子的常用熟知方法。另一种这样的方法是遗传比特分析(Genetic BitAnalysis),其中设计与邻接侧翼基因组DNA序列和插入DNA序列两者重叠的DNA寡核苷酸。将所述寡核苷酸固定化于微孔板的孔中。在对目的区域的PCR(使用一种在插入序列中的引物和一种在邻接侧翼基因组序列中的引物)之后,可将单链PCR产物与固定化的寡核苷酸杂交,并充当模板用于单碱基延伸反应,所述反应使用DNA聚合酶和对预期的下一个碱基具有特异性的标记的ddNTP。可通过将荧光信号的量进行定量而完成结合产物的分析。荧光信号表明由于成功的扩增、杂交和单碱基延伸所致的插入/侧翼序列的存在。
另一种方法是如由Winge(Innov.Pharma.Tech.00:18-24,2000)描述的焦磷酸测序(Pyrosequencing)技术。在这种方法中,设计与邻接基因组DNA和插入DNA连接重叠的寡核苷酸。设计与来自目的区域的单链PCR产物杂交的寡核苷酸(一种引物在插入序列中,而一种在侧翼基因组序列中),并在DNA聚合酶、ATP、硫酸化酶(sulfurylase)、萤光素酶、腺苷三磷酸双磷酸酶(apyrase)、腺苷5'磷酰硫酸(adenosine 5'phosphosulfate)和萤光素的存在下孵育。单独添加DNTP,且其掺入导致光信号,对该光信号进行测量。光信号表明由于成功的扩增、杂交和单碱基或多碱基延伸而导致的转基因插入/侧翼序列的存在。
荧光偏振是另一种可用于检测本发明的实施方案的扩增子的方法。遵循这种方法,设计与基因组侧翼和插入DNA连接重叠的寡核苷酸。将寡核苷酸杂交于来自目的区域的单链PCR产物(一种引物在插入DNA中,一种在侧翼基因组DNA序列中),并在DNA聚合酶和荧光标记的ddNTP的存在下孵育。单碱基延伸导致ddNTP的掺入。使用荧光计,可将荧光标记的ddNTP的掺入测量为在偏振方面的变化。在偏振方面的变化表明由于成功的扩增、杂交和单碱基延伸所致的转基因插入/侧翼序列的存在。
(PE Applied Biosystems,Foster City,Calif.)是检测和量化DNA序列的存在的方法。简言之,设计与基因组侧翼和插入DNA连接重叠的FRET寡核苷酸探针。在热稳定性聚合酶和dNTP存在下循环FRET探针和PCR引物(一种引物在插入DNA序列中,一种在侧翼基因组序列中)。在特异性扩增过程中,Taq DNA聚合酶校对机制使FRET探针上的荧光模块从淬灭模块释放。荧光信号表明由于成功的扩增和杂交所致的侧翼/转基因插入序列的存在。
已描述了分子信标用于多核苷酸序列检测。简言之,设计与侧翼基因组和插入DNA连接重叠的FRET寡核苷酸探针。FRET探针的独特结构导致其含有保持荧光和淬灭模块接近的二级结构。在热稳定性聚合酶和dNTP存在下循环FRET探针和PCR引物(一种引物在插入DNA序列中,一种在侧翼基因组序列中)。在成功的PCR扩增之后,FRET探针对靶序列的杂交导致探针二级结构的去除以及荧光和淬灭模块的空间分离。其结果为荧光信号。荧光信号表明由于成功的扩增和杂交所致的侧翼基因组/转基因插入序列的存在。
已经披露了棉花基因组中对于插入为优良的一个位置,本主题发明的实施方案还包括在这个基因组位置大致附近(general vicinity)包含至少一个非棉花事件pDAB4468.19.10.3插入物的棉花种子和/或棉花植物。一个选项是用不同的插入物取代来自本文中例示的棉花事件pDAB4468.19.10.3的插入物。一般而言,在特定的实施方案中采用例如靶向同源重组。这种类型的技术是例如WO 03/080809A2及其相应的已公开美国申请(US 20030232410)的主题。因而,本主题发明的实施方案包括植物和植物细胞,其包含侧翼为本文中鉴定的侧翼序列(SEQ ID NO:1的bp 1-1354和SEQ ID NO:2的bp 169-2898)的全部或可识别部分的异源插入物(代替或与多个拷贝的aad-12或pat基因一起)。也可以这种/这些方式将aad-12或pat基因的一个或一些另外的拷贝作为插入的靶。
包括了下述实施例以说明实践本发明实施方案的程序,并且演示本发明的某些优选实施方案。这些实施例不应视为限制性的。本领域技术人员应理解下述实施例中披露的技术代表用于说明其实践的优选模式的具体途径。然而,根据本披露,本领域技术人员应当理解,可在这些具体实施方案中做出许多变化,仍然获得相似或类似的结果而不背离本发明的精神和范围。除非另行指明,所有百分比按重量计,所有溶剂混合物比例按体积计,除非另行指明。
除非另行指明,使用下述缩写:
bp 碱基对
℃ 摄氏度
DNA 脱氧核糖核酸
EDTA 乙二胺四乙酸
kb 千碱基
μg 微克
μL 微升
mL 毫升
M 摩尔质量
PCR 聚合酶链式反应
PTU 植物转录单元或表达盒
SDS 十二烷基硫酸钠
SSC 含有氯化钠和柠檬酸纳的混合物、pH 7.0的缓冲溶液
TBE 含有Tris碱、硼酸和EDTA的混合物、pH 8.3的缓冲溶液
实施例
实施例1:事件特异性测定法
开发了事件特异性测定法,以在育种群体中检测棉花事件pDAB4468.19.10.3植物的存在。棉花事件pDAB4468.19.10.3含有二元载体pDAB4468的T-链(图1)。为了棉花事件pDAB4468.19.10.3的特异性检测,根据位于5’(SEQ ID NO:1)或3’(SEQ ID NO:2)插入物-植物连接中的DNA序列设计了两套引物和探针(图2)。将用于棉花事件pDAB4468.19.10.3的事件特异性测定法之一设计为使用两种引物和由Applied Biosystems(ABI)合成、其5’端含有VIC报道子的靶特异性MGB探针,特异性地检测跨越5’整合连接的77bp DNA片段(SEQ ID NO:3)。将用于棉花事件pDAB4468.19.10.3的第二事件特异性测定法设计为使用两种特异性引物和由ABI合成、在其5’端含有VIC报道子的靶特异性MGB探针,特异性地检测跨越3’整合连接的90bp DNA片段(SEQ ID NO:4)。针对其他的AAD12和PAT棉花事件以及非转基因棉花品种(Coker310)测试了这种用于棉花事件pDAB4468.19.10.3的检测方法的特异性。所有测定法以双重型式运行,其中测定法设计为检测已知的单拷贝棉花特异性内源参考基因Sah7(GenBank:AY117065.1)。
实施例1.1:gDNA分离
使用改良Qiagen Dneasy 96植物DNA试剂盒TM(Qiagen,Valencia,CA)提取基因组DNA。每个样品使用6个新鲜棉花叶子的子叶盘进行gDNA提取。用Pico GreenTM方法,根据销售商的说明书(Molecular Probes,Eugene,OR)将gDNA定量。将样品以1/5稀释度用无DNA酶的水进行稀释。
实施例1.2:测定法和结果
设计特异性引物和探针,用于棉花事件pDAB4468.19.10.3特异性测定法。这些试剂与以下列出的条件一起使用,以检测在棉花事件pDAB4468.19.10.3内的转基因插入物。表1列出了为了检测棉花事件pDAB4468.19.10.3的专门开发的引物和探针序列。
表1:PCR引物和探针
用于扩增的多重PCR条件如下:1X Roche PCR缓冲液,0.4μM事件特异性正向引物,0.4μM事件特异性反向引物,0.4μM引物IC_Sah7F,0.4μM引物IC_Sah7R,0.2μM事件特异性探针,0.2μM IC_Sah7Pr探针,0.1%PVP,2μL 1:5稀释的gDNA,总反应10μl。使用以下条件扩增所述混合物:i)在95℃持续10分钟,ii)在95℃持续10秒,iii)在55℃持续30秒,iv)重复步骤ii-iii持续40个循环,v)保持在40℃。在Roche480上进行实时PCR。数据分析是基于通过LightCycler 480软件确定的交叉点(Cp值)的测量,其为当在荧光方面的变化率达到其最大值时的PCR循环数。
针对含有aad12和pat PTU的不同棉花事件以及非转基因棉花品种,以具有棉花特异性内源参考基因IC Sah7(GenBank:AY117065.1)的多重型式对用于棉花事件pDAB4468.19.10.3的检测方法进行测试。这些测定法特异性地检测棉花事件pDAB4468.19.10.3,并且没有自对照(即,所述不同棉花事件和非转基因棉花品种Coker310)产生或扩增任何假阳性结果。所述事件特异性引物和探针可以用于检测棉花事件pDAB4468.19.10.3,并且这些条件和试剂适用于接合性分析。
已经说明并描述了本发明的原理,本领域的技术人员应当清楚的是,可以在布置和细节方面修改本发明而不背离这样的原理。我们主张所有这些修改均在所附权利要求书的精神和范围之内。
Claims (2)
1.一种在包含棉花DNA的样品中检测棉花事件如以ATCC登录号PTA-12457保藏的棉花种子中显示的pDAB4468.19.10.3的方法,所述方法包括:
(a)使所述样品与长度至少为10bp的第一引物以及长度至少为10bp的第二引物接触,所述第一引物与如SEQ ID NO:1的bp 1-1354所示的侧翼序列或其互补序列选择性地结合,所述第二引物与如SEQ ID NO:1的bp 1355-1672所示的插入序列或其互补序列选择性地结合;并且测定在所述引物之间产生的扩增子;或者
(b)使所述样品与长度至少为10bp的第一引物、以及长度至少为10bp的第二引物接触,所述第一引物与如SEQ ID NO:2的bp 1-168所示的插入序列或其互补序列选择性地结合,所述第二引物与如SEQ ID NO:2的bp 169-2898所示的侧翼序列或其互补序列选择性地结合;并且测定在所述引物之间产生的扩增子。
2.权利要求1的方法,其中所述包含棉花DNA的样品从包含事件pDAB4468.19.10.3、已经以ATCC登录号PTA-12457保藏的棉花植物获得。
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