CN111726983B - bZIP转录因子调节烟碱向降烟碱的转化 - Google Patents
bZIP转录因子调节烟碱向降烟碱的转化 Download PDFInfo
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Abstract
本文提供了一种减少烟碱向降烟碱转化的方法。所述方法包括将至少一种碱性区域/亮氨酸拉链(bZIP)型转录因子抑制剂施用于需要其的生物体。本文还提供了一种减少烟碱向降烟碱转化的方法，所述方法包括使烟碱N‑脱甲基酶(NND)的启动子上的bZIP型转录因子结合位点突变。本文还提供了一种减少烟碱向降烟碱转化的方法，所述方法包括使植物基因组突变以敲除至少一种bZIP型转录因子。
Description
相关申请
本申请要求于2017年12月7日提交的美国临时申请序列号62/595,983的权益，其全部公开内容通过该引用并入本文。
序列表
本申请含有序列表，该序列表已通过EFS-Web以ASCII格式提交，并通过引用以其全文并入本文。序列表的ASCII副本创建于2018年12月4日，名称为13177N-2183US.txt，且大小为12千字节。
技术领域
本发明涉及调节烟碱(nicotine)向降烟碱(nornicotine)转化的制品和方法。特别地，本公开的主题涉及用于调节烟碱向降烟碱转化的转录因子及其使用方法。
背景技术
普通烟草(Nicotiana tabacum，common tobacco)是一种起源于大约20万年前的天然异源四倍体。母本S基因组来自美花烟草(N.sylvestris)的祖先，且父本T基因组来自绒毛状烟草(N.tomentosiformis)的亲缘植物。烟碱是大多数栽培烟草品种中积累的主要生物碱。在过去的数十年中，在烟碱生物合成途径中的结构基因的分离和表征方面取得重大进展。茉莉酸(JA)是烟碱生物合成的主要诱导子。JA响应转录因子(TF)属于两个主要家族，APETALA2/乙烯响应因子(AP2/ERF)和碱性螺旋-环-螺旋(bHLH)，并且已知其在烟碱生物合成途径中诱导编码关键酶的基因的表达。
烟碱和其他托烷类生物碱(诸如天仙子胺和东莨菪碱)是在根部合成的，并通过木质部转运到叶片。已经分离多种转运子，并表征了它们在植物中生物碱的转运和液泡摄取(vacuolar sequestration)中的作用。在烟草中，许多属于多药及毒性化合物外排(MATE)家族的转运子(包括MATE1/2和茉莉酮酸酯诱导的生物碱转运子(JAT1/2))参与烟碱的转运和液泡摄取。然而，对这些转运子的调控中涉及的TF的研究不够彻底。
除烟碱外，烟草植物积聚另外三种吡啶生物碱，即降烟碱、新烟碱和去氢新烟碱。降烟碱是通过酶促过程从烟碱衍生而来的去甲基烟碱(不含甲基)。降烟碱也是在烟草材料的固化和加工过程中产生的N-亚硝基降烟碱(NNN)的前体。更具体地说，在收获后加工过程中，降烟碱与亚硝基化剂发生化学反应以形成NNN。由于NNN属于一类与吸烟有关的致癌物质，称为烟草特有亚硝胺(TSNA)，因此非常需要减少烟草制品中的TSNA。
有两种可能减少TSNA的方法。一种是减少整体烟碱含量；另一种是消除烟碱向降烟碱的转化。烟碱向降烟碱的转化由烟碱N-脱甲基酶(NND)(细胞色素P450酶的一个小家族)催化。在烟草中烟碱向降烟碱的转化中，已鉴定出三个NND基因：CYP82E4v1(起源于绒毛状烟草)、CYP82E5v2(起源于绒毛状烟草)和CYP82E10(起源于美花烟草)。CYP82E4v1(E4)在衰老叶片中的烟碱向降烟碱转化中起主要作用，而据报道CYP82E10(E10)的表达在根中且CYP82E5(E5)在根和叶中均起作用。然而，到目前为止，尚未鉴定出涉及调节烟碱向降烟碱转化的转录因子(TF)(即，E4、5和10基因的转录调节因子)。因此，尽管这些参与降烟碱生物合成的烟碱转运子和酶的生物化学和分子表征已取得重大进展，调节这些基因的潜在分子机制仍有待阐明。
因此，需要调节烟碱向降烟碱转化的制品和方法。
发明内容
本公开的主题满足若干或所有上述需求，这对于本领域普通技术人员在研究本文件中所提供的信息之后将变得显而易见。
发明内容描述本公开的主题的若干个实施方案，并且在许多情况下列出这些实施方案的变化和置换。发明内容仅仅是众多和变化的实施方案的示例。提及给定实施方案的一个或多个代表性特征同样是示例性的。这种实施方案通常可以具有或不具有所提到的特征；同样，那些特征可以应用于本公开的主题的其他实施方案，无论是否在发明内容中列出。为避免过多的重复，发明内容未列出或建议这些特征的所有可能组合。
在一些实施方案中，本公开的主题包括一种减少烟碱向降烟碱转化的方法，所述方法包含向需要其的生物体施用至少一种碱性区域/亮氨酸拉链(bZIP)型转录因子抑制剂。在一些实施方案中，至少一种bZIP转录因子抑制剂选自由C组bZIP转录因子抑制剂、S组bZIP转录因子抑制剂及其组合组成的组。在一些实施方案中，至少一种bZIP转录因子抑制剂选自由NtbZIP1a抑制剂、NtbZIP1b抑制剂、NtbZIP2a抑制剂、NtbZIP2b抑制剂及其组合组成的组。在一些实施方案中，至少一种bZIP转录因子抑制剂包含NtbZIP1a抑制剂和NtbZIP1b抑制剂。在一些实施方案中，至少一种bZIP转录因子抑制剂包含NtbZIP2a抑制剂和NtbZIP2b抑制剂。在一些实施方案中，至少一种bZIP转录因子抑制剂包含NtbZIP1a抑制剂、NtbZIP1b抑制剂、NtbZIP2a抑制剂和NtbZIP2b抑制剂。
在一些实施方案中，至少一种bZIP转录因子抑制剂选自由反义寡核苷酸、miRNA、siRNA、锁核酸(LNA)核苷酸及其组合组成的组。在一个实施方案中，至少一种bZIP转录因子抑制剂包含bZIP转录因子的反义寡核苷酸，所述bZIP转录因子选自由NtbZIP1a、NtbZIP1b、NtbZIP2a、NtbZIP2b及其组合组成的组。
在一些实施方案中，本文还提供了一种减少烟碱向降烟碱转化的方法，所述方法包含使烟碱N-脱甲基酶(NND)的启动子上的碱性区域/亮氨酸拉链(bZIP)型转录因子结合位点突变。在一些实施方案中，NND选自由CYP82E4v1、CYP82E5v2和CYP82E10组成的组。在一个实施方案中，NND为CYP82E4v1。在另一个实施方案中，CYP82E4v1的启动子上的bZIP结合位点是具有TACGTC的预突变序列的A/G盒。在另一个实施方案中，突变的结合位点具有序列TGCGTC。在一些实施方案中，突变的结合位点是通过定点诱变形成的。在一些实施方案中，所述方法还包括将至少一种bZIP型转录因子抑制剂施用于需要其的生物体。在一个实施方案中，至少一种bZIP转录因子抑制剂选自由NtbZIP1a抑制剂、NtbZIP1b抑制剂、NtbZIP2a抑制剂、NtbZIP2b抑制剂及其组合组成的组。
在一些实施方案中，本文还提供了一种减少烟碱向降烟碱转化的方法，所述方法包括使植物基因组突变以敲除至少一种碱性区域/亮氨酸拉链(bZIP)型转录因子。在一些实施方案中，至少一种bZIP转录因子选自由C组bZIP转录因子、S组bZIP转录因子及其组合组成的组。在一些实施方案中，至少一种bZIP转录因子选自由NtbZIP1a、NtbZIP1b、NtbZIP2a、NtbZIP2b及其组合组成的组。在一些实施方案中，至少一种bZIP转录因子选自由NtbZIP1a和NtbZIP2a、NtbZIP1b和NtbZIP2b及其组合组成的组。在一些实施方案中，所述方法还包括将至少一种bZIP型转录因子抑制剂施用于需要其的生物体。在一个实施方案中，至少一种bZIP转录因子抑制剂选自由NtbZIP1a抑制剂、NtbZIP1b抑制剂、NtbZIP2a抑制剂、NtbZIP2b抑制剂及其组合组成的组。
在研究本文件中的描述、附图和非限制性实施例之后，本公开的主题的其他特征和优点对于本领域普通技术人员将变得显而易见。
附图简述
当考虑到下面的详细描述时，将更好地理解本公开的主题，并且那些以上所述以外的特征、方面和优点将变得显而易见。这样的详细描述参考以下附图，其中：
图1示出说明八种不同烟草组织的转录组数据的分层簇分析的图。每种组织基于表达形成不同的簇。
图2示出说明烟草及其祖先中不同TF家族的分布的图。
图3示出说明在不同组织中的烟碱生物合成途径中的TF基因和结构基因的共表达分析的图。TF和结构基因基于它们的表达分为8个不同的模块(色彩标识的侧边栏)。黑色模块含有大部分烟碱生物合成途径的结构基因。YF，花蕾；MF，成熟的花；YL，嫩叶；ML，成熟叶片；SL，衰老的叶片。
图4示出说明NtbZIP1 a/b和NND在不同烟草组织中的表达的图。
图5示出说明烟草中的bZIP家族的图像。NtbZIP1a和NtbZIP1b用*表示。
图6示出对比NtbZIP1a和1b的核苷酸和氨基酸序列的图像。
图7示出说明烟草叶片中NtbZIP1a的瞬时过表达诱导CYP82E4v1、CYP82E5v2和CYP82E10的表达的图。
图8示出说明NtbZIP1a显著激活烟草细胞中的CYP82E4v1和CYP82E10启动子的图。
图9示出说明NtbZIP1a通过结合至A/G盒激活烟草细胞中的CYP82E4v1启动子的图。
图10示出说明NtbZIP1a和b显著激活烟草细胞中的CYP82E4v1启动子的图。
图11示出说明烟草植物的打顶下调NtbZIP和NND的表达的图。
图12A-C示出说明NtbZIP1a在烟草植物中的过表达的图和图像。(A)对照和三个转基因株系(株系#4、5和9)的基因组DNA PCR和cDNA PCR，分别证实了抗生素选择标记新霉素磷酸转移酶II(npt II；Kan)的整合和表达。(B)实时定量(qRT-PCR)分析，显示NtbZIP1和E4在对照(EV)和转基因株系(株系#4、5和9)中的相对表达。(C)代谢分析，显示对照和转基因株系(T0或第一代转基因植物)中烟碱向降烟碱的转化。
图13示出说明NtbZIP2a和2b的氨基酸序列比对的图像。
图14示出说明NtbZIP2a和b在烟草组织中具有相似表达模式的图。
图15示出说明NtbZIP2与NtbZIP1协同作用以激活烟草细胞中的E4启动子的图。
图16A-B示出说明使用酵母双杂交试验的NtbZIP1和NtbZIP2的蛋白质-蛋白质相互作用的图像。(A)酵母双杂交试验，显示NtbZIP1和NtbZIP2之间的蛋白质-蛋白质相互作用。在缺少亮氨酸、色氨酸、组氨酸和腺嘌呤(-leu-trp-his-ade)的合成缺陷型(syntheticdrop-out，SD)培养基上的菌落生长表明蛋白质(bZIP)之间的相互作用。(B)NtbZIP1和NtbZIP2的示意图。bZIP结构域由“阴影”矩形指示。数字表示氨基酸。
尽管本公开内容易于进行各种修改和替代形式，其特定实施方案已经在附图中以示例的方式示出，并且在下文中进行详细描述。然而，应当理解，对具体实施方式的描述并非旨在将本公开限制为涵盖落入由所附权利要求书限定的本公开的精神和范围内的所有修改、等同形式和替代形式。
定义
除非另有定义，本文中使用的所有技术和科学术语具有与本公开所属领域的普通技术人员通常所理解的相同含义。类似于或等同于本文描述的那些方法和材料的任何方法和材料都可以用于本公开的实践或测试中，包括以下描述的方法和材料。
遵循长期的专利法惯例，当在本申请(包括权利要求)中使用时，术语“一(a)”、“一(an)”和“该(the)”指的是“一或多”。因此，例如，提到“一细胞(a cell)”包括细胞的复数，依此类推。
术语“包含(comprising)”、“包括(including)”和“具有(having)”旨在是包括性的，并且意味着除所列要素之外，可能有额外的要素。
除非另有说明，在说明书和权利要求书中使用的表示成分、性质(诸如反应条件)等的量的数字在任何情况下均应理解为由术语“约”修饰。因此，除非有相反的指示，本说明书和权利要求书中列出的数字参数是近似值，其可以根据试图通过本公开的主题获得的期望性质而变化。
如本文所用，术语“约”在指代质量、重量、时间、体积、浓度、百分比等的值或量时，意在涵盖在一些实施方案中为相对于指定量的±50％、在一些实施方案中为相对于指定量的±40％、在一些实施方案中为相对于指定量的±30％、在一些实施方案中为相对于指定量的±20％、在一些实施方案中为相对于指定量的±10％、在一些实施方案中为相对于指定量的±5％、在一些实施方案中为相对于指定量的±1％、在一些实施方案中为相对于指定量的±0.5％、以及在一些实施方案中为相对于指定量的±0.1％的变化，因为这种变化适合于进行所公开的方法。
如本文所使用的，范围可以表示为从“约”一个特定值和/或到“约”另一特定值。还应理解，本文公开了许多值，并且除了值本身之外，每个值在本文中还公开为“约”该特定值。例如，如果公开值“10”，则也公开“约10”。还应理解，还公开了两个特定单元之间的每个单元。例如，如果公开了10和15，则还公开了11、12、13和14。
除非另外通过做出所引用的组合的上下文指定或明确地暗示相反情况，本文所使用的方法或处理步骤的所有组合可以以任何顺序进行。
发明详述
在本文件中阐述了本公开的主题的一个或多个实施方案的细节。在研究本文件中提供的信息之后，对本文件中描述的实施方案以及其他实施方案的修改对于本领域普通技术人员将是显而易见的。提供本文中提供的信息，尤其是所描述的示例性实施方案的具体细节，主要是为了清楚地理解，并且由此不会理解为不必要的限制。如有冲突，将以本文件的说明(包括定义)为准。
本公开的主题涉及调节烟碱向降烟碱转化的制品和方法。在一些实施方案中，所述制品包括一种或多种转录因子(TF)抑制剂。在一个实施方案中，例如，所述制品包括一种或多种碱性区域/亮氨酸拉链(bZIP)型转录因子的抑制剂。在另一个实施方案中，bZIP型转录因子来源于烟草。在另一个实施方案中，本发明人在烟草中鉴定出的133个bZIP型转录因子中，被分为十个亚组：A、B、C、D、E、F、G、H和S，合适的bZIP型转录因子包括亚组S中的27种bZIP中的至少一种、亚组C中的6种bZIP中的至少一个、其他bZIP 63同源物或其组合。在某些实施方案中，S亚组bZIP转录因子包括但不限于NtbZIP1a(SEQ ID NO：1和2)、NtbZIP1b(SEQ ID NO：3和4)或其组合；和/或C亚组bZIP转录因子包括但不限于NtbZIP2a(SEQ IDNO：5)、NtbZIP2b(SEQ ID NO：6)或其组合。
一种或多种转录因子抑制剂包括但不限于反义寡核苷酸、miRNA、siRNA、锁核酸(LNA)核苷酸或其组合。在一些实施方案中，这些抑制剂提供了RNAi介导的bZIP型转录因子的敲低/沉默。如本领域技术人员将理解的，转录因子抑制剂的特定序列/结构是基于特定转录因子的序列。因此，如本领域技术人员还将理解的，反义寡核苷酸和/或LNA可使用本文提供的bZIP型转录因子序列通过任何合适的方法形成。例如，在一个实施方案中，抑制剂包括与互补bZIP型转录因子具有100％序列同源性的反义寡核苷酸。在另一个实施方案中，转录因子抑制剂包括与bZIP型转录因子具有100％序列同源性的反义寡核苷酸，所述bZIP型转录因子与NtbZIP1a、NtbZIP1b、NtbZIP2a和/或NtbZIP2b互补。在这种实施方案中，转录因子抑制剂在烟草中提供RNAi介导的NtbZIP1a、NtbZIP1b、NtbZIP2a和/或NtbZIP2b的敲低/沉默。
在一些实施方案中，本文还提供了一种调节烟草植物或其他含烟碱的生物体中烟碱向降烟碱转化的方法。在一个实施方案中，所述方法包括将一种或多种本文公开的bZIP抑制剂施用于含烟碱的生物体。施用这些一种或多种bZIP抑制剂减少或消除烟碱向降烟碱的转化。可以以单一类型的bZIP转录因子或以bZIP转录因子的组合施用一种或多种抑制剂。例如，在一个实施方案中，所述方法包括施用一种或多种S bZIP型转录因子的抑制剂或C bZIP型转录因子的抑制剂。在另一个实施方案中，所述方法包括施用一种或多种S bZIP型转录因子的抑制剂和一种或多种C bZIP型转录因子的转录因子。在另一个实施方案中，所述方法包括向生物体施用一种或多种NtbZIP1a、NtbZIP1b、NtbZIP2a和/或NtbZIP2b的抑制剂。在某些实施方案中，抑制S bZIP型转录因子和C bZIP型转录因子两者对减少或消除烟碱向降烟碱的转化具有协同作用。
本文公开的方法包括施用单一类型的抑制剂或抑制剂的任何合适的组合，其对于每个被抑制的bZIP转录因子可以是相同的或不同的。例如，在一个实施方案中，所述方法包括施用NtbZIP1a、NtbZIP1b、NtbZIP2a和/或NtbZIP2b的反义寡核苷酸。在另一个实施方案中，所述方法包括施用一个bZIP转录因子(诸如NtbZIP1a)的反义寡核苷酸，以及另一个bZIP转录因子(诸如NtbZIP1b)的LNA核苷酸。如本领域技术人员将理解的，尽管以上针对bZIP转录因子和转录因子抑制剂的某些组合进行了讨论，本公开内容并不限于此并且可包括TF和TF抑制剂的任何其他合适的组合。
额外地或可选地，所述方法可包括bZIP型转录因子敲除和/或bZIP型转录因子在烟碱N-脱甲基酶(NND)的启动子上结合位点的突变。例如，在一个实施方案中，所述方法包括编辑植物基因组以敲除NtbZIP1a、NtbZIP1b、NtbZIP2a和/或NtbZIP2b。基因组编辑可通过任何合适的方法(诸如但不限于CRISPR/Cas9介导的基因组编辑)进行。在另一个实施方案中，所述方法包括通过定点诱变将称为A/G盒(TACGTC)的E4启动子中的bZIP结合元件突变为TGCGTC。尽管以上针对E4启动子中的特定突变进行了讨论，但是如本领域技术人员将理解的，本公开并不限于此并且包括E4、E5和/或E10启动子中的任何其他突变，以减少或消除bZIP型转录因子对相应NND的激活。
本文公开的TF抑制剂的施用、TF敲除和/或结合位点突变减少或消除bZIP型转录因子对NND的激活，这减少或消除烟碱向降烟碱的转化。与包括E4、E5和E10突变体的现有制品相反，本文公开的制品控制E4、E5和E10的表达以减少或消除烟碱向降烟碱的转化。通过减少或消除烟碱向降烟碱的转化，本文公开的制品和方法降低通常含有致癌降烟碱的产品(诸如但不限于烟草产品)的有害作用。
通过以下具体但非限制性的实施例进一步说明本公开的主题。以下示例可以包括代表与本公开的主题有关的开发和实验过程中在各个时间收集的数据的数据汇编。
实施例
实施例1
本实施例描述了对不同烟草组织(包括叶(嫩叶、成熟的叶片和衰老的叶片)、根、茎、花(花蕾和成熟的花)和蒴果)的转录组数据集的分析，以生成共表达网络。首先，进行了分层簇分析，揭示每一单独的组织类型均表现出独特的表达模式(图1)。接下来，鉴定编码烟草及其祖先中所有主要TF家族的基因。烟草基因组比其祖先含有更多的TF基因，并且属于MYB、AP2/ERF和bHLH家族的TF数量明显高于其他家族(图2)。鉴于此，基于它们在不同组织中的表达模式，然后将烟碱生物合成途径中的TF和结构基因分组为8个不同模块(色彩标识的侧边栏)(图3)。“黑色”模块特别有趣，因为在该模块中发现了大多数烟碱生物合成基因以及许多TF基因。许多这些TF属于MYB、bHLH、bZIP和ERF家族。如以下实施例2-4中所述，这些TF在烟草中烟碱生物合成中的作用是通过对其的分离和功能表征来建立的。
实施例2
本实施例描述来自烟草的两个bZIP型转录因子(图4)，其调节烟碱向降烟碱的转化，可以用于减少与吸烟有关的致癌物质，烟草特有亚硝胺(TSNA)。
bZIP TF的特征在于保守的亮氨酸拉链基序，其介导用于DNA结合的二聚体形成。在植物中，bZIP TF调节包括病原体防御、光和胁迫信号传导、种子成熟和花的发育的过程。许多bZIP因子，尤其是烟草中的bZIP因子，未得到很好的表征。通过共表达和簇分析，在本文中鉴定与E4、E5和E10共表达的两个bZIP TF基因。这些烟草bZIP TF被称为NtbZIP1a和NtbZIP1b。
NtbZIP1 a/b与CYP82E4v1(参与烟碱向降烟碱转化的主要NND酶)相比，表现出相似的表达模式，并在花和衰老叶片中高度表达(图4)。如图5所示，在烟草中鉴定出133个bZIP，并将其分为十个亚组：A、B、C、D、E、F、G、H、I和S，其中NtbZIP1 a/b属于S亚组。在玉米中，S组bZIP的表达是由创伤、寒冷和干旱胁迫诱导的。参考图6，还发现NtbZIP1a和b在核苷酸和氨基酸水平上的相同性超过97％。不希望受理论的束缚，据信这两个同源的bZIP来自烟草的两个祖先：美花烟草和绒毛状烟草。
确定两个bZIP TF后，测试NtbZIP1a的过表达是否导致E4、5和10的上调。在CaMV35S启动子和rbcS终止子的控制下，将NtbZIP1a克隆至pCAMBIA2300(二元载体)中。将二元载体(空对照和NtbZIP1a)导入农杆菌(Agrobacterium)中，并使用转化的农杆菌浸润烟草叶片。从农杆菌浸润的叶盘中分离出的总RNA用于cDNA合成，并使用实时定量PCR(qRT-PCR)检测NtbZIP1a、NtbZIP1b、E4、5和10的转录水平。普遍表达的管家基因(微管蛋白基因)在qRT-PCR中用作内部对照。结果显示，当NtbZIP1a瞬时高表达时，内源性NtbZIP1b、E4、5和10被上调(约3-10倍)，表明NtbZIP1a诱导NtbZIP1b，E4、5和10的表达，因此可能是这些基因的转录激活因子(图7)。
接下来，测试NtbZIP1a是否可以与其潜在靶基因的启动子结合。分离出E4、5和10的启动子(每个编码基因的5′非翻译区的大约1.0kb片段)，并将各个启动子与萤火虫荧光素酶报告基因融合。在CaMV 35S启动子和rbcS终止子的控制下，将NtbZIP1a基因克隆至pBlueScript(pBS)载体中。将质粒电穿孔导入烟草原生质体中。NtbZIP1a显著诱导了由E4和E10启动子控制的荧光素酶基因表达，但没有诱导E5启动子控制的荧光素酶基因表达，这表明NtbZIP1a可以直接激活E4和E10基因，这可能是通过与它们的启动子的结合(图8)。NtbZIP1a似乎未结合到用于激活实验的E5的1.0kb启动子区域。但是，如上所述，NtbZIP1a的过表达导致E5连同E4和10上调。启动子激活实验表明与E5基因有两种可能的NtbZIP1a调节关系：(1)NtbZIP1a与1.0kb启动子片段之外的位点结合，或(2)NtbZIP1a通过烟草中的另一种未知激活因子间接激活E5(例如，NtbZIP1a激活另一激活因子，其转而激活E5)。
为了支持NtbZIP1a直接与E4启动子结合以调节反式激活的可能性，通过定点诱变将E4启动子中的bZIP结合元件(称为A/G盒(TACGTC))突变为TGCGTC。如上所述，将突变的启动子与荧光素酶报告基因融合。然后，如上所述，使用突变报告质粒和NtbZIP1a表达载体进行反式激活实验。结果表明，在突变启动子的控制下，NtbZIP1a不能激活荧光素酶基因的表达(图9)。该实验表明，E4启动子通过A/G盒结合因子(最可能是NtbZIP1a)被激活。
参考图10，如对于NtbZIP1a所述，也使用NtbZIP1b表达载体和E4启动子-荧光素酶报告质粒进行反式激活实验。NtbZIP1b也以与NtbZIP1a类似的水平激活E4启动子。
最终，由于已经确定烟草打顶的农业实践(去除腋生枝)诱导烟碱的产生，本发明人分析来自打顶或未打顶的烟草植物的转录组数据。更具体地，在24小时后从对照(未打顶)和打顶的植物中收集叶片样品。从未打顶和打顶的叶片分离的RNA用于cDNA合成和qRT-PCR分析。结果表明，与未打顶的植物相比，打顶导致bZIP1 a/b以及E4、5和10的表达降低约70-90％，这表明打顶负调节烟草中bZIP1 a/b和NND的表达(图11)。结果还表明，bZIP1 a/b和NND在烟草中协调表达，如基因调节因子及其靶基因通常那样。
基于以上实施例，据信NtbZIP1a和1b作为激活因子参与三个NND基因的调节。NtbZIP1a/b的减少或失活可导致降烟碱的减少。
实施例3
本实施例描述了过表达NtbZIP1a的转基因株系的形成以及这些转基因株系对内源E4表达的影响。
为了形成转基因株系，将含有在CaMV 35S启动子和rbcS终止子控制下的NtbZIP1a的pCAMBIA2300(二元载体)导入农杆菌中，并用转化的农杆菌感染烟草叶盘。从农杆菌感染的叶盘中产生了20多个过表达NtbZIP1a的转基因株系。从对照和三个转基因株系中分离出的基因组DNA用于通过PCR扩增抗生素选择标记新霉素磷酸转移酶II(nptII；kan)来验证植物的转基因状态。从对照和三个转基因株系的叶片中分离的总RNA用于cDNA合成。RT-PCR用于验证nptII(kan)基因在转基因植物中的表达(图12A)。实时定量PCR(qRT-PCR)用于检测NtbZIP1a和E4的转录水平(图12B)。普遍表达的管家基因(微管蛋白基因)在qRT-PCR中用作内部对照。
本实施例的结果表明，与对照相比，NtbZIP1a在转基因植物中的表达显著更高。当NtbZIP1a高表达时，内源性E4表达被上调(约6-8倍)，表明NtbZIP1a诱导E4的表达，并因此可能是E4基因的转录激活因子。另外，代谢分析表明，与对照相比，转基因烟草叶片中烟碱向降烟碱的转化率更高(图12C)。用于计算烟碱向降烟碱的转化率的公式为：
这三个株系分析中的两个显示烟碱向降烟碱的转化率更高。由于代谢分析是用独立的T0(第一代转基因植物)隔离种群进行的，代谢结果可能会有所不同。
实施例4
如以上实施例2中所述，本发明人表征了NtbZIP1a和b，两个属于S组bZIP因子的NtbZIP。在拟南芥中，已知S组bZIP因子与某些C组因子相互作用以调节基因表达。鉴于这种相互作用，本发明人鉴定了与S组因子相互作用的C组成员拟南芥bZIP 63的烟草同源物。在烟草中，有两个bZIP 63同源物，在这里被称为NtbZIP2a和b，它们具有大于95％的氨基酸相同性(图13)。不希望受到理论的束缚，据信NtbZIP2a和b来源于两个烟草祖先美花烟草和绒毛状烟草，并且在功能上是冗余的。
根据转录组学分析，NtbZIP2a和b在烟草花、叶、茎和根中具有相似的表达模式(图14)。基于前述讨论，本发明人假设NtbZIP2a和b也单独和/或与NtbZIP1a和b协调调节E4/5/10。因此，单独地或与NtbZIP1a组合地测试了NtbZIP2a对E4启动子的反式激活。更具体地，将E4启动子与萤火虫荧光素酶报告基因融合，并且在CaMV 35S启动子和rbcs终止子的控制下，将bZIP TF克隆至pBS载体中。结果表明，NtbZIP1a和NtbZIP2a分别单独地激活E4启动子；然而，当NtbZIP1a和2a都被共表达时，与单独由每种因子诱导的相比，E4启动子的反式激活显著增加(图15)。
接下来，为了确定NtbZIP1a/b是否与NtbZIP2a相互作用，发明人进行了酵母杂交试验。酵母细胞在缺少亮氨酸、色氨酸、组氨酸和腺嘌呤的合成缺陷型(SD)培养基(SD-leu-trp-his-ade)上的生长表明NtbZIP1a/b与NtbZIP2a相互作用(图16A；1和2)。另外，NtbZIP2与其自身相互作用以形成同型二聚体(图16A；3)。NtbZIP1和NtbZIP2的bZIP结构域如图16B所示。
尽管本实施例仅测试了NtbZIP2a对E4启动子的活性，但未测试其对E5或E10启动子的活性，但相信这两个NtbZIP2因子都是E4/5/10基因的激活因子。即，本实施例表明C组NtbZIP2a和b是E4/5/10基因的两个先前未表征的调节物。另外，本实施例表明，NtbZIP1和NtbZIP2在激活E4(可能是E5和10)启动子中是协同的。特别地，不希望受理论的束缚，据信与单独的NtbZIP1或NtbZIP2相比，NtbZIP2a和/或b与NtbZIP1a和/或b相互作用以增强DNA结合能力并显著提高E4/5/10启动子的激活。
总之，在本文中表征了作为E4/5/10基因的正调节物的两个S组和两个C组NtbZIP因子。NtbZIP1a和2a对E4启动子的反式激活是加性的。因此，不希望受到理论的束缚，据信使这些基因中的一个或全部失活的敲除方法降低E4/5/10基因的表达。
参考文献
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尽管本公开内容易于进行各种修改和替代形式，其特定实施方案已经在附图中以示例的方式示出，并且在下文中进行详细描述。然而，应当理解，对具体实施方式的描述并非旨在将本公开限制为涵盖落入由所附权利要求书限定的本公开的精神和范围内的所有修改、等同形式和替代形式。
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Leu Asn Pro Leu Phe Gln Ala Met Ser Glu Ile Ser Ser Met Val Met
340 345 350
Pro Ser Tyr Ser Gly Ser Pro Ser Asp Thr Ser Ala Asp Ala Ala Val
355 360 365
Pro Val Gln Asp Asp Pro Lys His His Tyr Tyr Gln Gln Pro Pro Asn
370 375 380
Asn Leu Met Pro Thr His Asp Pro Arg Ile Gln Asn Gly Met Val Asp
385 390 395 400
Val Pro Pro Ile Glu Asn Val Glu Gln Asn Pro Ala Thr Ala Ala Val
405 410 415
Gly Gly Asn Lys Met Gly Arg Thr Thr Ser Met Gln Arg Val Ala Ser
420 425 430
Leu Glu His Leu Gln Lys Arg Ile Arg Gly Glu Val Ser Ser Cys Gly
435 440 445
Thr Gln Gly Arg Gly Glu Gln
450 455
<210> 6
<211> 442
<212> PRT
<213> Nicotiana tabacum
<400> 6
Met Asp Arg Val Phe Ser Val Asp Asp Asp Ile Gly Asp His Phe Trp
1 5 10 15
Ser Thr Pro Pro Thr Ala Asp Leu Gly Val Asp Ser Pro Thr Thr Ala
20 25 30
Thr Ala Ala Ser Tyr Ser Lys Met Met Asn Arg Ser Ser Ser Glu Trp
35 40 45
Ala Phe Gln Arg Phe Leu Gln Glu Ala Thr Ala Ala Gly Thr Ser Thr
50 55 60
Ser Ser His Pro Gln Pro Pro Thr Met Thr Ala Ser Ser Ser Ser Ser
65 70 75 80
Ser His Gln Asn Asp Val Val Glu Ile Lys Asp Glu Asn Leu Ser Thr
85 90 95
Pro Asn Leu Asn Pro Ser Thr Ala Leu Asn Ser Lys Pro Ala Ser Ser
100 105 110
Phe Gly Leu Thr Pro Pro Pro Asn Ile Ala Val Asp Ser Glu Glu Tyr
115 120 125
Gln Ala Phe Leu Lys Ser Gln Leu His Leu Ala Cys Ala Ala Val Ala
130 135 140
Leu Thr Arg Gly Lys Ser Leu Asn Pro Gln Asp Ser Gly Ser Thr Ala
145 150 155 160
His Asp Lys Gly Ser Glu Thr Ala Ser Ala Ala Gln Ser Gly Ser His
165 170 175
Glu Met Ala Lys Ile Gln Asp Lys Asp Ala Gly Gly Pro Val Gly Ile
180 185 190
Pro Ser Leu Pro Pro Val Gln Lys Lys Pro Val Val Gln Val Arg Ser
195 200 205
Thr Thr Ser Gly Ser Ser Arg Glu Gln Ser Asp Asp Asp Glu Ala Glu
210 215 220
Gly Glu Ala Glu Thr Thr Gln Gly Met Asp Pro Ala Asp Ala Lys Arg
225 230 235 240
Val Arg Arg Met Leu Ser Asn Arg Glu Ser Ala Arg Arg Ser Arg Arg
245 250 255
Arg Lys Gln Ala His Leu Thr Glu Leu Glu Thr Gln Val Ser Gln Leu
260 265 270
Arg Val Glu Asn Ser Ser Leu Leu Lys Arg Leu Thr Asp Ile Ser Gln
275 280 285
Lys Tyr Asn Glu Ala Ala Val Asp Asn Arg Val Leu Lys Ala Asp Val
290 295 300
Glu Thr Leu Arg Ala Lys Val Lys Met Ala Glu Glu Thr Val Lys Arg
305 310 315 320
Val Thr Gly Leu Asn Pro Leu Phe Gln Ala Met Ser Glu Ile Ser Ser
325 330 335
Met Val Met Pro Ser Tyr Ser Gly Ser Pro Ser Asp Thr Ser Ala Asp
340 345 350
Ala Ala Val Pro Val Gln Asp Asp Pro Lys His His Tyr Tyr Gln Gln
355 360 365
Pro Pro Asn Asn His Met Pro Thr Asn Asp Pro Arg Ile Gln Asn Gly
370 375 380
Met Val Asp Val Pro Pro Ile Glu Asn Val Gln Gln Asn Pro Ala Thr
385 390 395 400
Ala Ala Val Gly Gly Asn Lys Met Gly Arg Thr Ala Ser Met Gln Arg
405 410 415
Val Ala Ser Leu Glu His Leu Gln Lys Arg Ile Arg Gly Glu Ile Ser
420 425 430
Ser Cys Gly Thr Gln Gly Arg Gly Glu Gln
435 440
Claims (3)
1.一种减少烟草植物中烟碱向降烟碱转化的方法，所述方法包含使烟碱N-脱甲基酶的启动子上的碱性区域/亮氨酸拉链型转录因子结合位点突变，其中所述碱性区域/亮氨酸拉链型转录因子如选自SEQ ID NO:2和4组成的组中的氨基酸序列所示，且其中所述烟碱N-脱甲基酶选自CYP82E4v1和CYP82E10组成的组，且其中所述碱性区域/亮氨酸拉链型转录因子结合位点从TACGTC突变为TGCGTC。
2.权利要求1的方法，其中所述烟碱N-脱甲基酶为CYP82E4v1。
3.权利要求2的方法，其中所述突变的结合位点是通过定点诱变形成的。
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201762595983P | 2017-12-07 | 2017-12-07 | |
| US62/595,983 | 2017-12-07 | ||
| PCT/US2018/064317 WO2019113360A1 (en) | 2017-12-07 | 2018-12-06 | bZIP TRANSCRIPTION FACTORS REGULATE CONVERSION OF NICOTINE TO NORNICOTINE |
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| Publication Number | Publication Date |
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| CN111726983A CN111726983A (zh) | 2020-09-29 |
| CN111726983B true CN111726983B (zh) | 2024-05-07 |
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| CN201880086430.6A Active CN111726983B (zh) | 2017-12-07 | 2018-12-06 | bZIP转录因子调节烟碱向降烟碱的转化 |
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| Country | Link |
|---|---|
| US (2) | US11773401B2 (zh) |
| EP (1) | EP3720274A4 (zh) |
| CN (1) | CN111726983B (zh) |
| AR (1) | AR113489A1 (zh) |
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| WO (1) | WO2019113360A1 (zh) |
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| BR112021024528A2 (pt) * | 2019-06-05 | 2022-01-18 | Reynolds Tobacco Co R | Fatores de transcrição bzip regulam a conversão de nicotina em nornicotina e reduzem os níveis de precursores específicos de tabaco (tsna) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2009064771A2 (en) * | 2007-11-12 | 2009-05-22 | North Carolina State University | Alteration of tobacco alkaloid content through modification of specific cytochrome p450 genes |
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| US20070006335A1 (en) | 2004-02-13 | 2007-01-04 | Zhihong Cook | Promoter, promoter control elements, and combinations, and uses thereof |
| US8088975B2 (en) * | 2006-10-27 | 2012-01-03 | Ceres, Inc. | Phenylpropanoid related regulatory protein-regulatory region associations |
| AU2006216715A1 (en) * | 2005-02-22 | 2006-08-31 | Ceres Inc. | Modulating plant alkaloids |
| WO2006091194A1 (en) * | 2005-02-23 | 2006-08-31 | North Carolina State University | Alteration of tobacco alkaloid content through modification of specific cytochrome p450 genes |
| US8153863B2 (en) * | 2007-03-23 | 2012-04-10 | New York University | Transgenic plants expressing GLK1 and CCA1 having increased nitrogen assimilation capacity |
| KR101611847B1 (ko) * | 2010-01-15 | 2016-04-14 | 노쓰 캐롤라이나 스테이트 유니버시티 | 담배에서의 니코틴 합성을 최소화하기 위한 조성물 및 방법 |
| US10415050B2 (en) * | 2014-05-08 | 2019-09-17 | Philip Morris Products S.A. | Reduction of nicotine to nornicotine conversion in plants |
| CN105602961B (zh) * | 2015-09-07 | 2018-10-23 | 中国农业科学院烟草研究所 | 突变的烤烟cyp82e4基因及其应用 |
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- 2018-12-06 US US16/770,906 patent/US11773401B2/en active Active
- 2018-12-06 CN CN201880086430.6A patent/CN111726983B/zh active Active
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009064771A2 (en) * | 2007-11-12 | 2009-05-22 | North Carolina State University | Alteration of tobacco alkaloid content through modification of specific cytochrome p450 genes |
Non-Patent Citations (3)
| Title |
|---|
| Non-Functionalization of Two CYP82E Nicotine N-Demethylase Genes Abolishes Nornicotine Formation in Nicotiana langsdorffii;Phattharaporn Pakdeechanuan等，;《Plant and Cell Physiology》;20121203;第53卷(第12期);2044页第1段 * |
| RNA interference (RNAi)-induced suppression of nicotine demethylase activity reduces levels of a key carcinogen in cured tobacco leaves;Ramsey S Lewis等;《Plant Biotechnol J》;20080214;第6卷(第4期);摘要、第347页第2段 * |
| 周骏等.烟碱去甲基酶.《应用生物技术降低烟草中特有N-亚硝胺》.北京：科学技术文献出版社,2016, * |
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| Publication number | Publication date |
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| EP3720274A4 (en) | 2021-09-22 |
| AR113489A1 (es) | 2020-05-06 |
| US20210171967A1 (en) | 2021-06-10 |
| BR112020011386A2 (pt) | 2020-12-01 |
| CN111726983A (zh) | 2020-09-29 |
| US11773401B2 (en) | 2023-10-03 |
| WO2019113360A1 (en) | 2019-06-13 |
| US20230416766A1 (en) | 2023-12-28 |
| EP3720274A1 (en) | 2020-10-14 |
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