CN1196753A - 根皮层特异性基因启动子 - Google Patents
根皮层特异性基因启动子 Download PDFInfo
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- CN1196753A CN1196753A CN96197068A CN96197068A CN1196753A CN 1196753 A CN1196753 A CN 1196753A CN 96197068 A CN96197068 A CN 96197068A CN 96197068 A CN96197068 A CN 96197068A CN 1196753 A CN1196753 A CN 1196753A
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Abstract
本发明的分离的DNA分子包含DNA启动子序列,所说的启动子序列指导植物细胞中下游异源DNA片段的根皮层特异性转录。DNA构建体包含表达盒,所说的表达盒在5′—3′的方向上包含本发明的启动子和定位在此启动子下游并与之可操作性地结合的异源DNA片段。转化的植物(如烟草)包含含有异源DNA构建体的转化的植物细胞,所说的DNA构建体包含以上所述的表达盒。
Description
本发明是由政府以国家科学基金MCB-9206506资助的。政府对本发明可以具有一定的权利。
发明领域
本发明涉及组织特异性的基因启动子,特别是涉及在植物根皮层中具有活性的启动子。
发明背景
启动子是位于转录基因侧翼的DNA序列,并且如果要把侧翼基因转录成信使RNA,RNA聚合酶必须与启动子结合。启动子可以由许多不同的调节元件组成,这些调节元件影响以不同方式可操作性地与所说的启动子结合的结构基因。例如,调节基因可以增强或抑制所结合的结构基因的表达,对该基因进行发育调节或者有助于该基因的组织特异性调节。使用重组DNA方法进行启动子的修饰可能获得选择性的基因表达模式。例如参见Old和Primrose,基因操作原理(第4版,1989)。
植物启动子的一个例子是在用于矮牵牛属的小亚基核酮糖-1,5-二磷酸羧化酶的基因的侧翼发现的启动子。参见美国专利4,962,028。另一个例子是含有小麦Em基因5′侧翼区的启动子。参见EPO申请335528。还有一个例子是在EPO申请0 330 479中公开的诱导胁迫的调节元件。
尽管根部基因表达的调节作用在植物发育过程中具有重要的作用,但是相对来说这方面的工作做得很少。这种缺陷部分是由于缺乏容易鉴别的根特异性生化功能(可能很容易对这些基因进行克隆和研究)。Evans等(分子基因遗传学214,153-157(1988))试图从豌豆分离根特异性cDNA克隆,但没有成功,得到的结论为根特异性mRNA(如果存在)仅以非常低的水平存在于根mRNA群体中。Fuller等(美国科学院学报80,2594-2598(1983))已经克隆并定性了大量的根瘤特异性基因。比较所说的DNA序列转录起始处的5′端表明在这三个检验的基因中存在重复的8核苷酸。遗憾的是对于大多数豆科(Leguminaceae)缺乏有效的转化/再生***,阻碍了这些顺式作用序列的功能分析。Bogusz等(自然331,178-180(1988))通过不形成瘤的植物的血红蛋白基因与紧密相关的形成瘤的植物种类的血红蛋白基因的同源性分离了在不形成瘤的植物的根中特异表达的血红蛋白基因。Keller和Lamb(基因和发育3,1639-1646(1989))分离了在侧根起始过程中表达的编码细胞壁的富含羟脯氨酸的糖蛋白的基因。Lerner和Raikhel(植物生理学91,124-129(1989))最近报道了大麦根特异性凝集素的克隆和定性。
许多植物病原体和害虫破坏植物的根,造成严重的作物损害和损失。最常损害的根组织是根皮层,这是一层主要由贮藏薄壁组织组成的层,这些贮藏薄壁组织在表皮层下并围绕根的中央维管束。根皮层可能还包含厚壁组织、分泌细胞、树脂道、其它结构和细胞类型。根皮层的细胞与地上茎的皮层细胞具有形态和发育的相似性。
为了使用遗传工程技术经外源基因的表达将有用的性状传递给植物,需要各种组织特异性的启动子以便使新的性状能够在合适的植物组织中进行选择性表达。本发明是以我们与这个问题有关的持续研究为基础的。
发明概述
本发明是以识别烟草RD2(TobRD2)启动子为基础的,这种启动子指导相关基因的根皮层特异性表达。本发明的第一个方面是指导植物细胞中下游异源DNA片段的根皮层特异性转录的分离的DNA分子,这种分离的DNA分子具有选自(a)由本文提供的SEQ ID NO:1-9和(b)在严格的条件下和SEQ ID NO:1-9任一之序列杂交并指导植物细胞中下游异源DNA片段的根皮层特异性转录的DNA序列组成的组中的序列。
本发明的另一个方面是包含烟草RD2启动子和位于启动子的下游并可操作性地与启动子连接的异源DNA片段的表达盒。
本发明的另一个方面是一种表达盒,这种表达盒包含根皮层特异性启动子和异源DNA片段、从本文所提供的SEQ ID NO:1-9中选择的根皮层特异性启动子的序列和在严格的条件下和SEQ ID NO:1-9任一之序列杂交并指导根皮层特异性转录的DNA序列。
本发明的另一方面还包括含有以上所述的表达盒的植物细胞、由这样的植物细胞产生转化植物的方法以及含有这样转化的植物细胞的转化植物。
图的简单描述
图1A显示7天大的幼苗通过原位杂交在烟草根横断面中定位的烟草RD2转录物。
图1B显示7天大的幼苗通过原位杂交在烟草根纵切面中定位的烟草RD2转录物。
图2是TobRD2的5′区的2010个碱基对序列(SEQ ID NO:1)。
图3图解显示了用于检验指导根皮层特异性基因表达的RD2启动子的能力的TobRD2启动子/葡糖苷酸酶(GUS)构建体。
图4是总结了用嵌合报道基因构建体转化的植物的根(实心柱)、叶片(斜纹柱)、茎(点柱)中β-葡糖苷酸酶(GUS)活性的柱形图,结果如表1所示。此图显示出用基因构建体转化的植物的活性,其中使用了不同的启动子(CaMV35S;Δ2.00;Δ1.50;Δ1.40;Δ1.25;Δ0.80;Δ0.70;Δ0.60;Δ0.30),同时只使用载体pBI101.3作为对照。以pmolMU/μg蛋白质/分钟表示GUS活性。
图5A是总结了用嵌合报道基因构建体转化的烟草植物的根、叶片中相对β-葡糖苷酸酶(GUS)活性的柱形图,结果如表1所示,其中使用了不同的启动子(CaMV35S;Δ2.00;Δ1.50;Δ1.40;Δ1.25;Δ0.80;Δ0.70;Δ0.60;Δ0.30),同时只使用载体pBI101.3作为对照。以pmolMU/μg蛋白质/分钟表示GUS活性,所示的相对活性是根活性/叶活性。
图5B是总结了用嵌合报道基因构建体转化的烟草植物的根、茎中相对β-葡糖苷酸酶(GUS)活性的柱形图,结果如表1所示,其中使用了不同的启动子(CaMV35S;Δ2.00;Δ1.50;Δ1.40;Δ1.25;Δ0.80;Δ0.70;Δ0.60;Δ0.30),同时只使用载体pBI101.3作为对照。以pmolMU/μg蛋白质/分钟表示GUS活性,所示的相对活性是根活性/茎活性。
图6A是光学显微照片,显示出GUS活性在由报道基因(GUS)(由Δ2.0启动子驱动)转化的烟草植物根的横切面上的组化定位。
图6B是光学显微照片,显示出GUS活性在由报道基因(GUS)(由Δ2.0启动子驱动)转化的烟草植物根的根尖上的组化定位。
发明的详细描述
本文只是以单链形式在5′-3′方向从左到右显示了核苷酸序列。本文以IUPAC-IUB生物化学命名法委员会推荐的形式表示了核苷酸。
表达抑制或者杀死特殊的害虫或病原体的肽的转基因植物提供了减少作物危害和损失的方法。例如,在转基因玉米中的苏云金芽孢杆菌蛋白质的表达提供了对欧洲玉米螟虫的抗性。然而,转基因在所有植物组织中的表达(组成型表达)是不利的,因为它能使非靶有机体暴露于转基因蛋白,并且还会增加用于对转基因蛋白具有抗性的病原体和害虫发育的选择性压力。转基因在整个植物中的高水平的表达也可能对植物的成长和产量有负面影响。另一个策略是只在受特定害虫或者病原体影响的器官或组织中表达有毒的肽。由于缺乏已定性的根特异性启动子,实施这一针对危害植物根的害虫和病原体的策略受到阻碍。
当在RNA聚合酶和基因启动子之间形成稳定的复合体时,基因的转录就开始了。在所有转录单元开始的启动子一般大约为100个碱基对长,并且位置紧靠着转录起始位点的上游。例如参见,Maniatis等,科学236:1238(1987)。启动子在“强度”上可以变化,即它们精确有效地开始转录的能力是可变的。认为RNA聚合酶全酶能覆盖紧靠着转录区上游大约50个碱基对的区域。有时通过与启动子区域邻近结合的辅助蛋白可以增加转录起始的强度,所说的启动子区紧靠着所说的转录DNA的上游。例如参见Singer和Berg,基因和基因组,140-145,大学科学图书,MillValley,CA(1991)。
本发明的根皮层特异性启动子的具体例子是具有与SEQ ID NO:1-9(下文将更详细地讨论)任一之序列相应的序列的DNA分子。显而易见,可以制备其它的比前述的序列更长或者更短的或具有最小添加、缺失、或者取代的烟草RD2 5′侧翼区域的序列片段,这些序列也将携带TobRD2根皮层特异性启动子,所有这些序列都包含在本发明之内。本发明的另一个方面包括从其它烟草基因或者从下文所述的非烟草植物中分离的启动子,这些启动子与烟草RD2启动子同源,并且能够指导细胞中下游异源DNA片段的根皮层特异性转录。
本文使用的TobRD2启动子指具有与在烟草RD2基因转录区5′端发现的DNA连续片段相同(或者基本上同源)的序列的DNA分子。本文给出的SEQ ID NO:1提供了在紧靠着TobRD2基因中转录开始的5’端发现的2kb区域的序列。TobRD2启动子在紧靠着TobRD2转录区的5′端包括至少100个碱基对的区域、150个碱基对区域或者优选的200个碱基对区域,并且指导根皮层特异性表达。本文使用的“基本上同源”的区域至少有75%,更为优选的是80%、85%、90%,或者甚至95%的同源性。
本文所使用的根皮层特异性启动子是优先指导根皮层组织中可操作性结合的基因表达的启动子,这是相对于叶片或茎组织或者根的其它组织中的表达而言。
其它植物的根皮层特异性启动子序列包括那些与紧靠着转录DNA区上游的烟草RD2启动子的大约100个碱基片段具有至少大约75%同源性(更为优选的是80%、85%、90%,或者甚至95%同源性)的序列,并且所说的序列能够指导植物细胞中下游异源DNA片段的根皮层特异性转录。其它植物的根皮层特异性启动子包括那些与本文的SEQ ID No:1-9所限定的TobRDa启动子的连续部分具有至少大约75%同源性(更为优选的是80%、85%、90%,或者甚至95%同源性)的序列,并且所说的序列能够指导植物细胞中下游异源DNA片段的根皮层特异性转录。
允许异源DNA序列与本文给出的DNA序列杂交的高度严格的杂交条件是本领域公知的。例如,可以在25%甲酰胺、5X SSC、5X Denhardt溶液、100μg/ml单链DNA、5%硫酸葡聚糖中42℃下将这些序列与本文公开的DNA杂交,洗涤条件为25%甲酰胺、5X SSC、0.1%SDS在42℃下洗涤15分钟,以便使具有60%同源性的序列杂交。更为严格的条件具有更严格的洗涤条件:0.3M NaCl、0.03M柠檬酸钠、0.1%SDS、60℃或者甚至70℃,使用标准的原位杂交测定。(参见Sambrook等,分子克隆,实验室手册(第2版1989)(冷泉港实验室))。一般来说,编码根皮层特异性启动子并与编码本文公开的烟草RD2根皮层特异性启动子的DNA序列杂交的植物DNA序列与编码本文公开的烟草RD2根皮层特异性启动子的DNA序列具有至少大约75%、80%、85%、90%或者甚至95%或更多的同源性。
本发明的根皮层特异性启动子在指导转化植物的转基因进行组织特异性表达时是有用的。这种组织特异性转基因的表达在提供对由侵害植物根的害虫和病原体造成的损害的抗性方面是有用的。此外,当根皮层为光合产物的主要积累器官时,意在改变储存的碳水化合物的转基因的表达可以由这些启动子指导。对根皮层特异性表达具有特殊重要性的外源基因包含:编码与重金属(如金属硫蛋白)结合的蛋白质的基因;编码对土壤中存在的害虫和病原体有抗性的蛋白质的基因;编码对热、盐(盐度)和干旱具有抗性的蛋白质的基因;编码用于脱盐化的蛋白质的基因;编码将植物储藏的化合物代谢成另外优选的产物或形式的蛋白质的基因。
组织特异性启动子也可以用于将选择的组织位点上杀虫剂原转化成活性形式。Hsu等(杀虫剂科学,44,9(1995))报道了包含根特异性启动子TobRB7和β-葡糖苷酸酶基因的嵌合基因在优先将根中的杀虫剂原转化成活性形式方面的用途。将灭活的杀虫剂原(羟甲基草氨酰的葡糖苷酸)施用到叶片上,然后通过植物韧皮部将其运输到根部,在此由葡糖苷酸酶将其转化成有活性的杀线虫剂形式。
另外,根皮层特异性启动子在组织学上是有用的,可以用来通过使用报道基因(如β-葡糖苷酸酶)鉴定或者染色根皮层组织。
本文使用的“可操作性地结合”的术语是指DNA序列包含在单一DNA分子上,这些DNA序列被结合以便一个DNA序列的功能可以受另一个DNA序列影响。这样当启动子能够影响基因(即此基因在所说的启动子的转录控制下)的表达时,所说的启动子可操作性地与该基因结合。所说的启动子在该基因的“上游”,也可以说该基因在启动子的“下游”。
本发明的DNA构建体或″表达盒″在5′-3′的转录方向上包含本发明的启动子、与所说的启动子可操作性地结合的的异源DNA片段、可有可无的转录和翻译的终止区(如终止信号和多腺苷酸化区)。所有这些调控区都应该能够在转化细胞中起作用。3′终止区可以来源于与转录起始区相同的或不同的基因。
植物可以分为不含有叶绿素的植物(如真菌)和含有叶绿素的植物(如绿藻、苔);进而可以分成含有叶绿素和维管组织的植物(如蕨类、裸子植物、松类、单子叶植物和双子叶植物)。后一种植物群包括那些可能具有根、茎和叶的植物。本文所用的术语“植物”包括上文所述的所有有机体。本文所用的术语“天然植物DNA”是指从没有经过基因工程改造的或非转化的植物(例如通过选育而产生的植物品种)中分离出的DNA。
本文所用的术语异源基因或者异源DNA片段是指用于经遗传工程技术转化细胞的基因或者DNA片段,并且这种基因不可能是细胞中天然存在的。结构基因是基因的一部分,它们包含编码蛋白质、多肽或其部分的DNA片段,可能包含核糖体结合位点和/或翻译起始密码子,但是缺乏启动子。此术语也可以指天然存在于细胞中的,但是经人工引入的结构基因的拷贝。结构基因可以编码通常在植物细胞中不存在的蛋白质,所说的细胞是此基因或者与此基因可操作性结合的启动子一起引入的细胞。与本发明用于在植物种类中表达的启动子可操作性结合的基因可以来源于染色体基因、cDNA、合成基因、或者它们的结合物。本文所用的术语异源DNA片段也包含编码非蛋白质产物的DNA片段(如核酶或反义RNAs)。反义RNAs是公知的(例如参见美国专利4,801,540(Calgene公司))。
本发明使用的植物中的重要基因包括那些影响多种表型和非表型特征的基因。在这些表型特征中是蛋白质(如酶)提供了对各种环境胁迫的抗性,所说的环境胁迫包括但不限于由脱水(原因是热、盐、干旱)、除草剂、有毒的金属、痕量元素、害虫和病原体引起的胁迫。抗性可以归因于靶位点的变化、宿主细胞中靶蛋白质量的增加、与保护宿主免受胁迫的产物生物合成途径有关的一种或多种酶量的增加等等。可以从原核生物或者真核生物、细菌、真菌(例如酵母、病毒、植物和哺乳动物)中获得结构基因,或者可以全部或部分合成结构基因。具体的基因包含具有glyphosphate抗性的3-enolpyruvylphosphoshikinate合成酶基因、腈水解酶、脯氨酸和谷氨酰胺生物合成途径的基因和金属硫蛋白。
与本发明的启动子可操作性地结合的结构基因可以是那些编码对昆虫有毒的蛋白质的基因,如对昆虫有毒的苏云金芽孢杆菌晶体蛋白。美国专利4,853,331公开了编码对鞘翅目(Coleoptera)有毒的苏云金芽孢杆菌毒素的DNA序列和这个序列的变体(其中保留了编码毒性的部分)(也参见美国专利4,918,006和4,910,136)(本文引用的所有美国专利文献的全部公开内容本文一并参考)。PCT申请WO 90/02804公开了苏云金芽孢杆菌的能够使植物对鳞翅目(Lepidoptera)有毒的基因序列。PCT申请WO 89/04868公开了用载体转化的转基因植物,所说的载体能促进苏云金芽孢杆菌晶体蛋白毒素的表达,并且此载体的序列可以与本发明结合使用。PCT申请WO90/06999公开了编码对鳞翅目有活性的苏云金芽孢杆菌晶体蛋白毒素的DNA。美国专利4,918,006公开了另一个编码杀虫晶体蛋白的基因序列。编码其它昆虫毒素的基因序列的例子是在美国专利4,940,840和PCT申请WO 90/07001中公开的编码壳多糖酶(例如EC-3.2.1.14)的基因序列。在美国专利申请08/007,998中公开了编码能诱导线虫孔蛋白(这种蛋白在产生抗根线虫的转基因植物方面是有用的)的基因。在美国专利4,948,734和5,093,120(Edwards等)中公开了能够产生有活性的抗线虫的多肽毒素的苏云金芽孢杆菌的菌株。
当所说的基因表达产物定位在非细胞质的细胞区室中时,可以构建这样的结构基因,即包含编码使产物转移到特定的位点(如细胞质膜)或分泌到细胞的胞质或外部环境中的特殊氨基酸序列的区域。文献中描述了各种分泌性前导序列、膜整合序列以及指导所说的肽表达产物进入特定位点的转移序列。例如参见,Cashmore等,生物技术(1985)3:803-808,Wickner和Lodish,科学(1985)230:400-407。
可以以DNA构建体提供所说的表达盒,这种DNA构建体也具有至少一个复制***。为了方便起见,通常含有在大肠杆菌中有功能的复制***,例如ColE1、pSC101、pACYC184等等。以这种方式,在每次操作后的各阶段都可以对所得的构建体进行克隆和测序,并检测操作的正确性。此外,可以使用广泛宿主范围的复制***,例如P-1不亲和性质粒的复制***(例如pRK290),或者以此代替大肠杆菌复制***。除了复制***外,可以至少含有一个标记,这种标记可以在一个或多个宿主中是有用的,或者对于不同的宿主使用不同的标记。也就是说,在原核宿主中选择时可以使用一个标记,而在真核宿主(特别是植物宿主)中选择时使用另一个标记。所说的标记可以提供保护以防止杀生物剂(例如抗菌素、毒素、重金属等等)的危害,所说的标记可以通过将原养型传递给营养缺陷的宿主而提供互补作用;或者所说的标记可以通过在植物中产生新的化合物而提供可见的表型。可以使用的基因例子包含新霉素磷酸转移酶(NPTII)、潮霉素磷酸转移酶(HPT)、氯霉素乙酰转移酶(CAT)、腈水解酶和庆大霉素抗性基因。为了进行植物宿主的选择,非限定的合适的标记的例子是β-葡糖苷酸酶(GUS)(产生靛蓝)、荧光素酶(产生可见光)、NPTII(产生卡那霉素抗性或G418抗性)、HPT(提供潮霉素抗性)和突变aroA基因(提供草甘膦抗性)。
通过合适的复制***的限制性酶切和特定的构建体或片段在可使用的位点的***将包含各种构建体、表达盒、标记等的各种片段连续地引入。在连接和克隆后,可以分离所说的DNA构建体以便用于进一步的操作。文献中通过举例详细地说明了这些技术。例如参见,Maniatis等,分子克隆:实验手册,冷泉港实验室,冷泉港,纽约,(1982)。
载体是可复制的DNA构建体。可以用于通过本发明的DNA构建体转化植物组织的载体包括土壤杆菌属载体和冲击载体,以及适合用于DNA介导的转化载体。含有本发明DNA构建体的根癌土壤杆菌细胞(其中所说的DNA构建体包含Ti质粒)在产生转化植物的各种方法中是有用的。用根癌土壤杆菌感染植物细胞以便产生转化的植物细胞,然后由转化的植物细胞再生植物。
用于本发明的大量的土壤杆菌属的载体***是已知的。例如,美国专利4,459,355公开了用含有Ti质粒的土壤杆菌菌株转化敏感植物(包括双子叶植物)的方法。美国专利4,795,855公开了使用土壤杆菌载体转化木本植物。此外,美国专利4,940,838(Schilperoort等)公开了在本发明中有用的二元土壤杆菌载体(即这样一种载体,在这种载体中土壤杆菌包含一种含有Ti质粒的vir区但没有T-DNA区的质粒,同时包含第二种含有T-DNA区但没有vir区的质粒)。
携带本发明DNA构建体的微颗粒(这种微颗粒适用于对植物细胞的冲击转化),也在产生本发明的转化植株上是有用的。将所说的微颗粒推进到植物细胞以便产生转化的植物细胞,并且从这些转化的植物细胞再生植株。任何合适的冲击细胞转化方法和仪器都可以用于实现本发明。在Sanford和Wolf的美国专利4,945,050和Agracetus欧洲专利申请出版物0 270 356(题目为“花粉介导的植物转化”)公开了仪器和方法的例子。当使用冲击转化方法时,可以将表达盒掺入到能够在将要转化的细胞中复制的质粒中。在这些***中适合使用的微颗粒的例子包括1-5μm金粉球。通过任何合适的技术(如通过沉淀)将所说的DNA构建体沉积在这种微颗粒上。
转化的宿主细胞是已经由包含本文公开的DNA序列的构建体(使用重组DNA技术产生的)转化或转染的细胞。可以使用本发明的DNA构建体通过DNA介导的植物细胞原生质体转化,接下来按照本领域公知的方法从所说的转化原生质体再生植株而完成对植物的转化。
本文公开的启动子序列可以用于在任何能够使用此启动子的植物种类(即该植物的RNA聚合酶能够与本文公开的启动子序列结合)中表达异源DNA序列。适于用本发明的DNA构建体转化的植物种类的例子包括单子叶植物和双子叶植物,包括但不限于烟草、大豆、土豆、棉花、甜菜、向日葵、胡萝卜、芹菜、亚麻、甘蓝和其它的十字花科植物、胡椒、番茄、柑桔、大豆、草莓、莴苣、玉米、苜蓿、燕麦、小麦、水稻、大麦、高粱和canola。这样,可以用本发明的DNA构建体转化的植物种类的例子是双子叶植物,可以用本发明的DNA构建体转化的更具体的植物种类是茄科的成员。
任何尔后能够进行无性繁殖(或者通过器官再生或者通过胚再生)的植物组织都可以用本发明的载体转化。本文所用的术语“器官再生”是指一种方法,通过这种方法能够由分生组织中心先后发育成枝条和根;本文所用的术语“胚再生”是一种方法,通过这种方法能够由体细胞或配子以协同的方式(而不是先后)发育出枝条和根。选择的特定组织将取决于能够使用的(并且最好是适合于)所转化的特定植物种类的无性繁殖***。组织靶的例子包含叶盘、花粉、胚、子叶、胚轴、大配子体、愈伤组织、现有的分生组织(例如顶端分生组织、腋芽和根分生组织)和诱导的分生组织(如子叶分生组织和胚轴分生组织)。
下面的实施例是为了具体说明本发明的各种特定的实施方案,而不是用来限制本发明的。
实施例1
分离基因组根皮层特异性RD2基因
在EMBL 3 SP6/T7λ载体(Clon Tech,Pale Alto,CA)中构建由烟草幼苗分离的DNA的烟草(Nicotania tabacun)基因组文库。使用TobRD2cDNA(Conkling等,植物生理93,1203(1990))作为探针,从初级文库中分离含有烟草RD2基因的基因组克隆。在K802细菌细胞上筛选了总数为1.2×107个重组噬菌体。将噬菌斑转移到尼龙膜(Magnagraph)上,并通过高压灭菌(10分钟,重力循环)固定所说的DNA。所有的杂交都在65℃下于含水溶液(5X SSC[750mM氯化钠,75mM柠檬酸钠],5XDenhardt[0.1%各种ficoll,BSA,聚乙烯吡咯烷酮],0.5%SDS,100 mg/ml变性的鲑精DNA)中进行16小时。将滤液在60℃下于0.2X SSC和0.1%SDS中洗涤。
通过筛选1.2X107个重组噬菌体鉴定了与TobRD2 cDNA杂交的13个基因组克隆。分离这些克隆并通过限制图谱进一步定性。通过Rachwitz等(基因,30:195(1984))的快速作图方法构建限制性图谱。测定一个与TobRD2 cDNA同源的克隆的全部序列,并确定其启动子。通过比较TobRD2 cDNA和基因组克隆的序列确定所说的转译区5′端的基因组克隆区。测定这一非翻译区的序列,并鉴定推定的启动子的TATAA框。在植物启动子中,TATAA框一般为从转录起始位点开始的第-35至-29核苷酸。使用引物延伸实验鉴定了转录的5′端。
图2给出了TohRD2 cDNA转录区上游的2010个碱基对区(SEQ IDNO:1)。此序列包含转录区推测的起点(在第2000核苷酸处)和所说的启动子的TATAA框(核苷酸1971-1975)。
实施例2
核酸测序
将分离的基因组克隆(实施例1)的限制片段亚克隆到bluescript(pBSKS II+或pBS KS II+;Stratagene,La Jolla,CA)载体上。通过核酸外切酶III和S1核酸酶消化获得用于每个克隆和两条DNA链的一系列单向缺失(Henikoff,基因28,351(1984))。通过双脱氧链终止法(Sanger等,美国科学院学报,74,5463(1977))使用测序酶(美国生物化学药品公司,Cleveland,OH)测定此DNA序列。在所有情况下,对两条DNA链进行了测序。
实施例3
原位杂交
为了确定TobRD2 mRNA转录物在根各种组织中的空间分布,在非转化植物中进行原位杂交。使用如Meyerowitz(植物分子生物学报道5,242(1987))和Smith等(植物分子生物学报道5,237(1987))所述的技术将TobRD2的反义链与根组织中的TobRD2 mRNA进行原位杂交。将7天大的烟草(Nicotania tabacum)幼苗根固定在磷酸缓冲的戊二醛中,包埋在Paraplast Plus(Monoject公司,St.Louis,MO)中,并将其切割成8毫米厚的段以便获得横切面和纵切面。将在35S-ATP存在下体外合成的反义TobRDa转录物用作探针。经碱处理使标记的RNA水解以便在使用前获得平均长度为100-200个碱基的片段。
42℃下在50%甲酰胺中杂交16小时,每毫升杂交溶液中含有大约5X106个/分钟(cpm)标记的RNA。曝光后,冲洗胶片并在明场和暗场显微镜下观察。
如图1A和1B所示,杂交信号定位在根皮层细胞中。比较同一切片在明场和暗场下的图象表明TobRD2转录物定位在根皮层的薄壁组织细胞中。在表皮或中柱中没有观察到杂交信号。
实施例4
嵌和基因的构建
通过聚合酶链反应(PCR)构建一系列启动子缺失。模板是经核酸外切酶III/S1核酸酶消化(实施例2)产生的TobRD2基因组克隆的5′侧翼区的各种缺失片段。
使用同一套寡核苷酸引物扩增所有的模板。一个引物是修饰的噬菌体M13的前引物(例如参见Sanger等,美国科学院学报74,5463(1977));此寡核苷酸5′端包含HindIII识别序列以及使得限制酶能够更有效切割的附加的5′序列。设计另一个引物,使之在5′末端具有BamHI位点(以及用于有效切割的附加核苷酸),并且此引物与TobRD2的16核苷酸序列同源,所说的TobRD2在ATG起始密码子的5′端有22个碱基(即此引物与SEQ IDNO:1的1973-1988碱基同源)。
所说的PCR扩增反应包含模板质粒DNA(5-10ng);反应缓冲液(50mMKCl;10mM Tris-HCl;pH 9.0[25℃];0.1%Triton X-100;1.5mMMgCl2);各种dATP、dGTP、dTTP、dCTP各为0.25mM;每种引物40ng;1.25单位的Taq DNA聚合酶(Promega,Madison,WS)。
94℃下持续1分钟的PCR循环使模板变性,46℃下持续1分钟将引物退火,在72℃持续5分钟使链延长。将此循环重复40次,最后的延长循环增加10分钟。在程控热循环仪(PCT-100,M.J.Research)中进行PCR扩增。
用Hind III和Bam HI消化扩增的产物,并将其克隆到土壤杆菌二元载体pBI 101.3(R.Jefferson等,EMBO J.6,3901-3907(1987))的Hind III和Bam HI位点上。此载体包含β-葡糖苷酸酶(GUS)报道基因和nptII选择标记(其侧面为T-DNA边缘序列)。
实施例5
植物转化方法
基本上如An等(Belvin和R.Schilperoot编辑,植物分子生物学手册Martinus Nijhoff,Dordrecht,荷兰,pp A3-1-19(1988))的描述将嵌合报道基因构建体引入到携带缓和Ti-质粒(LBA4404)的土壤杆菌宿主中,所说的质粒能够提供(in trans)vir功能(其对T-DNA转移和在植物基因组中的整合是必要的)。经三亲本配对或电感受态的土壤杆菌细胞的电穿孔(这是本领域公知的技术)将构建体引入到所说的宿主中。如An等:植物生理学81,301-305(1986)的描述将烟草(SR1)进行叶盘转化和植株再生。选择具有卡那霉素抗性的植株进行进一步分析。
实施例6
转基因植物中GUS测定方法
在转化植物的根、茎和叶的切面上进行组化染色。在底物进行短暂真空渗入后,37℃下将这些外植体组织在1mM5-溴-4-氯-3-吲哚基-B-D-葡萄糖醛酸化物(X-Gluc)、25mM磷酸钠缓冲液(pH 7.0)、0.5%DMSO中过夜温育。表达GUA活性的组织能切割这种底物,因此染成蓝色。
如Jefferson等(EMBO J.6,3901-3907(1987))的描述进行荧光GUS测定,以便定量GUS的表达水平。37℃下,在1mM 4-甲基散形基-B-D-葡萄糖醛酸化物(MUG)存在下温育根、叶和茎的细胞提取物。以0、5、10、15和20分钟的间隔取样。通过加入0.2M碳酸钠终止酶促反应。用10nM和100nM MUG校准荧光计。按照Bradford的方法(Anal.Biochem.72,248(1976))测定样品中的蛋白浓度。
实施例7
嵌合基因构建体能够指导组织特异性基因表达
为了确定TobRD2基因的2010碱基对序列(SEQ ID No:1)是否包括指导在根皮层的薄壁组织细胞中特异性表达的启动子元件,构建了嵌合基因。通过聚合酶链反应扩增1988碱基对区(SEQ ID No:2)并在GUS报道基因的5’端进行克隆(如上所述)。将嵌合基因引入烟草(如上所述),分析转基因植物表达GUS的能力(如上所述)。
表1的25-33行(转化体325II1-325IV5)显示了9个独立转化体(即每个转化体是独立转化过程的产物)的分析结果。发现Δ2.0启动子(SEQ ID NO:2)能够指导高水平的基因表达(比一般称作“强”启动子的CaMV35S启动子大约高4倍)(图4)。在叶片或茎中没有检测到以比对照高的水平表达的报道基因(参见显示出表1的平均活性的图4、5A和5B)。GUS活性基本上限于根,并且如图6所示尤其是限于根的皮层。使用驱动GUS的Δ2.0启动子(在pBI101.3中)转化图6所示的植物。
将多个独立的转化叶盘置于培养皿中。表1中转化体的命名表明启动子/编号的培养皿/独立的转化体的编号。这样,325II1指使用Δ2.0启动子、在培养皿II中来自叶盘1的转化体;而101.I1是指使用pBI101.3(无启动子的GUS作为对照)的转化和培养皿I中的编号为1的转化体。在表I中,字首121指使用pBI121(带有GUS的CaMV35S启动子);325是指具有GUS的Δ2.0启动子(SEQ ID NO:2);484是指具有GUS的Δ1.4启动子(SEQ ID NO:3);421是指具有GUS的Δ1.3启动子(SEQ ID NO:4);428是指具有GUS的Δ1.0启动子(SEQ ID NO:5);490是指具有GUS的Δ0.7启动子(SEQ ID NO:6);491是指具有GUS的Δ0.6启动子(SEQ ID NO:7);492是指具有GUS的Δ0.5启动子(SEQ ID NO:8);495是指具有GUS的Δ0.2启动子(SEQ ID NO:9)。″R-GUS″是指根组织中的GUS活性;″L-GUS″是指叶组织中的GUS活性;和″S-GUS″是指茎组织中的GUS活性。R/L是指根/叶的相对GUS活性;R/S是指根/茎的相对GUS活性。GUS活性以pmolMU/μg蛋白/分钟表示。
表1
TobRD2启动子分析
转化体 | R-GUS活性 | 平均值 | L-GUS活性 | 平均值 | S-GUS活性 | 平均值 | R/L | R/L平均值 | R/S | R/S平均值 |
101.I1 | 0.19 | 0.56 | 0.23 | 0.33 | 0.22 | 0.36 | 0.83 | 1.67 | 0.86 | 1.51 |
101.I2 | 0.12 | 0.14 | 0.15 | 0.86 | 0.80 | |||||
101.I3 | 0.13 | 0.35 | 0.32 | 0.37 | 0.41 | |||||
101.I4 | 0.73 | 0.46 | 0.24 | 1.59 | 3.04 | |||||
101.II1 | 0.44 | 0.31 | 1.42 | |||||||
101.II3 | 0.59 | 02.3 | 0.47 | 2.57 | 1.26 | |||||
101.II4 | 0.86 | 0.41 | 0.34 | 2.10 | 2.53 | |||||
101.II5 | 0.64 | 0.36 | 0.33 | 1.78 | 1.94 | |||||
101.III1 | 0.69 | 0.24 | 0.42 | 2.88 | 1.64 | |||||
101.III3 | 0.25 | 0.19 | 0.21 | 1.32 | 1.19 | |||||
101.III4 | 0.71 | 0.37 | 0.27 | 1.92 | 2.63 | |||||
101.III5 | 0.15 | 0.13 | 0.21 | 1.15 | 0.71 | |||||
101.IV1 | 0.21 | 0.10 | 0.13 | 2.10 | 1.62 | |||||
101.IV2 | 0.27 | 0.24 | 0.23 | 1.13 | 1.17 | |||||
101.IV3 | 0.88 | 0.42 | 0.57 | 2.10 | 1.54 | |||||
101.IV4 | 0.75 | 0.35 | 0.67 | 2.14 | 1.12 | |||||
101.IV5 | 1.88 | 0.98 | 1.02 | 1.92 | 1.84 | |||||
121.I5 | 3.00 | 10.50 | 3.65 | 14.36 | 2.25 | 5.81 | 0.82 | 0.71 | 1.33 | 1.69 |
121.IV1 | 24.67 | 30.79 | 11.96 | 0.80 | 2.06 | |||||
121.IV2 | 9.20 | 11.66 | 5.33 | 0.79 | 1.73 | |||||
121.IV4 | 12.13 | 15.61 | 7.42 | 0.78 | 1.63 | |||||
121.4 | 3.50 | 10.10 | 2.08 | 0.35 | 1.68 | |||||
表1
TobRD2启动子分析
325II1 | 35.30 | 32.15 | 0.54 | 0.46 | 0.61 | 0.78 | 65.37 | 67.19 | 57.87 | 50.17 |
325II2 | 24.94 | 0.24 | 0.35 | 103.92 | 71.26 | |||||
325II4 | 13.64 | 0.17 | 0.23 | 80.24 | 59.30 | |||||
325II5 | 38.09 | 0.64 | 59.52 | |||||||
325III1 | 45.31 | 0.38 | ||||||||
325III2 | 34.05 | 0.44 | ||||||||
325III5 | 55.81 | 0.76 | 0.77 | 73.43 | 72.48 | |||||
325IV1 | 16.51 | 0.68 | 0.94 | 24.28 | 17.56 | |||||
325IV5 | 25.71 | 0.46 | 1.95 | 55.89 | 13.18 | |||||
484I1 | 61.75 | 36.68 | 0.46 | 0.67 | 74.41 | 53.68 | ||||
484I3 | 59.72 | |||||||||
484I4 | 72.35 | |||||||||
484I5 | 56.58 | |||||||||
484V2 | 38.32 | 0.78 | 0.86 | 49.13 | 44.56 | |||||
484V3 | 23.66 | 0.31 | 2.29 | 76.32 | 10.33 | |||||
484III3 | 63.28 | |||||||||
484III4 | 42.91 | 0.87 | 0.98 | 49.32 | 43.79 | |||||
484II4 | 15.80 | 0.43 | 0.27 | 36.74 | 58.52 | |||||
484V4 | 58.25 | 0.46 | 0.48 | 126.63 | 121.35 | |||||
484V1 | 26.86 | 0.81 | 1.27 | 33.16 | 21.15 | |||||
484V5 | 8.53 | 0.42 | 0.34 | 20.31 | 25.09 | |||||
484IV5 | 17.83 | 0.51 | 0.29 | 34.96 | 61.48 | |||||
484IV3 | 14.05 | 0.35 | 0.34 | 40.14 | 41.32 | |||||
484IV2 | 32.33 | 0.32 | 0.51 | 101.03 | 63.39 |
表1
TobRD2启动子分析
484II3 | 10.18 | 0.13 | 0.16 | 78.31 | 63.63 | |||||
484II5 | 33.51 | 0.55 | 0.63 | 60.93 | 53.19 | |||||
484II2 | 52.54 | 0.43 | 0.79 | 122.19 | 66.51 | |||||
484II1 | 8.50 | 0.04 | 0.11 | 212.50 | 77.27 | |||||
421IV4 | 25.04 | 31.87 | 0.82 | 0.81 | 2.27 | 1.01 | 30.54 | 40.54 | 11.03 | 36.78 |
421V4 | 46.31 | 0.82 | 56.48 | |||||||
421II4 | 79.23 | 0.96 | 1.89 | 82.53 | 41.92 | |||||
421III3 | 17.00 | 0.45 | 1.09 | 37.78 | 15.60 | |||||
421II3 | 19.07 | 0.42 | 0.37 | 45.40 | 51.54 | |||||
421I1 | 27.67 | 0.72 | 0.64 | 38.43 | 43.23 | |||||
421I3 | 74.45 | 2.27 | 1.44 | 32.80 | 51.70 | |||||
421II2 | 43.36 | 0.88 | 0.56 | 49.27 | 77.43 | |||||
421I4 | 8.41 | |||||||||
421V1 | 32.32 | 0.94 | 1.34 | 34.38 | 24.12 | |||||
421V2 | 5.07 | 0.43 | 0.13 | 11.79 | 39.00 | |||||
421IV3 | 4.52 | 0.17 | 0.37 | 26.59 | 12.22 | |||||
428I5 | 20.62 | 38.64 | 0.98 | 0.66 | 0.83 | 0.65 | 21.04 | 72.65 | 24.84 | 47.43 |
428I2 | 15.05 | 0.97 | 0.25 | 15.52 | 60.20 | |||||
428III3 | 69.87 | 1.10 | 63.52 | |||||||
428III1 | 30.97 | 0.52 | 0.36 | 59.56 | 86.03 | |||||
428V2 | 54.66 | 0.24 | 227.75 | |||||||
428V1 | 85.71 | 0.98 | 1.25 | 87.46 | 68.57 | |||||
428IV4 | 4.15 | 0.29 | 14.31 |
表1
TobRD2启动子分析
428IV5 | 26.42 | 0.43 | 1.10 | 61.44 | 24.02 | |||||
428V3 | 1.58 | 0.16 | 0.17 | 9.88 | 9.29 | |||||
428V2 | 25.60 | 0.34 | 75.29 | |||||||
428III5 | 90.36 | 0.86 | 0.98 | 105.07 | 92.20 | |||||
490II4 | 9.38 | 22.77 | 0.54 | 0.75 | 41.65 | 36.11 | ||||
490II5 | 9.67 | 0.35 | 0.65 | 27.63 | 14.88 | |||||
490I1 | 33.62 | 0.93 | 2.02 | 36.15 | 16.64 | |||||
490I2 | 34.66 | 0.98 | 1.13 | 35.37 | 30.67 | |||||
490I3 | 4.58 | |||||||||
490III2 | 76.74 | |||||||||
490III4 | 58.75 | 1.07 | 12.1 | 54.91 | 48.55 | |||||
490III5 | 6.65 | 0.21 | 0.09 | 31.67 | 73.89 | |||||
490IV2 | 12.24 | |||||||||
490II1 | 8.09 | 0.22 | 0.21 | 36.77 | 38.52 | |||||
490IV4 | 20.19 | 0.35 | 0.52 | 57.69 | 38.83 | |||||
490IV5 | 17.57 | 0.34 | 0.57 | 51.68 | 30.82 | |||||
490IV3 | 18.11 | |||||||||
490I5 | 23.03 | 0.78 | 0.93 | 29.53 | 24.78 | |||||
490V5 | 8.27 | 0.15 | 0.19 | 55.13 | 43.53 | |||||
491I2 | 8.31 | 39.76 | 0.50 | 0.63 | 53.70 | 45.85 | ||||
491II3 | 6.73 | |||||||||
491II4 | 13.01 | 0.23 | 0.19 | 56.57 | 68.47 | |||||
491V5 | 87.40 |
表1
TobRD2启动子分析
491IV1 | 77.12 | 1.02 | 1.34 | 75.61 | 57.55 | |||||
491IV3 | 49.20 | 0.98 | 1.23 | 50.20 | 40.00 | |||||
491III1 | 18.84 | 0.32 | 0.34 | 58.88 | 55.41 | |||||
491III2 | 30.82 | 0.47 | 0.58 | 65.57 | 53.14 | |||||
491III5 | 8.46 | 0.28 | 0.45 | 30.21 | 18.80 | |||||
491IV5 | 2.88 | |||||||||
491II5 | 8.55 | 0.22 | 0.31 | 28.86 | 27.58 | |||||
491IV4 | 165.77 | |||||||||
492V2 | 2.40 | 9.89 | 0.21 | 0.57 | 0.24 | 0.54 | 11.43 | 15.59 | 10.00 | 16.72 |
492V4 | 3.17 | 0.27 | 0.48 | 11.74 | 6.60 | |||||
492I3 | 4.40 | 0.87 | 0.35 | 5.06 | 12.57 | |||||
492I4 | 6.58 | 0.50 | 0.37 | 13.16 | 17.78 | |||||
492I5 | 10.26 | |||||||||
492III2 | 11.87 | 0.78 | 1.06 | 15.22 | 11.20 | |||||
492IV4 | 7.38 | |||||||||
492IV5 | 21.63 | |||||||||
492III5 | 11.39 | 0.61 | 0.32 | 18.67 | 35.59 | |||||
492IV1 | 20.38 | 0.81 | 0.94 | 25.16 | 21.68 | |||||
492II3 | 12.15 | 0.42 | 0.53 | 28.93 | 22.92 | |||||
492III1 | 7.03 | 0.64 | 0.58 | 10.98 | 12.12 | |||||
495I1 | 3.58 | 5.83 | 0.37 | 0.41 | 0.43 | 0.54 | 9.68 | 17.98 | 8.33 | 13.35 |
495I3 | 16.41 | 0.59 | 0.74 | 27.81 | 22.18 | |||||
495I4 | 3.20 | 0.17 | 0.17 | 18.82 | 18.82 |
表1
TobRD2启动子分析
495I5 | 5.96 | 0.32 | 0.34 | 18.63 | 17.53 | |||||
495II2 | 8.49 | 0.54 | 0.52 | 15.72 | 16.33 | |||||
495III2 | 5.12 | 0.40 | 0.77 | 12.80 | 6.65 | |||||
495IV1 | 5.57 | 0.21 | 0.45 | 26.52 | 12.38 | |||||
495IV2 | 9.74 | 0.75 | 1.03 | 12.99 | 9.46 | |||||
495IV3 | 2.64 | 0.14 | 0.31 | 18.66 | 8.52 | |||||
495IV4 | 1.20 | |||||||||
495V1 | 3.67 | |||||||||
495V2 | 2.38 | |||||||||
495V3 | 7.60 | |||||||||
495V4 | 6.10 | 0.56 | 0.62 | 10.89 | 9.84 |
实施例8
5′启动子-缺失对报道基因活性表达的影响
以基本上与上文实施例7描述的相同的方式进行下列实验,只是用作启动子的TobRD2侧翼区的长度发生变化以便研究侧翼区的各部分如何影响GUS的表达。
在2010碱基对TobRD2序列(SEQ ID NO:1)上游区产生用作启动子序列的一系列7嵌套5′-缺失突变。图3以图解的方式显示了这些缺失突变体,分别称为Δ2.0(SEQ ID NO:2);Δ1.4(SEQ ID NO:3);Δ1.3(SEQ ID NO:4);Δ1.0(SEQ ID NO:5);Δ0.7(SEQ ID NO:6);Δ0.6(SEQ ID NO:7);Δ0.5(SEQ ID NO:8)和Δ0.2(SEQ ID NO:9)。
通过土壤杆菌介导的叶盘转化方法(如实施例4的描述)将实施例3描述的并且含有Δ2.00启动子(SEQ ID NO:2)或截短的启动子(SEQ ID NOs:3-9)的嵌合基因构建体引入到烟草中。单独使用土壤杆菌载体pBI101.3作为对照,CaMV35S启动子用来提供参照标准物。分析再生植株根、叶和茎的GUS活性(表1:图4)。
图4给出了根、叶和茎中GUS活性的图解说明,其中使用了全长TobRD2启动子、启动子缺失系列、花椰菜花叶病毒35S[CaMV35S)启动子、和作为对照的载体pBI101.3。如图4所示,发现试验的6个启动子能赋予高水平的根皮层特异性表达:Δ2.00(SEQ ID NO:2);Δ1.4(SEQ ID NO:3);Δ1.3(SEQ ID NO:4);Δ1.0(SEQ ID NO:5);Δ0.7(SEQ ID NO:6);Δ0.6(SEQ ID NO:7)。图4显示出表1的平均值。
图4还显示出一个长度大约50个碱基对区的缺失(比较Δ0.6(SEQ IDNO:7)和Δ0.5(SEQ ID NO:8))大大降低了GUS表达水平。然而,此结果表明由Δ0.5启动子(SEQ ID NO:8)提供的GUS在根组织中的表达水平与由CaMV35S启动子引起的GUS表达水平相同。由Δ0.2启动子(SEQ ID NO:9)提供的根皮层中的GUS表达大约为由CaMV35S启动子提供的GUS表达的一半。
图5A和5B还说明了报道基因使用TobRD2启动子的表达的器官特异性特征。在所有试验的实例中GUS活性在根中严格地表达,而在同一转化烟草植株的茎或者叶中只能检测到微乎其微的活性(如果有的话)。当在用Δ0.60和Δ0.30启动子转化的根中检测的GUS活性水平等于或少于由CaMV35S启动子提供的GUS水平时(图4),图5A和5B说明了由Δ0.60和Δ0.30启动子指导的表达是根特异性的,而在茎和叶中的表达微乎其微,这一点与由CaMV35S启动子指导的表达不同。
前面的实施例是用来具体说明本发明,而不是用来限制本发明的。下面的权利要求限定本发明,其中包括这些权利要求的等同物。
序列表(1)一般信息:
(i)申请人:Conkling.Mark A.
Mendu,Nandini
Song,Wen
(ii)发明题目:根皮层特异性基因启动子
(iii)序列数:9
(iv)通讯地址:
(A)地址:Kenneth D.Sibley;Bell.Seltzer,Park和Gibson
(B)街道:Post Office Drawer 34009
(C)城市:Charlotte
(D)州:北卡罗来纳
(E)国家:美国
(F)邮编代码:28234
(v)计算机可读形式:
(A)介质类型:软盘
(B)计算机:IBM PC兼容机
(C)操作***:PC-DOS/MS-DOS
(D)软件:PatentIn Release #1.0.Version #1.30
(vi)当前申请数据:
(A)申请号:
(B)申请日期:
(C)分类:
(viii)律师/代理人信息:
(A)姓名:Sibley,Kenneth D.
(B)登记号:31,665
(C)证书号:5051-294
(ix)电讯信息:
(A)电话:919-420-2200
(B)传真:919-881-3175
(2)SEQ ID NO:1信息:
(i)序列特征:
(A)长度:2010个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:DNA(基因组)
(xi)序列描述:SEQ ID NO:1CTCGAGGATC TAAATTGTGA GTTCAATCTC TTCCCTATTG GATTGATTAT CCTTTCTTTT 60CTTCCAATTT GTGTTTCTTT TTGCCTAATT TATTGTGTTA TCCCCTTTAT CCTATTTTGT 120TTCTTTACTT ATTTATTTGC TTCTATGTCT TTGTACAAAG ATTTAAACTC TATGGCACAT 180ATTTTAAAGT TGTTAGAAAA TAAATTCTTT CAAGATTGAT GAAAGAACTT TTTAATTGTA 240GATATTTCGT AGATTTTATT CTCTTACTAC CAATATAACG CTTGAATTGA CGAAAATTTG 300TGTCCAAATA TCTAGCAAAA AGGTATCCAA TGAAAATATA TCATATGTGA TCTTCAAATC 360TTGTGTCTTA TGCAAGATTG ATACTTTGTT CAATGGAAGA GATTGTGTGC ATATTTTTAA 420AATTTTTATT AGTAATAAAG ATTCTATATA GCTGTTATAG AGGGATAATT TTACAAAGAA 480CACTATAAAT ATGATTGTTG TTGTTAGGGT GTCAATGGTT CGGTTCGACT GGTTATTTTA 540TAAAATTTGT ACCATACCAT TTTTTTCGAT ATTCTATTTT GTATAACCAA AATTAGACTT 600TTCGAAATCG TCCCAATCAT GTCGGTTTCA CTTCGGTATC GGTACCGTTC GGTTAATTTT 660CATTTTTTTT TAAATGTCAT TAAAATTCAC TAGTAAAAAT AGAATGCAAT AACATACGTT 720CTTTTATAGG ACTTAGCAAA AGCTCTCTAG ACATTTTTAC TGTTTAAAGG ATAATGAATT 780AAAAAACATG AAAGATGGCT AGAGTATAGA TACACAACTA TTCGACAGCA ACGTAAAAGA 840AACCAAGTAA AAGCAAAGAA AATATAAATC ACACGAGTGG AAAGATATTA ACCAAGTTGG 900GATTCAAGAA TAAAGTCTAT ATTAAATATT CAAAAAGATA AATTTAAATA ATATGAAAGG 960AAACATATTC AATACATTGT AGTTTGCTAC TCATAATCGC TAGAATACTT TGTGCCTTGC 1020TAATAAAGAT ACTTGAAATA GCTTAGTTTA AATATAAATA GCATAATAGA TTTTAGGAAT 1080TAGTATTTTG AGTTTAATTA CTTATTGACT TGTAACAGTT TTTATAATTC CAAGGCCCAT 1140GAAAAATTTA ATGCTTTATT AGTTTTAAAC TTACTATATA AATTTTTCAT ATGTAAAATT 1200TAATCGGTAT AGTTCGATAT TTTTTCAATT TATTTTTATA AAATAAAAAA CTTACCCTAA 1260TTATCGGTAC AGTTATAGAT TTATATAAAA ATCTACGGTT CTTCAGAAGA AACCTAAAAA 1320TCGGTTCGGT GCGGACGGTT CGATCGGTTT AGTCGATTTT CAAATATTCA TTGACACTCC 1380TAGTTGTTGT TATAGGTAAA AAGCAGTTAC AGAGAGGTAA AATATAACTT AAAAAATCAG 1440TTCTAAGGAA AAATTGACTT TTATAGTAAA TGACTGTTAT ATAAGGATGT TGTTACAGAG 1500AGGTATGAGT GTAGTTGGTA AATTATGTTC TTGACGGTGT ATGTCACATA TTATTTATTA 1560AAACTAGAAA AAACAGCGTC AAAACTAGCA AAAATCCAAC GGACAAAAAA ATCGGCTGAA 1620TTTGATTTGG TTCCAACATT TAAAAAAGTT TCAGTGAGAA AGAATCGGTG ACTGTTGATG 1680ATATAAACAA AGGGCACATT GGTCAATAAC CATAAAAAAT TATATGACAG CTACAGTTGG 1740TAGCATGTGC TCAGCTATTG AACAAATCTA AAGAAGGTAC ATCTGTAACC GGAACACCAC 1800TTAAATGACT AAATTACCCT CATCAGAAAG CAGATGGAGT GCTACAAATA ACACACTATT 1860CAACAACCAT AAATAAAACG TGTTCAGCTA CTAAAACAAA TATAAATAAA TCTATGTTTG 1920TAAGCACTCC AGCCATGTTA ATGGAGTGCT ATTGCCTGTT AACTCTCACT TATAAAATAG 1980TAGTAGAAAA AATATGAACC AAAACACAAC 2010(2)SEQ ID NO:2信息:
(i)序列特征:
(A)长度:1988个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:DNA(基因组)
(xi)序列描述:SEQ ID NO:2CTCGAGGATC TAAATTGTGA GTTCAATCTC TTCCCTATTG GATTGATTAT CCTTTCTTTT 60CTTCCAATTT GTGTTTCTTT TTGCCTAATT TATTGTGTTA TCCCCTTTAT CCTATTTTGT 120TTCTTTACTT ATTTATTTGC TTCTATGTCT TTGTACAAAG ATTTAAACTC TATGGCACAT 180ATTTTAAAGT TGTTAGAAAA TAAATTCTTT CAAGATTGAT GAAAGAACTT TTTAATTGTA 240GATATTTCGT AGATTTTATT CTCTTACTAC CAATATAACG CTTGAATTGA CGAAAATTTG 300TGTCCAAATA TCTAGCAAAA AGGTATCCAA TGAAAATATA TCATATGTGA TCTTCAAATC 360TTGTGTCTTA TGCAAGATTG ATACTTTGTT CAATGGAAGA GATTGTGTGC ATATTTTTAA 420AATTTTTATT AGTAATAAAG ATTCTATATA GCTGTTATAG AGGGATAATT TTACAAAGAA 480CACTATAAAT ATGATTGTTG TTGTTAGGGT GTCAATGGTT CGGTTCGACT GGTTATTTTA 540TAAAATTTGT ACCATACCAT TTTTTTCGAT ATTCTATTTT GTATAACCAA AATTAGACTT 600TTCGAAATCG TCCCAATCAT GTCGGTTTCA CTTCGGTATC GGTACCGTTC GGTTAATTTT 660CATTTTTTTT TAAATGTCAT TAAAATTCAC TAGTAAAAAT AGAATGCAAT AACATACGTT 720CTTTTATAGG ACTTAGCAAA AGCTCTCTAG ACATTTTTAC TGTTTAAAGG ATAATGAATT 780AAAAAACATG AAAGATGGCT AGAGTATAGA TACACAACTA TTCGACAGCA ACGTAAAAGA 840AACCAAGTAA AAGCAAAGAA AATATAAATC ACACGAGTGG AAAGATATTA ACCAAGTTGG 900GATTCAAGAA TAAAGTCTAT ATTAAATATT CAAAAAGATA AATTTAAATA ATATGAAAGG 960AAACATATTC AATACATTGT AGTTTGCTAC TCATAATCGC TAGAATACTT TGTGCCTTGC 1020TAATAAAGAT ACTTGAAATA GCTTAGTTTA AATATAAATA GCATAATAGA TTTTAGGAAT 1080TAGTATTTTG AGTTTAATTA CTTATTGACT TGTAACAGTT TTTATAATTC CAAGGCCCAT 1140GAAAAATTTA ATGCTTTATT AGTTTTAAAC TTACTATATA AATTTTTCAT ATGTAAAATT 1200TAATCGGTAT AGTTCGATAT TTTTTCAATT TATTTTTATA AAATAAAAAA CTTACCCTAA 1260TTATCGGTAC AGTTATAGAT TTATATAAAA ATCTACGGTT CTTCAGAAGA AACCTAAAAA 1320TCGGTTCGGT GCGGACGGTT CGATCGGTTT AGTCGATTTT CAAATATTCA TTGACACTCC 1380TAGTTGTTGT TATAGGTAAA AAGCAGTTAC AGAGAGGTAA AATATAACTT AAAAAATCAG 1440TTCTAAGGAA AAATTGACTT TTATAGTAAA TGACTGTTAT ATAAGGATGT TGTTACAGAG 1500AGGTATGAGT GTAGTTGGTA AATTATGTTC TTGACGGTGT ATGTCACATA TTATTTATTA 1560AAACTAGAAA AAACAGCGTC AAAACTAGCA AAAATCCAAC GGACAAAAAA ATCGGCTGAA 1620TTTGATTTGG TTCCAACATT TAAAAAAGTT TCAGTGAGAA AGAATCGGTG ACTGTTGATG 1680ATATAAACAA AGGGCACATT GGTCAATAAC CATAAAAAAT TATATGACAG CTACAGTTGG 1740TAGCATGTGC TCAGCTATTG AACAAATCTA AAGAAGGTAC ATCTGTAACC GGAACACCAC 1800TTAAATGACT AAATTACCCT CATCAGAAAG CAGATGGAGT GCTACAAATA ACACACTATT 1860CAACAACCAT AAATAAAACG TGTTCAGCTA CTAAAACAAA TATAAATAAA TCTATGTTTG 1920TAAGCACTCC AGCCATGTTA ATGGAGTGCT ATTGCCTGTT AACTCTCACT TATAAAATAG 1980TAGTAGAA 1988(2)SEQ ID NO:3信息:
(i)序列特征:
(A)长度:1372个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:DNA(基因组)
(xi)序列描述:SEQ ID NO:3TCATGTCGGT TTCACTTCGG TATCGGTACC GTTCGGTTAA TTTTCATTTT TTTTTAAATG 60TCATTAAAAT TCACTAGTAA AAATAGAATG CAATAACATA CGTTCTTTTA TAGGACTTAG 120CAAAAGCTCT CTAGACATTT TTACTGTTTA AAGGATAATG AATTAAAAAA CATGAAAGAT 180GGCTAGAGTA TAGATACACA ACTATTCGAC AGCAACGTAA AAGAAACCAA GTAAAAGCAA 240AGAAAATATA AATCACACGA GTGGAAAGAT ATTAACCAAG TTGGGATTCA AGAATAAAGT 300CTATATTAAA TATTCAAAAA GATAAATTTA AATAATATGA AAGGAAACAT ATTCAATACA 360TTGTAGTTTG CTACTCATAA TCGCTAGAAT ACTTTGTGCC TTGCTAATAA AGATACTTGA 420AATAGCTTAG TTTAAATATA AATAGCATAA TAGATTTTAG GAATTAGTAT TTTGAGTTTA 480ATTACTTATT GACTTGTAAC AGTTTTTATA ATTCCAAGGC CCATGAAAAA TTTAATGCTT 540TATTAGTTTT AAACTTACTA TATAAATTTT TCATATGTAA AATTTAATCG GTATAGTTCG 600ATATTTTTTC AATTTATTTT TATAAAATAA AAAACTTACC CTAATTATCG GTACAGTTAT 660AGATTTATAT AAAAATCTAC GGTTCTTCAG AAGAAACCTA AAAATCGGTT CGGTGCGGAC 720GGTTCGATCG GTTTAGTCGA TTTTCAAATA TTCATTGACA CTCCTAGTTG TTGTTATAGG 780TAAAAAGCAG TTACAGAGAG GTAAAATATA ACTTAAAAAA TCAGTTCTAA GGAAAAATTG 840ACTTTTATAG TAAATGACTG TTATATAAGG ATGTTGTTAC AGAGAGGTAT GAGTGTAGTT 900GGTAAATTAT GTTCTTGACG GTGTATGTCA CATATTATTT ATTAAAACTA GAAAAAACAG 960CGTCAAAACT AGCAAAAATC CAACGGACAA AAAAATCGGC TGAATTTGAT TTGGTTCCAA 1020CATTTAAAAA AGTTTCAGTG AGAAAGAATC GGTGACTGTT GATGATATAA ACAAAGGGCA 1080CATTGGTCAA TAACCATAAA AAATTATATG ACAGCTACAG TTGGTAGCAT GTGCTCAGCT 1140ATTGAACAAA TCTAAAGAAG GTACATCTGT AACCGGAACA CCACTTAAAT GACTAAATTA 1200CCCTCATCAG AAAGCAGATG GAGTGCTACA AATAACACAC TATTCAACAA CCATAAATAA 1260AACGTGTTCA GCTACTAAAA CAAATATAAA TAAATCTATG TTTGTAAGCA CTCCAGCCAT 1320GTTAATGGAG TGCTATTGCC TGTTAACTCT CACTTATAAA ATAGTAGTAG AA 1372(2)SEQ ID NO:4信息:
(i)序列特征:
(A)长度:1294个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:DNA(基因组)
(xi)序列描述:SEQ ID NO:4AAAAATAGAA TGCAATAACA TACGTTCTTT TATAGGACTT AGCAAAAGCT CTCTAGACAT 60TTTTACTGTT TAAAGGATAA TGAATTAAAA AACATGAAAG ATGGCTAGAG TATAGATACA 120CAACTATTCG ACAGCAACGT AAAAGAAACC AAGTAAAAGC AAAGAAAATA TAAATCACAC 180GAGTGGAAAG ATATTAACCA AGTTGGGATT CAAGAATAAA GTCTATATTA AATATTCAAA 240AAGATAAATT TAAATAATAT GAAAGGAAAC ATATTCAATA CATTGTAGTT TGCTACTCAT 300AATCGCTAGA ATACTTTGTG CCTTGCTAAT AAAGATACTT GAAATAGCTT AGTTTAAATA 360TAAATAGCAT AATAGATTTT AGGAATTAGT ATTTTGAGTT TAATTACTTA TTGACTTGTA 420ACAGTTTTTA TAATTCCAAG GCCCATGAAA AATTTAATGC TTTATTAGTT TTAAACTTAC 480TATATAAATT TTTCATATGT AAAATTTAAT CGGTATAGTT CGATATTTTT TCAATTTATT 540TTTATAAAAT AAAAAACTTA CCCTAATTAT CGGTACAGTT ATAGATTTAT ATAAAAATCT 600ACGGTTCTTC AGAAGAAACC TAAAAATCGG TTCGGTGCGG ACGGTTCGAT CGGTTTAGTC 660GATTTTCAAA TATTCATTGA CACTCCTAGT TGTTGTTATA GGTAAAAAGC AGTTACAGAG 720AGGTAAAATA TAACTTAAAA AATCAGTTCT AAGGAAAAAT TGACTTTTAT AGTAAATGAC 780TGTTATATAA GGATGTTGTT ACAGAGAGGT ATGAGTGTAG TTGGTAAATT ATGTTCTTGA 840CGGTGTATGT CACATATTAT TTATTAAAAC TAGAAAAAAC AGCGTCAAAA CTAGCAAAAA 900TCCAACGGAC AAAAAAATCG GCTGAATTTG ATTTGGTTCC AACATTTAAA AAAGTTTCAG 960TGAGAAAGAA TCGGTGACTG TTGATGATAT AAACAAAGGG CACATTGGTC AATAACCATA 1020AAAAATTATA TGACAGCTAC AGTTGGTAGC ATGTGCTCAG CTATTGAACA AATCTAAAGA 1080AGGTACATCT GTAACCGGAA CACCACTTAA ATGACTAAAT TACCCTCATC AGAAAGCAGA 1140TGGAGTGCTA CAAATAACAC ACTATTCAAC AACCATAAAT AAAACGTGTT CAGCTACTAA 1200AACAAATATA AATAAATCTA TGTTTGTAAG CACTCCAGCC ATGTTAATGG AGTGCTATTG 1260CCTGTTAACT CTCACTTATA AAATAGTAGT AGAA 1294(2)SEQ ID NO:5信息:
(i)序列特征:
(A)长度:1030个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:DNA(基因组)
(xi)序列描述:SEQ ID NO:5GGAAACATAT TCAATACATT GTAGTTTGCT ACTCATAATC GCTAGAATAC TTTGTGCCTT 60GCTAATAAAG ATACTTGAAA TAGCTTAGTT TAAATATAAA TAGCATAATA GATTTTAGGA 120ATTAGTATTT TGAGTTTAAT TACTTATTGA CTTGTAACAG TTTTTATAAT TCCAAGGCCC 180ATGAAAAATT TAATGCTTTA TTAGTTTTAA ACTTACTATA TAAATTTTTC ATATGTAAAA 240TTTAATCGGT ATAGTTCGAT ATTTTTTCAA TTTATTTTTA TAAAATAAAA AACTTACCCT 300AATTATCGGT ACAGTTATAG ATTTATATAA AAATCTACGG TTCTTCAGAA GAAACCTAAA 360AATCGGTTCG GTGCGGACGG TTCGATCGGT TTAGTCGATT TTCAAATATT CATTGACACT 420CCTAGTTGTT GTTATAGGTA AAAAGCAGTT ACAGAGAGGT AAAATATAAC TTAAAAAATC 480AGTTCTAAGG AAAAATTGAC TTTTATAGTA AATGACTGTT ATATAAGGAT GTTGTTACAG 540AGAGGTATGA GTGTAGTTGG TAAATTATGT TCTTGACGGT GTATGTCACA TATTATTTAT 600TAAAACTAGA AAAAACAGCG TCAAAACTAG CAAAAATCCA ACGGACAAAA AAATCGGCTG 660AATTTGATTT GGTTCCAACA TTTAAAAAAG TTTCAGTGAG AAAGAATCGG TGACTGTTGA 720TGATATAAAC AAAGGGCACA TTGGTCAATA ACCATAAAAA ATTATATGAC AGCTACAGTT 780GGTAGCATGT GCTCAGCTAT TGAACAAATC TAAAGAAGGT ACATCTGTAA CCGGAACACC 840ACTTAAATGA CTAAATTACC CTCATCAGAA AGCAGATGGA GTGCTACAAA TAACACACTA 900TTCAACAACC ATAAATAAAA CGTGTTCAGC TACTAAAACA AATATAAATA AATCTATGTT 960TGTAAGCACT CCAGCCATGT TAATGGAGTG CTATTGCCTG TTAACTCTCA CTTATAAAAT 1020AGTAGTAGAA 1030(2)SEQ ID NO:6信息:
(i)序列特征:
(A)长度:722个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:DNA(基因组)
(xi)序列描述:SEO ID NO:6GTACAGTTAT AGATTTATAT AAAAATCTAC GGTTCTTCAG AAGAAACCTA AAAATCGGTT 60CGGTGCGGAC GGTTCGATCG GTTTAGTCGA TTTTCAAATA TTCATTGACA CTCCTAGTTG 120TTGTTATAGG TAAAAAGCAG TTACAGAGAG GTAAAATATA ACTTAAAAAA TCAGTTCTAA 180GGAAAAATTG ACTTTTATAG TAAATGACTG TTATATAAGG ATGTTGTTAC AGAGAGGTAT 240GAGTGTAGTT GGTAAATTAT GTTCTTGACG GTGTATGTCA CATATTATTT ATTTAAAACTA 300GAAAAAACAG CGTCAAAACT AGCAAAAATC CAACGGACAA AAAAATCGGC TGAATTTGAT 360TTGGTTCCAA CATTTAAAAA AGTTTCAGTG AGAAAGAATC GGTGACTGTT GATGATATAA 420ACAAAGGGCA CATTGGTCAA TAACCATAAA AAATTATATG ACAGCTACAG TTGGTAGCAT 480GTGCTCAGCT ATTGAACAAA TCTAAAGAAG GTACATCTGT AACCGGAACA CCACTTAAAT 540GACTAAATTA CCCTCATCAG AAAGCAGATG GAGTGCTACA AATAACACAC TATTCAACAA 600CCATAAATAA AACGTGTTCA GCTACTAAAA CAAATATAAA TAAATCTATG TTTGTAAGCA 660CTCCAGCCAT GTTAATGGAG TGCTATTGCC TGTTAACTCT CACTTATAAA ATAGTAGTAG 720AA 722(2)SEQ ID NO:7信息:
(i)序列特征:
(A)长度:574个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:DNA(基因组)
(xi)序列描述:SEQ ID NO:7AGGTAAAATA TAACTTAAAA AATCAGTTCT AAGGAAAAAT TGACTTTTAT AGTAAATGAC 60TGTTATATAA GGATGTTGTT ACAGAGAGGT ATGAGTGTAG TTGGTAAATT ATGTTCTTGA 120CGGTGTATGT CACATATTAT TTATTAAAAC TAGAAAAAAC AGCGTCAAAA CTAGCAAAAA 180TCCAACGGAC AAAAAAATCG GCTGAATTTG ATTTGGTTCC AACATTTAAA AAAGTTTCAG 240TGAGAAAGAA TCGGTGACTG TTGATGATAT AAACAAAGGG CACATTGGTC AATAACCATA 300AAAAATTATA TGACAGCTAC AGTTGGTAGC ATGTGCTCAG CTATTGAACA AATCTAAAGA 360AGGTACATCT GTAACCGGAA CACCACTTAA ATGACTAAAT TACCCTCATC AGAAAGCAGA 420TGGAGTGCTA CAAATAACAC ACTATTCAAC AACCATAAAT AAAACGTGTT CAGCTACTAA 480AACAAATATA AATAAATCTA TGTTTGTAAG CACTCCAGCC ATGTTAATGG AGTGCTATTG 540CCTGTTAACT CTCACTTATA AAATAGTAGT AGAA 574(2)SEQ ID NO:8信息:
(i)序列特征:
(A)长度:523个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:DNA(基因组)
(xi)序列描述:SEQ ID NO:8GTAAATGACT GTTATATAAG GATGTTGTTA CAGAGAGGTA TGAGTGTAGT TGGTAAATTA 60TGTTCTTGAC GGTGTATGTC ACATATTATT TATTAAAACT AGAAAAAACA GCGTCAAAAC 120TAGCAAAAAT CCAACGGACA AAAAAATCGG CTGAATTTGA TTTGGTTCCA ACATTTAAAA 180AAGTTTCAGT GAGAAAGAAT CGGTGACTGT TGATGATATA AACAAAGGGC ACATTGGTCA 240ATAACCATAA AAAATTATAT GACAGCTACA GTTGGTAGCA TGTGCTCAGC TATTGAACAA 300ATCTAAAGAA GGTACATCTG TAACCGGAAC ACCACTTAAA TGACTAAATT ACCCTCATCA 360GAAAGCAGAT GGAGTGCTAC AAATAACACA CTATTCAACA ACCATAAATA AAACGTGTTC 420AGCTACTAAA ACAAATATAA ATAAATCTAT GTTTGTAAGC ACTCCAGCCA TGTTAATGGA 480GTGCTATTGC CTGTTAACTC TCACTTATAA AATAGTAGTA GAA 523(2)SEQ ID NO:9信息:
(i)序列特征:
(A)长度:220个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:DNA(基因组)
(xi)序列描述:SEQ ID NO:9TAAAGAAGGT ACATCTGTAA CCGGAACACC ACTTAAATGA CTAAATTACC CTCATCAGAA 60AGCAGATGGA GTGCTACAAA TAACACACTA TTCAACAACC ATAAATAAAA CGTGTTCAGC 120TACTAAAACA AATATAAATA AATCTATGTT TGTAAGCACT CCAGCCATGT TAATGGAGTG 180CTATTGCCTG TTAACTCTCA CTTATAAAAT AGTAGTAGAA 220
Claims (23)
1.一种分离的DNA分子,这种DNA分子指导植物细胞中下游异源DNA片段的根皮层特异性转录,所说的分离DNA分子具有选自下组的序列:
(a)本文提供的SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQID NO:8和SEQ ID NO:9;和
(b)能够在严格的洗涤条件(0.3M NaCl、0.03M柠檬酸钠、0.1%SDS、60℃)下与具有上文(a)序列的分离DNA分子杂交的DNA序列,这种DNA序列能够指导植物细胞中下游异源DNA片段的根皮层特异性转录。
2.一种包含表达盒的DNA构建体,这种构建体在5′-3′的方向上包含烟草RD2启动子和定位在此启动子下游并与之可操作性地结合的异源DNA片段。
3.一种包含表达盒的DNA构建体,这种构建体在5′-3′的方向上包含根皮层特异性启动子和定位在此启动子下游并与之可操作性地结合的异源DNA片段,所说的根皮层特异性启动子具有选自下组的序列:
(a)本文提供的SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQID NO:8和SEQ ID NO:9;和
(b)能够在严格的洗涤条件(0.3M NaCl、0.03M柠檬酸钠0.1%SDS、60℃)下与具有上文(a)序列的分离DNA分子杂交的DNA序列,这种DNA序列能够指导植物细胞中下游异源DNA片段的根皮层特异性转录。
4.按照权利要求3的DNA构建体,其中所说的DNA构建体还含有质粒。
5.按照权利要求3的DNA构建体,其中所说的异源DNA片段是编码杀虫蛋白的基因。
6.按照权利要求4的DNA构建体,其中所说的异源DNA片段编码对昆虫有毒的苏云金芽孢杆菌(Bacillus thuringiensis)晶体蛋白的基因。
7.一种含有权利要求3的DNA构建体的植物细胞。
8.一种产生转化的植物的方法,这种方法包括由权利要求7的植物细胞再生植株。
9.一种包含权利要求3的DNA构建体的根癌土壤杆菌(Agrobacteriumtumefaciens)细胞,其中所说的DNA构建体还包含Ti质粒。
10.一种产生转化植物的方法,这种方法包括用按照权利要求9的根癌土壤杆菌感染植物细胞以便产生转化的植物细胞,然后由所说的转化的植物细胞再生植株。
11.一种携带按照权利要求3的DNA构建体的微颗粒,其中所说的微颗粒适合用于植物细胞的冲击转化。
12.一种产生转化植物的方法,这种方法包括把按照权利要求11的微颗粒推进到植物细胞中以便产生转化的植物细胞,然后由所说的转化的植物细胞再生植株。
13.一种含有按照权利要求3的DNA构建体的植物细胞原生质体。
14.一种产生转化植物的方法,这种方法包括由按照权利要求13的植物细胞原生质体再生植株。
15.一种包含转化的植物细胞的转化植物,这种转化的植物细胞包含异源DNA构建体,所说的构建体在5′-3′的方向上包含根皮层特异性启动子和定位在此启动子下游并与之可操作性地结合的异源DNA片段,所说的启动子指导所说的异源DNA片段的根皮层特异性转录。
16.按照权利要求15的转化植物,其中所说的根皮层特异性启动子是烟草RD2启动子,这种启动子指导植物细胞中下游异源DNA片段的根皮层特异性转录。
17.按照权利要求15的转化植物,其中所说的启动子具有选自下组的序列:
(a)本文提供的SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQID NO:8和SEQ ID NO:9;和
(b)能够在严格的洗涤条件(0.3M NaCl、0.03M柠檬酸钠、0.1%SDS、60℃)下与具有上文(a)序列的分离DNA分子杂交的DNA序列,这种DNA序列能够指导植物细胞中下游异源DNA片段的根皮层特异性转录。
18.按照权利要求15的转化植物,其中所说的植物是双子叶植物。
19.按照权利要求15的转化植物,其中所说的植物是单子叶植物。
20.按照权利要求15的转化植物,其中所说的植物是烟草(Nicotianatabacum)。
21.一种分离的DNA分子,这种DNA分子实质上包含指导植物细胞中下游异源DNA片段的根皮层特异性转录的启动子,并且具有选自由本文提供的SEQ ID NO:1-9组成的组中的序列。
22.一种包含表达盒的DNA构建体,这种构建体在5′-3′的方向上包含按照权利要求21的启动子和定位在此启动子下游并与之可操作性地结合的异源DNA片段。
23.一种包含转化植物细胞的转化植物,所说的转化植物细胞包含按照权利要求22的DNA构建体。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/508,786 US5837876A (en) | 1995-07-28 | 1995-07-28 | Root cortex specific gene promoter |
US08/508,786 | 1995-07-28 |
Publications (2)
Publication Number | Publication Date |
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CN1196753A true CN1196753A (zh) | 1998-10-21 |
CN1154740C CN1154740C (zh) | 2004-06-23 |
Family
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Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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CNB961970685A Expired - Lifetime CN1154740C (zh) | 1995-07-28 | 1996-07-24 | 根皮层特异性基因启动子 |
Country Status (13)
Country | Link |
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US (1) | US5837876A (zh) |
EP (1) | EP0842285B1 (zh) |
JP (1) | JP3565562B2 (zh) |
CN (1) | CN1154740C (zh) |
AT (1) | ATE348891T1 (zh) |
AU (1) | AU709691B2 (zh) |
BR (1) | BR9609958A (zh) |
CA (2) | CA2228046C (zh) |
DE (1) | DE69636782T2 (zh) |
ES (1) | ES2279524T3 (zh) |
HK (1) | HK1016653A1 (zh) |
NZ (1) | NZ313474A (zh) |
WO (1) | WO1997005261A1 (zh) |
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GB9706381D0 (en) * | 1997-03-27 | 1997-05-14 | Cambridge Advanced Tech | Improvements relating to the specificity of gene expression |
US6586661B1 (en) * | 1997-06-12 | 2003-07-01 | North Carolina State University | Regulation of quinolate phosphoribosyl transferase expression by transformation with a tobacco quinolate phosphoribosyl transferase nucleic acid |
AU2002300895B2 (en) * | 1997-06-12 | 2006-03-16 | North Carolina State University | Regulation of quinolate phosphoribosyl transferase expression |
GB9725556D0 (en) * | 1997-12-03 | 1998-02-04 | Ciba Geigy Ag | Organic compounds |
US6518486B1 (en) | 1998-06-12 | 2003-02-11 | University Of Guelph | Enhanced storage organ production in plants |
WO2001090387A2 (en) * | 2000-05-24 | 2001-11-29 | The University Of British Columbia | Gene regulatory region that promotes root-specific transcription and its uses |
KR20030029885A (ko) * | 2000-08-30 | 2003-04-16 | 노쓰 캐롤라이나 스테이트 유니버시티 | 단백질 함량을 변화시키는 분자 미끼를 포함하는유전자이식 식물 |
EP1317182A1 (en) * | 2000-09-11 | 2003-06-11 | Minos Biosystems Limited | Pest control system |
AU2002230691A1 (en) * | 2000-11-07 | 2002-05-21 | North Carolina State University | Putrescine-n-methyltransferase promoter |
EA200400013A1 (ru) * | 2001-06-08 | 2004-06-24 | Вектор Тобакко Лтд. | Модификация содержания никотина и нитрозамина в табаке |
US6730832B1 (en) * | 2001-09-10 | 2004-05-04 | Luis Mayan Dominguez | High threonine producing lines of Nicotiana tobacum and methods for producing |
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US4943674A (en) * | 1987-05-26 | 1990-07-24 | Calgene, Inc. | Fruit specific transcriptional factors |
US4962028A (en) * | 1986-07-09 | 1990-10-09 | Dna Plant Technology Corporation | Plant promotors |
US5229292A (en) * | 1986-07-28 | 1993-07-20 | Stine Seed Farm, Inc. | Biological control of insects using pseudomonas strains transformed with bacillus thuringiensis insect toxingene |
US5231020A (en) * | 1989-03-30 | 1993-07-27 | Dna Plant Technology Corporation | Genetic engineering of novel plant phenotypes |
US4943647A (en) * | 1989-03-30 | 1990-07-24 | Akzo America Inc. | Preparation of hexaalkyldisilthiane |
US5097025A (en) * | 1989-08-01 | 1992-03-17 | The Rockefeller University | Plant promoters |
US5177308A (en) * | 1989-11-29 | 1993-01-05 | Agracetus | Insecticidal toxins in plants |
GB9005945D0 (en) * | 1990-03-16 | 1990-05-09 | Cambridge Advanced Tech | Plant promoter |
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1995
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1996
- 1996-07-24 CA CA002228046A patent/CA2228046C/en not_active Expired - Lifetime
- 1996-07-24 DE DE69636782T patent/DE69636782T2/de not_active Expired - Lifetime
- 1996-07-24 JP JP50770397A patent/JP3565562B2/ja not_active Expired - Lifetime
- 1996-07-24 AU AU65959/96A patent/AU709691B2/en not_active Expired
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DE69636782D1 (de) | 2007-02-01 |
WO1997005261A1 (en) | 1997-02-13 |
EP0842285A1 (en) | 1998-05-20 |
JPH11510056A (ja) | 1999-09-07 |
CN1154740C (zh) | 2004-06-23 |
ATE348891T1 (de) | 2007-01-15 |
CA2228046A1 (en) | 1997-02-13 |
AU6595996A (en) | 1997-02-26 |
CA2350843C (en) | 2007-10-16 |
HK1016653A1 (en) | 1999-11-05 |
JP3565562B2 (ja) | 2004-09-15 |
BR9609958A (pt) | 1999-02-02 |
EP0842285B1 (en) | 2006-12-20 |
CA2350843A1 (en) | 1997-02-13 |
EP0842285A4 (en) | 2003-06-11 |
NZ313474A (en) | 1999-01-28 |
DE69636782T2 (de) | 2007-10-18 |
US5837876A (en) | 1998-11-17 |
AU709691B2 (en) | 1999-09-02 |
ES2279524T3 (es) | 2007-08-16 |
CA2228046C (en) | 2003-04-08 |
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