CN1258593C - 腐胺-n-甲基转移酶启动子 - Google Patents
腐胺-n-甲基转移酶启动子 Download PDFInfo
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Abstract
公开了腐胺-N-甲基转移酶启动子,尤其是从烟草中分离的启动子以及含有所述启动子的重组核酸、含有所述重组核酸的表达载体以及用所述表达载体产生的转基因植物。也公开了它们的使用方法。
Description
相关申请
本申请要求于2000年11月7日申请的美国临时专利申请顺序号60/246,488的权益;所述临时专利申请的公开内容通过引用全部结合到本文中。
发明领域
本发明涉及可用于植物的根特异性启动子及其使用方法、含有所述根特异性启动子的构建体以及用这类启动子产生的转基因植物。
发明背景
启动子一般定义为位于转录的基因上游或下游、并且RNA聚合酶将侧翼基因转录为信使RNA时必须与之结合的核酸序列。启动子可以由许多不同的调节元件构成,所述调节元件以不同的方式影响与所述启动子操作上连接的结构基因。例如,调节元件可以增强或阻抑相连结构基因的表达,对该基因进行发育调节或引起该基因的组织特异性调节。采用重组DNA方法对启动子进行修饰,可以达到可能任选模式的基因表达。参见例如Old and Primrose,Principles of GeneManipulation(4th Ed.,1989)。
表达抑制或杀死特定害虫或病原体的肽的转基因植物,提供了一种减少作物病害和损失的方法。例如苏云金芽孢杆菌(Bacillusthuringiensis)蛋白在转基因玉米中的表达提供对欧洲玉米螟的抗性。然而,转基因在所有的植物组织中表达(组成型表达)可能有多个缺点,因为它可能使非靶生物体接触转基因蛋白并且可能增加产生抗所述转基因蛋白的病原体和害虫的选择压力。在整株植物中高水平的转基因表达也可能负面影响植物的生长和产量。一种可供选择的策略是只在受特定害虫或病原体影响的器官或组织中表达毒性肽。针对侵袭植物根的害虫和病原体实施这一策略,由于缺乏经表征的根特异性启动子而受到阻碍。
当在RNA聚合酶和基因启动子之间形成稳定的复合物时,启动基因的转录。启动子一般位于所有转录单位的起始处,通常长度约100个碱基对,一般紧接转录起始位点的上游。参见例如Maniatis等,Science 236:1238(1987)。启动子在其“强度”方面有所变化;也就是说,在其准确有效地起始转录的能力方面有所变化。认为RNA聚合酶全酶覆盖约50个碱基、紧接所转录区上游的区域。在某些情况下,转录起始的强度可以为邻近位于紧接所转录DNA上游的启动子区结合的辅助蛋白所增强。参见例如Singer和Berg,Genes and Genomes,140-145,University Science Books,Mill Valley,Cailf.(1991)。
Conkling和Yamamoto的美国专利第5,459,252号描述了RB7根启动子。
Conkling、Mendu和Song的美国专利第5,837,876号描述了RD2根皮层特异性启动子,也称为NtQPT1启动子。
发明概述
本发明的第一个方面是指导下游异源DNA区段在植物细胞中根特异性转录的分离的DNA分子。所述启动子为腐胺-N-甲基转移酶(PMT)启动子,例如烟草PMT启动子或NtPMT1启动子。本发明启动子的实例包括具有选自以下序列并且指导下游异源DNA区段在植物细胞中根特异性转录的分离的DNA分子:(a)本文提供的SEQ ID NO:1-11和(b)与SEQ ID NO:1-11中任一个或其互补序列杂交(最好在严格性条件下)的DNA序列。
本发明的再一方面是一种包含烟草PMT启动子和位于所述启动子下游且与其操作上连接的异源DNA区段的表达盒。
本发明的再一方面是一种包含根特异性启动子和异源DNA区段的表达盒,所述根特异性启动子的序列如本文中所述。
本发明的其它方面是包含上述表达盒的植物细胞、从这类植物细胞产生转化植物的方法以及包含这类转化植物细胞的转化植物。
附图简述
图1显示在叶片(第1栏)、茎(第2栏)和根(第3栏)中由所述PMT启动子指导的平均GUS表达水平。
优选实施方案的详细描述
在本文中核苷酸序列仅仅以从左至右为5′→3′方向的单链表示。本文根据IUPAC-IUB Biochemical Nomenclature Commission(IUPAC-IUB生物化学命名委员会)建议的方式表示各种核苷酸。
本发明根特异性启动子的具体实例是具有对应于SEQ ID NO:1-11中所示序列中任一个的序列的DNA分子,所有这些将在下文更为详细地论述。显然可以制备也将携带TobPMT根特异性启动子、来自烟草PMT5′侧翼区、比前述序列更长或更短或者具有对其进行的微小添加、缺失或取代的其它序列片段,所有这些都包括在本发明中。本发明的再一方面包括从其它烟草基因分离的启动子,或者从除烟草以外的如下所述的植物中分离的、与所述烟草PMT启动子同源并且能够指导下游异源DNA区段在植物细胞中的根特异性转录的启动子。
本文所用的TobPMT启动子是指具有与烟草PMT基因转录区5′存在的DNA连续区段相同或基本同源的序列的DNA分子。本文中给出的SEQ ID NO:1提供紧接在TobPMT基因中的转录起始位点5′存在的区域的序列。TobPMT启动子包括紧接TobPMT转录区5′的至少100个碱基对区、150个碱基对区、或者最好是200个碱基对区并且指导根特异性表达。本文所用的“基本同源的”区是与所述核酸序列有至少75%同源性,更优选为80%、85%、90%或者甚至95%同源性。
本文所用的根特异性启动子是与在叶组织或茎组织或者植物的其它组织中的表达相比、优先指导操作上连接的DNA、核酸或基因在根组织中表达的启动子。
来自其它植物的根特异性启动子序列包括与紧接烟草PMT DNA转录区上游烟草PMT启动子的大约100个碱基区段有至少约75%同源性(且更优选有80%、85%、90%或者甚至95%同源性)并且能够指导下游异源DNA区段在植物细胞中的根特异性转录的那些根特异性启动子序列。来自其它植物的根特异性启动子包括与本文中以SEQ ID NO:1-11限定的TobPMT启动子连续部分有至少约75%同源性(且更优选有80%、85%、90%或者甚至95%同源性)并且能够指导下游异源DNA区段在植物细胞中的根特异性转录的那些根特异性启动子。通过运用合适的比较算法或者通过目测检测,同源性百分率可以通过将参比序列例如SEQ ID NO:1-11与另一待测序列进行比较来确定。在一个实施方案中,在长度至少约20-50个残基的序列区内存在大致同一性。对于序列比较,通常将一个序列用作参比序列,然后将其与待测序列进行比较。当运用序列比较算法时,将待测序列和参比序列输入计算机中,必要时指定亚序列坐标,然后指定序列算法程序参数。根据所指定的程序参数,序列比较算法则计算与参比序列相比待测序列的序列同一性百分率。例如通过Smith和Waterman,Adv.Appl.Math.2:482(1981)的局部同源性算法、Needleman和Wunsch,J.Mol.Biol.48:443(1970)的同源性序列比对算法、Pearson和Lipman,Proc.Nat′l.Acad.Sci.USA85:2444(1988)的相似性搜索方法、这些算法的计算机工具(theSoftware Package(Wisconsin Genetics软件包)中的GAP、BESTFIT、FASTA和TFASTA,Genetics Computer Group,575 Science Dr.,Madison,Wis.),或者通过目测检测(一般参见Ausubel等,参见上文),可以进行比较序列的最佳比对。有用算法的一个实例是PILEUP。PILEUP运用渐近配对序列比对创建与一组相关序列的多序列比对,以显示相关性和序列同一性百分率。它也绘出进化树或分枝图(dendogram),以显示用来创建序列比对的聚类关系。PILEUP采用的Feng和Doolittle,J.Mol.Evol.35:351-360(1987)的渐近序列比对方法的简化方法。所使用的方法类似于Higgins和Sharp,CABIOS5:151-153(1989)描述的方法。所述程序可以比对多达300个序列,每个序列最大长度为5,000个核苷酸或氨基酸。所述多序列比对法始于两个最为相似序列的配对比对,产生一组两个经比对的序列。然后该组序列与下一个最相关的序列或下一组经比对的序列进行比对。通过两个个别序列的配对比对的简单延伸,将两组序列进行比对。通过一系列渐近配对序列比对,完成最终的序列比对。通过指定具体序列及其序列比较区的氨基酸或核苷酸坐标并且指定程序参数,运行该程序。例如,采用以下参数:缺省空位加权(default gap weight)(3.00)、缺省空位长度加权(default gap lengthweight)(0.10)和加权末端空位(weighted end gap),可以将参比序列与其它待测序列进行比较,以确定序列同一性关系百分率。适用于测定序列同一性百分率和序列相似性百分率的算法的另一个实例是BLAST算法,该算法描述于Altschul等,J.Mol.Biol.215:403-410(1990)。用于进行BLAST分析的软件公众可通过the National Center forBiotechnology Information(http://www.ncbi.nlm.nih.gov/)得到。该算法包括首先通过鉴定在查询序列中与数据库序列中相同长度的字串比对时或者匹配或者满足某些正数值最低分值T的长度W的短字串来鉴定高分序列配对(HSP)。T被称为邻近字串最低分值(Altschul等,参见上文)。这些原始邻近字串命中作为起始搜索以发现含有它们的更长HSP的种子。只要累积序列比对分值可以增加,字串命中则以两个方向沿每个序列延伸。对于核苷酸序列,使用参数M(一对匹配残基的奖分(reward score for a pair of matching residues);总是>0)和N(错配残基的罚分(penalty score for mismatching residues;总是<0),计算累积分值。对于氨基酸序列,使用打分矩阵来计算累积分值。字串命中在每个方向的延伸当遇到下述情况之一时终止:累积序列比对分值比其获得的最大值低数量X;累积分值为零或零以下,这是由于一个或多个负打分残基比对的累积所致;或者到达每个序列的末端。BLAST算法参数W、T和X决定所述比对的灵敏度和速度。BLASTN程序(用于核苷酸序列)使用的缺省字长(wordlength)(W)为11,期望值(Expectation)(E)为10,M=5,N=-4,并且比较两条链。对于氨基酸序列而言,BLASTP程序使用的缺省字长(W)为3,期望值(E)为10,使用BLOSUM62打分矩阵(参见Henikoff和Henikoff,Proc.Natl.Acad.Sci.USA89:10915(1989))。
允许DNA同源序列与本文中给出的DNA序列杂交的高严格性杂交条件是本领域众所周知的。例如,这类序列与本文中公开的DNA的杂交可以在25%甲酰胺、5X SSC、5X Denhardt氏溶液、100μg/ml单链DNA和5%硫酸葡聚糖中于42℃进行,洗涤条件为25%甲酰胺、5X SSC、0.1%SDS、42℃,达15分钟,允许约60%同源性的序列杂交。更严格的条件为:洗涤严格性为0.3M NaCl、0.03M柠檬酸钠、0.1%SDS于60℃或者甚至70℃,使用标准原位杂交测定。(参见Sambrook等,Molecular Cloning,A Laboratory Manual(2d Ed.1989)(Cold Spring Harbor Laboratory))。一般而言,编码根特异性启动子并且与本文中公开的编码烟草PMT根特异性启动子的DNA序列杂交的植物DNA序列,与本文中公开的编码烟草PMT根特异性启动子的DNA序列有至少75%、80%、85%、90%或者甚至95%同源性或更高同源性。
本发明的根特异性启动子可用来指导转基因在转化植物中的组织特异性表达。这样的组织特异性转基因表达可用来提供针对由侵袭植物根的害虫和病原体引起的病害的抗性。另外,由于根是光合产物贮藏物的主要贮藏器官,因此设计用以改变所贮藏糖类的转基因的表达可以由这样的启动子来指导。对于根特异性表达而言,特别感兴趣的外源基因包括编码结合重金属的蛋白质(例如金属硫蛋白)、赋予对土传害虫和病原体抗性的蛋白质、赋予对热、盐(盐碱化)和干旱抗性的蛋白质、用于脱盐的蛋白质以及将植物贮藏化合物代谢为替代的优选产物或形式的蛋白质的那些外源基因。
本发明的根特异性启动子可用来在“分子农业”应用中表达蛋白质或肽。这样的蛋白质或肽包括但不限于工业用酶、抗体、治疗药物和营养制品。这样的根特异性表达当植物为根用作物例如甜菜时特别有用。
本发明的根特异性启动子也可用来表达将会减少或抑制天然基因在植物中表达的寡核苷酸。这样的寡核苷酸可以长4、6或8个核苷酸至40、80或100个核苷酸或者更长,并且可以编码反义寡核苷酸、核酶、有义抑制因子或抑制天然基因表达的其它产物。
组织特异性启动子也可以用来使原杀虫剂(pro-pesticide)在所选组织部位转化为活性形式。Hsu等Pestic.Sci.,44,9(1995)报道,应用包含根特异性启动子TobRB7和B-葡糖醛酸糖苷酶基因的嵌合基因使原杀虫剂优先在根中转化为活性形式。将无活性原杀虫剂(羟甲基草氨酰的葡糖苷酸)施用于叶面,然后通过植物韧皮部运输至根,在此通过葡糖醛酸糖苷酶将其转化为活性杀线虫剂形式。
另外,根皮层特异性启动子可用于组织学目的,以便用诸如B-葡糖醛酸糖苷酶的报道基因来鉴定根皮层组织或使其染色。
本文所用的术语“操作上连接的”是指在单个DNA分子中所含的DNA序列当连接时使得一个序列受另一个序列的影响。因此,将启动子与基因操作上连接,则其能够影响该基因的表达(即该基因处于该启动子的转录控制之下)。所述启动子被认为位于所述基因的“上游”,也可以说所述基因位于所述启动子的“下游”。
本发明的DNA构建体或“表达盒”以5′→3′的转录方向包括一个本发明启动子、一个与所述启动子操作上连接的异源DNA区段和任选的转录和翻译终止区例如终止信号区和/或聚腺苷酸化区。这些调节区最好能够在转化细胞中操作。所述3′终止区可以来源于同一基因如转录起始区或者来源于不同的基因。
植物可以分为缺乏叶绿素的植物(例如真菌)和含有叶绿素的植物(例如绿藻、苔藓);并且可以进一步分为含有叶绿素并且具有维管组织的植物(例如蕨类植物、裸子植物、松柏类植物、单子叶植物和双子叶植物)。后一组植物包括其中可以有根、茎和叶的那些植物。本文所用的术语“植物”包括所有这样的上述生物体。本文所用的术语“天然植物DNA”是指从非遗传改变或未转化的植物(例如通过选择育种产生的植物变种)中分离的DNA。
本文所用的术语异源基因或异源DNA区段是指通过遗传工程技术转化细胞所用的并且在所述细胞中可能天然不存在的基因(或DNA区段)。结构基因是包含编码蛋白质、肽或其部分的DNA区段的那些基因部分,可能包括核糖体结合位点和/或翻译起始密码子,但缺乏启动子。所述术语也可以指在细胞内天然存在的、但为人工引入的结构基因拷贝。结构基因可以编码植物细胞中非正常存在的蛋白质,其中所述基因是被引入的或者同与其操作上连接的启动子联合引入的。可以与本发明启动子操作上连接的、供在植物种中表达用的基因可以来源于染色体基因、cDNA、合成基因或它们的组合。本文所用的术语异源DNA区段也包括编码非蛋白产物例如核酶或反义RNA的DNA区段。反义RNA是众所周知的(参见例如美国专利第4,801,540号(Calgene,Inc.))。
可供本发明在植物中使用的感兴趣基因包括影响各种各样的表型和非表型特性的那些基因。在所述表型特性中有提供对各种环境逆境抗性的蛋白质例如酶,所述逆境包括但不限于由于脱水(由于热、盐度或干旱引起的)、杀虫剂、有毒金属、微量元素、害虫和病原体引起的逆境。抗性可能是由于靶部位中的变化、靶蛋白量在宿主细胞中的增加、参与保护宿主抵抗所述逆境的产物生物合成途径的一种或多种酶量的增加等所致。结构基因可以得自原核生物或真核生物、细菌、真菌(例如得自酵母、病毒、植物和哺乳动物),或者可以全合成或部分合成的。说明性的基因包括草甘膦抗性3-烯醇丙酮酸磷酸莽草酸合酶基因、腈水解酶基因、脯氨酸和谷氨酰胺生物合成途径中的基因和金属硫蛋白基因。
与本发明启动子操作上连接的结构基因可以是编码对昆虫有毒的蛋白质例如对昆虫有毒的苏云金芽孢杆菌晶体蛋白的那些结构基因。编码对鞘翅目(Coleoptera)有毒的苏云金芽孢杆菌(B.thuringiensis)毒素的DNA序列和其中保留所述毒性的该序列变异体公开于美国专利第4,853,331号(也参见美国专利第4,918,006号和第4,910,136号)(本文引用的所有美国专利参考文献的公开内容通过引用全部结合到本文中)。使植物种对鳞翅目(Lepidoptera)有毒的来自苏云金芽孢杆菌的基因序列公开于PCT申请WO90/02804。PCT申请WO89/04868公开了用启动苏云金芽孢杆菌晶体蛋白表达的载体转化的转基因植物,所述序列可以与本发明联合使用。PCT申请WO90/06999公开了编码有效抵抗鳞翅目的苏云金芽孢杆菌晶体蛋白毒素的DNA。编码杀虫晶体蛋白的另一基因序列公开于美国专利第4,918,006号。编码其它昆虫毒素的示例性基因序列是编码壳多糖酶(例如EC-3.2.114)的基因序列,公开于美国专利第4,940,840号和PCT申请号WO90/07001。可用来产生抗根线虫的转基因植物的线虫诱导型孔蛋白(pore protein)的编码基因公开于美国专利申请顺序号08/007,998。产生有效抵抗线虫的多肽毒素的苏云金芽孢杆菌菌株公开于美国专利第4,948,734号和第5,093,120号。
当基因的表达产物位于除细胞质以外的细胞区室的情况下,可以构建结构基因以包括编码导致所述产物转运至特定部位例如细胞质膜或分泌至壁膜间隙或细胞外部环境中的特定氨基酸序列的区域。用于指导所述肽表达产物至特定部位的各种分泌前导序列、膜整合序列和转运序列在文献中有描述。参见例如Cashmore等,Biotechnology(1985)3:803-808,Wickner和Lodish,Science(1985)230:400-407。
所述表达盒可以在也具有至少一个复制系统的DNA构建体中提供。为方便起见,DNA构建体通常具有在大肠杆菌(Escherichia coli)中有功能的复制系统,例如ColEl、pSC101、pACYC184等。在该方法中,在每次操作后的每个步骤,可以将所得的构建体克隆、测序,以确定操作的正确性。另外,或者不用大肠杆菌复制系统,而可以使用广宿主范围型复制系统,例如P-1不亲和性质粒例如pRK290的复制系统。除所述复制系统以外,还可以存在至少一个标记,所述标记可用于一种或多种宿主中,或者不同的宿主使用不同的标记。也就是说,可以使用一种标记用于在原核宿主中进行选择,而可以使用另一种标记用于在真核宿主、尤其是在植物宿主中进行选择。所述标记可以提供抗杀生物剂例如抗生素、毒素、重金属等的保护作用;可以提供给原养型赋予营养缺陷型的工具;或者可以通过在植物中产生新型化合物而提供可见表型。可以使用的示例性基因包括新霉素磷酸转移酶(NPTII)基因、潮霉素磷酸转移酶(HPT)基因、氯霉素乙酰转移酶(CAT)基因、腈水解酶基因和庆大霉素抗性基因。对于植物宿主选择,合适标记的非限制性实例是β-葡糖醛酸糖苷酶(GUS)(提供indigo产生)、荧光素酶(提供可见光产生)、NPTII(提供卡那霉素抗性或G418抗性)、HPT(提供潮霉素抗性)和突变型aroA基因(提供草甘膦抗性)。
可以通过限制性酶切割合适的复制系统并且将特定构建体或片段插入到可利用的位点中,连续引入包含各种构建体、表达盒、标记等的各种片段。在连接和克隆后,可以将所述DNA构建体分离出来,供进一步操作使用。所有这些技术在文献中有大量的范例。参见例如Maniatis等,Molecular Cloning:A Laboratory Manual,Cold Spring HarborLaboratory,Cold Spring Harbor,N.Y.(1982)。
载体是可复制的DNA构建体。可以用来用本发明的DNA构建体转化植物组织的载体包括土壤杆菌属(Agrobacterium)载体和基因枪(ballistic)载体以及适合DNA介导的转化的载体。含有本发明DNA构建体的根癌土壤杆菌(Agrobacterium tumefaciens)细胞可用于产生转化植物的方法中,其中所述DNA构建体包含Ti质粒。用根癌土壤杆菌感染植物细胞,产生转化植物细胞,然后从转化植物细胞再生植株。
可用于实施本发明的多种土壤杆菌属载体系统是已知的。例如美国专利第4,459,355号公开了一种用含Ti质粒的土壤杆菌属菌株转化包括双子叶植物在内的敏感植物的方法。用土壤杆菌属载体转化木本植物公开于美国专利第4,795,855号。此外,Schilperoort等的美国专利第4,940,838号公开了一种可用于实施本发明的双元土壤杆菌属载体(即其中土壤杆菌含有一个具有Ti质粒vir区而无T-DNA区的质粒和第二个具有T-DNA区而无vir区的质粒的载体)。
携带本发明DNA构建体、适合于植物细胞的基因枪转化的微粒也可用于产生本发明的转化植物。将微粒推进植物细胞中,产生转化植物细胞,然后从转化植物细胞再生植株。任何合适的基因枪细胞转化方法和设备都可以用来实施本发明。示例性的设备和方法公开于Sanford和Wolf的美国专利第4,945,050号以及Agracetus的欧洲专利申请公布号0270356,题为“Pollen-mediated Plant Transformation(花粉介导的植物转化)”。当采用基因枪转化法时,可以将表达盒掺入到能够在待转化细胞中复制的质粒中。适合用于这类系统的微粒的实例包括1-5μm金微球。通过任何合适的技术,例如通过沉淀,可以使DNA构建体沉积在微粒上。
转化宿主细胞是已经采用重组DNA技术用含有本文中公开的DNA序列的构建体转化或转染了的细胞。按照本领域熟知的方法,通过DNA介导的植物细胞原生质体转化并且随后从转化原生质体再生植株,可以用本发明的DNA构建体转化各种植物种。
本文中公开的启动子序列可以用来在能够利用所述启动子的任何植物种(即其RNA聚合酶可与本文中公开的启动子序列结合的任何植物种)中表达异源DNA序列。适合于用本发明DNA构建体转化的植物种的实例既包括单子叶植物也包括双子叶植物,并且包括但不限于烟草、大豆、马铃薯、棉花、甜菜、向日葵、胡萝卜、芹菜、亚麻、卷心菜和其它十字花科植物、胡椒、番茄、柑桔树、菜豆、草莓、莴苣、玉米、苜蓿、燕麦、小麦、水稻、大麦、高粱和canola。因此可以用本发明DNA构建体转化的植物的说明性类别是双子叶植物,可以使用本发明的DNA构建体转化的植物的更具体的类别是茄科(Solanaceae)的成员。
能够随后克隆繁殖(无论是通过器官发生还是通过胚胎发生)的任何植物组织,都可以用本发明的载体进行转化。本文所用的术语“器官发生”是指从分生组织中心顺序发育芽和根的过程;本文所用的术语“胚胎发生”是指无论从体细胞还是从配子,芽和根以协同方式(非顺序的)一起发育的过程。所选的特定组织将根据特定待转化物种可利用的且是最适合的克隆繁殖系统而变化。示例性的组织靶包括叶盘片、花粉、胚、子叶、下胚轴、大配子体、愈伤组织、原有分生组织(例如顶端分生组织、腋芽和根分生组织)和诱导的分生组织(例如子叶分生组织和下胚轴分生组织)。
提供以下的实施例用以说明本发明的各种具体实施方案,所述实施例不应解释为限制本发明。
实施例1
NtPMT启动子的克隆
使用N.Hibi等(Plant Cell 6,723-725(1994))公布的PMT cDNA序列,我们设计了嵌套趋异PCR引物,供反向PCR用。用各种各样的限制性内切核酸酶切割烟草基因组DNA,在低DNA浓度下连接(以促进环化),然后将其用作供反向PCR用的模板。从已经用NdeI消化的基因组DNA获得最长的扩增产物。将该片段克隆并测序。与所述PMTcDNA的序列比较显示,在已知区中存在序列同一性,说明所扩增的产物事实上是所述PMT基因的5’侧翼区。NtPMT启动子的DNA序列如下:
GTATACCAAA AATCAATTCA ACCCCCAAAA CATAATACAA CCAATGTTAA
TGCAATATCT CTGCTGCTAT CACGAAGATA ATTGTAGCTC ACGAAAGTAG
GATACATTAT GTAGGTTACA TCACATAGAG GTAATCTAAA GCTCCCAATA
ATAAGATGTG TAATGTTGAT TATGTAGAAA TTTGCCAGGT TATTTAGAAT
AAACAAGAAG AGGAGAAAAA AAGTACAATT TACCTGAACT CTTCAATGTA
TCCTACAAAT AACCTAGACT TCATGGACGT CAGTTGTCAG TTTACTTTTG
TTTTAATGGT ACATCATTTG TCAAATACTT TATTTGGATA AAAACAGTTT
TGCCTAAGGA GTAAACAGAT CCGGAGTAAG AAAGCAGACG ATTAAAGCAA
TTTTTAAAAA AGGAGAGAGA AATTAATGAG CACACACATA TACTAGTGAA
ATTAGGGTAC TAATTTACTA ATAATTGCAC CGAGACAAAC TTATATTTTA
GTTCCAAAAT GTCAGTCTAA CCCTGCACGT TGTAATAAAT TTTTAACTCT
ATTATATTAT ATCGAGTTGC GCCCTCCACT CCTCGGTGTC CAAATTGTAT
TTAAATGCAT AGATGTTTAA TGGGAGTGTA CAGCAAGCTT TC
GGAAAATA
CAAACCATAA TACTTTCTCT TCTTCAATTT GTTTAGTTTA ATTTTGAAAA
TGGAAGTCAT ATCTACCAAC ACAAATGGCT CTACCATCTT CA
对应于Hibi等(参见上文)公布的PMT cDNA的序列以下划线显示。起始ATG以粗体显示。用作反向PCR引物的序列以斜体显示。
实施例2
转基因植物
按照标准技术,将实施例1中叙述的NtPMT1启动子(SEQ ID NO:1)与pBI101中的GUS基因融合,然后转化到烟草中。参见例如美国专利第5,837,876号中的实施例4-6。使用X-Gluc,使转基因烟草由于有GUS活性而被染色。转基因根因有GUS活性而被染色。
实施例3
缺失突变体
本发明PMT启动子的其它实施包括上述SEQ ID NO:1所示启动子的缺失突变体。这些包括如下文SEQ ID NO:2-6中所示、缺失5’区的突变体:
SEQ ID NO:2:
AATCAATTCA ACCCCCAAAA CATAATACAA CCAATGTTAA
TGCAATATCT CTGCTGCTAT CACGAAGATA ATTGTAGCTC ACGAAAGTAG
GATACATTAT GTAGGTTACA TCACATAGAG GTAATCTAAA GCTCCCAATA
ATAAGATGTG TAATGTTGAT TATGTAGAAA TTTGCCAGGT TATTTAGAAT
AAACAAGAAG AGGAGAAAAA AAGTACAATT TACCTGAACT CTTGAATGTA
TCCTACAAAT AACCTAGACT TCATGGACGT CAGTTGTCAG TTTACTTTTG
TTTTAATGGT ACATCATTTG TCAAATACTT TATTTGGATA AAAACAGTTT
TGCCTAAGGA GTAAACAGAT CCGGAGTAAG AAAGCAGACG ATTAAAGCAA
TTTTTAAAAA AGGAGAGAGA AATTAATGAG CACACACATA TACTAGTGAA
ATTAGGGTAC TAATTTACTA ATAATTGCAC CGAGACAAAC TTATATTTTA
GTTCCAAAAT GTCAGTCTAA CCCTGCACGT TGTAATAAAT TTTTAACTCT
ATTATATTAT ATCGAGTTGC GCCCTCCACT CCTCGGTGTC CAAATTGTAT
TTAAATGCAT AGATGTTTAA TGGGAGTGTA CAGCAAGCTT TCGGAAAATA
CAAACCATAA TACTTTCTCT TCTTCAATTT GTTTAGTTTA ATTTTGAAAA
TGGAAGTCAT ATCTACCAAC ACAAATGGCT CTACCATCTT CA
SEQ ID NO:3:
ACCCCCAAAA CATAATACAA CCAATGTTAA
TGCAATATCT CTGCTGCTAT CACGAAGATA ATTGTAGCTC ACGAAAGTAG
GATACATTAT GTAGGTTACA TCACATAGAG GTAATCTAAA GCTCCCAATA
ATAAGATGTG TAATGTTGAT TATGTAGAAA TTTGCCAGGT TATTTAGAAT
AAACAAGAAG AGGAGAAAAA AAGTACAATT TACCTGAACT CTTGAATGTA
TCCTACAAAT AACCTAGACT TCATGGACGT CAGTTGTCAG TTTACTTTTG
TTTTAATGGT ACATCATTTG TCAAATACTT TATTTGGATA AAAACAGTTT
TGCCTAAGGA GTAAACAGAT CCGGAGTAAG AAAGCAGACG ATTAAAGCAA
TTTTTAAAAA AGGAGAGAGA AATTAATGAG CACACACATA TACTAGTGAA
ATTAGGGTAC TAATTTACTA ATAATTGCAC CGAGACAAAC TTATATTTTA
GTTCCAAAAT GTCAGTCTAA CCCTGCACGT TGTAATAAAT TTTTAACTCT
ATTATATTAT ATCGAGTTGC GCCCTCCACT CCTCGGTGTC CAAATTGTAT
TTAAATGCAT AGATGTTTAA TGGGAGTGTA CAGCAAGCTT TC
GGAAAATA
CAAACCATAA TACTTTCTCT TCTTCAATTT GTTTAGTTTA ATTTTGAAAA
TGGAAGTCAT ATCTACCAAC ACAAATGGCT CTACCATCTT CA
SEQ ID NO:4:
CATAATACAA CCAATGTTAA
TGCAATATCT CTGCTGCTAT CACGAAGATA ATTGTAGCTC ACGAAAGTAG
GATACATTAT GTAGGTTACA TCACATAGAG GTAATCTAAA GCTCCCAATA
ATAAGATGTG TAATGTTGAT TATGTAGAAA TTTGCCAGGT TATTTAGAAT
AAACAAGAAG AGGAGAAAAA AAGTACAATT TACCTGAACT CTTGAATGTA
TCCTACAAAT AACCTAGACT TCATGGACGT CAGTTGTCAG TTTACTTTTG
TTTTAATGGT ACATCATTTG TCAAATACTT TATTTGGATA AAAACAGTTT
TGCCTAAGGA GTAAACAGAT CCGGAGTAAG AAAGCAGACG ATTAAAGCAA
TTTTTAAAAA AGGAGAGAGA AATTAATGAG CACACACATA TACTAGTGAA
ATTAGGGTAC TAATTTACTA ATAATTGCAC CGAGACAAAC TTATATTTTA
GTTCCAAAAT GTCAGTCTAA CCCTGCACGT TGTAATAAAT TTTTAACTCT
ATTATATTAT ATCGAGTTGC GCCCTCCACT CCTCGGTGTC CAAATTGTAT
TTAAATGCAT AGATGTTTAA TGGGAGTGTA CAGCAAGCTT TC
GGAAAATA
CAAACCATAA TACTTTCTCT TCTTCAATTT GTTTAGTTTA ATTTTGAAAA
TGGAAGTCAT ATCTACCAAC ACAAATGGCT CTACCATCTT CA
SEQ ID NO:5:
CCAATGTTAA
TGCAATATCT CTGCTGCTAT CACGAAGATA ATTGTAGCTC ACGAAAGTAG
GATACATTAT GTAGGTTACA TCACATAGAG GTAATCTAAA GCTCCCAATA
ATAAGATGTG TAATGTTGAT TATGTAGAAA TTTGCCAGGT TATTTAGAAT
AAACAAGAAG AGGAGAAAAA AAGTACAATT TACCTGAACT CTTGAATGTA
TCCTACAAAT AACCTAGACT TCATGGACGT CAGTTGTCAG TTTACTTTTG
TTTTAATGGT ACATCATTTG TCAAATACTT TATTTGGATA AAAACAGTTT
TGCCTAAGGA GTAAACAGAT CCGGAGTAAG AAAGCAGACG ATTAAAGCAA
TTTTTAAAAA AGGAGAGAGA AATTAATGAG CACACACATA TACTAGTGAA
ATTAGGGTAC TAATTTACTA ATAATTGCAC CGAGACAAAC TTATATTTTA
GTTCCAAAAT GTCAGTCTAA CCCTGCACGT TGTAATAAAT TTTTAACTCT
ATTATATTAT ATCGAGTTGC GCCCTCCACT CCTCGGTGTC CAAATTGTAT
TTAAATGCAT AGATGTTTAA TGGGAGTGTA CAGCAAGCTT TC
GGAAAATA
CAAACCATAA TACTTTCTCT TCTTCAATTT GTTTAGTTTA ATTTTGAAAA
TGGAAGTCAT ATCTACCAAC ACAAATGGCT CTACCATCTT CA
SEQ ID NO:6:
TGCAATATCT CTGCTGCTAT CACGAAGATA ATTGTAGCTC ACCAAAGTAG
GATACATTAT GTAGGTTACA TCACATAGAG GTAATCTAAA GCTCCCAATA
ATAAGATGTG TAATGTTGAT TATGTAGAAA TTTGCCAGGT TATTTAGAAT
AAACAAGAAG AGGAGAAAAA AAGTACAATT TACCTGAACT CTTGAATGTA
TCCTACAAAT AACCTAGACT TCATGGACGT CAGTTGTCAG TTTACTTTTG
TTTTAATGGT ACATCATTTG TCAAATACTT TATTTGGATA AAAACAGTTT
TGCCTAAGGA GTAAACAGAT CCGGAGTAAG AAAGCAGACG ATTAAAGCAA
TTTTTAAAAA AGGAGAGAGA AATTAATGAG CACACACATA TACTAGTGAA
ATTAGGGTAC TAATTTACTA ATAATTGCAC CGAGACAAAC TTATATTTTA
GTTCCAAAAT GTCAGTCTAA CCCTGCACGT TGTAATAAAT TTTTAACTCT
ATTATATTAT ATCGAGTTGC GCCCTCCACT CCTCGGTGTC CAAATTGTAT
TTAAATGCAT AGATGTTTAA TGGGAGTGTA CAGCAAGCTT TC
GGAAAATA
CAAACCATAA TACTTTCTCT TCTTCAATTT GTTTAGTTTA ATTTTGAAAA
TGGAAGTCAT ATCTACCAAC ACAAATGGCT CTACCATCTT CA
本发明PMT启动子的其它实例包括如以上SEQ ID NO:1给出的序列的其中缺失3’区的缺失突变体。实例包括以下的SEQ ID NO:7-9:
SEQ ID NO:7:
GTATACCAAA AATCAATTCA ACCCCCAAAA CATAATACAA CCAATGTTAA
TGCAATATCT CTGCTGCTAT CACGAAGATA ATTGTAGCTC ACGAAAGTAG
GATACATTAT GTAGGTTACA TCACATAGAG GTAATCTAAA GCTCCCAATA
ATAAGATGTG TAATGTTGAT TATGTAGAAA TTTGCCAGGT TATTTAGAAT
AAACAAGAAG AGGAGAAAAA AAGTACAATT TACCTGAACT CTTGAATGTA
TCCTACAAAT AACCTAGACT TCATGGACGT CAGTTGTCAG TTTACTTTTG
TTTTAATGGT ACATCATTTG TCAAATACTT TATTTGGATA AAAACAGTTT
TGCCTAAGGA GTAAACAGAT CCGGAGTAAG AAAGCAGACG ATTAAAGCAA
TTTTTAAAAA AGGAGAGAGA AATTAATGAG CACACACATA TACTAGTGAA
ATTAGGGTAC TAATTTACTA ATAATTGCAC CGAGACAAAC TTATATTTTA
GTTCCAAAAT GTCAGTCTAA CCCTGCACGT TGTAATAAAT TTTTAACTCT
ATTATATTAT ATCGAGTTGC GCCCTCCACT CCTCGGTGTC CAAATTGTAT
TTAAATGCAT AGATGTTTAA TGGGAGTGTA CAGCAAGCTT TC
SEQ ID NO:8:
GTATACCAAA AATCAATTCA ACCCCCAAAA CATAATACAA CCAATGTTAA
TGCAATATCT CTGCTGCTAT CACGAAGATA ATTGTAGCTC ACGAAAGTAG
GATACATTAT GTAGGTTACA TCACATAGAG GTAATCTAAA GCTCCCAATA
ATAAGATGTG TAATGTTGAT TATGTAGAAA TTTGCCAGGT TATTTAGAAT
AAACAAGAAG AGGAGAAAAA AAGTACAATT TACCTGAACT CTTGAATGTA
TCCTACAAAT AACCTAGACT TCATGGACGT CAGTTGTCAG TTTACTTTTG
TTTTAATGGT ACATCATTTG TCAAATACTT TATTTGGATA AAAACAGTTT
TGCCTAAGGA GTAAACAGAT CCGGAGTAAG AAAGCAGACG ATTAAAGCAA
TTTTTAAAAA AGGAGAGAGA AATTAATGAG CACACACATA TACTAGTGAA
ATTAGGGTAC TAATTTACTA ATAATTGCAC CGAGACAAAC TTATATTTTA
GTTCCAAAAT GTCAGTCTAA CCCTGCACGT TGTAATAAAT TTTTAACTCT
ATTATATTAT ATCGAGTTGC GCCCTCCACT CCTCGGTGTC CAAATTGTAT
TTAAATGCAT AGATGTTTAA TGGGAGTGTA
SEQ ID NO:9:
GTATACCAAA AATCAATTCA ACCCCCAAAA CATAATACAA CCAATGTTAA
TGCAATATCT CTGCTGCTAT CACGAAGATA ATTGTAGCTC ACGAAAGTAG
GATACATTAT GTAGGTTACA TCACATAGAG GTAATCTAAA GCTCCCAATA
ATAAGATGTG TAATGTTGAT TATGTAGAAA TTTGCCAGGT TATTTAGAAT
AAACAAGAAG AGGAGAAAAA AAGTACAATT TACCTGAACT CTTGAATGTA
TCCTACAAAT AACCTAGACT TCATGGACGT CAGTTGTCAG TTTACTTTTG
TTTTAATGGT ACATCATTTG TCAAATACTT TATTTGGATA AAAACAGTTT
TGCCTAAGGA GTAAACAGAT CCGGAGTAAG AAAGCAGACG ATTAAAGCAA
TTTTTAAAAA AGGAGAGAGA AATTAATGAG CACACACATA TACTAGTGAA
ATTAGGGTAC TAATTTACTA ATAATTGCAC CGAGACAAAC TTATATTTTA
GTTCCAAAAT GTCAGTCTAA CCCTGCACGT TGTAATAAAT TTTTAACTCT
ATTATATTAT ATCGAGTTGC GCCCTCCACT CCTCGGTGTC CAAATTGTAT
TTAAATGCAT AGATGTTTAA
本发明PMT启动子的另外的实例包括如以上SEQ ID NO:1给出的序列的其中缺失3’区和5’区的缺失突变体。实例包括以下的SEQ IDNO:10-11:
SEQ ID NO:10:
AATCAATTCA ACCCCCAAAA CATAATACAA CCAATGTTAA
TGCAATATCT CTGCTGCTAT CACGAAGATA ATTGTAGCTC ACGAAAGTAG
GATACATTAT GTAGGTTACA TCACATAGAG GTAATCTAAA GCTCCCAATA
ATAAGATGTG TAATGTTGAT TATGTAGAAA TTTGCCAGGT TATTTAGAAT
AAACAAGAAG AGGAGAAAAA AAGTACAATT TACCTGAACT CTTGAATGTA
TCCTACAAAT AACCTAGACT TCATGGACGT CAGTTGTCAG TTTACTTTTG
TTTTAATGGT ACATCATTTG TCAAATACTT TATTTGGATA AAAACAGTTT
TGCCTAAGGA GTAAACAGAT CCGGAGTAAG AAAGCAGACG ATTAAAGCAA
TTTTTAAAAA AGGAGAGAGA AATTAATGAG CACACACATA TACTAGTGAA
ATTAGGGTAC TAATTTACTA ATAATTGCAC CGAGACAAAC TTATATTTTA
GTTCCAAAAT GTCAGTCTAA CCCTGCACGT TGTAATAAAT TTTTAACTCT
ATTATATTAT ATCGAGTTGC GCCCTCCACT CCTCGGTGTC CAAATTGTAT
TTAAATGCAT AGATGTTTAA TGGGAGTGTA CAGCAAGCTT
SEQ ID NO:11:
ACCCCCAAAA CATAATACAA CCAATGTTAA
TGCAATATCT CTGCTGCTAT CACGAAGATA ATTGTAGCTC ACGAAAGTAG
GATACATTAT GTAGGTTACA TCACATAGAG GTAATCTAAA GCTCCCAATA
ATAAGATGTG TAATGTTGAT TATGTAGAAA TTTGCCAGGT TATTTAGAAT
AAACAAGAAG AGGAGAAAAA AAGTACAATT TACCTGAACT CTTGAATGTA
TCCTACAAAT AACCTAGACT TCATGGACGT CAGTTGTCAG TTTACTTTTG
TTTTAATGGT ACATCATTTG TCAAATACTT TATTTGGATA AAAACAGTTT
TGCCTAAGGA GTAAACAGAT CCGGAGTAAG AAAGCAGACG ATTAAAGCAA
TTTTTAAAAA AGGAGAGAGA AATTAATGAG CACACACATA TACTAGTGAA
ATTAGGGTAC TAATTTACTA ATAATTGCAC CGAGACAAAC TTATATTTTA
GTTCCAAAAT GTCAGTGTAA CCCTGCACGT TGTAATAAAT TTTTAACTCT
ATTATATTAT ATCGAGTTGC GCCCTCCACT CCTCGGTGTC CAAATTGTAT
TTAAATGCAT AGATGTTTAA TGGGAGTGTA
可以将所有的前述SEQ ID NO:2-11与如上所述感兴趣的异源核酸或DNA操作上连接,以产生重组核酸,可以将所述重组核酸插入到载体、进而插入到如上所述的植物细胞和转基因植物中,以引起异源核酸或DNA在所述植物中的表达。
实施例4
表达分析
图1显示在叶(第1栏)、茎(第2栏)和根(第3栏)中由所述PMT启动子指导的平均GUS表达水平。我们检查了所有48个独立转化子的数据。转化子之间在GUS水平方面存在相当大的变异。重要的是注意到,平均GUS活性水平(pmol MU/mg蛋白/min)对于根而言为544.82,对于茎而言为13.24(在根中约增加至40倍),而对于叶而言为0.26(在根中约增加至2000倍)。也注意到大多数叶(44/48)和茎(40/48)没有GUS活性。
上文说明了本发明,但不应解释为限制本发明。本发明仅受以下权利要求书的限定,并且本发明包括权利要求书的等同方案。
序列表
<110>Conkling,Mark A.
Li,Yan
<120>腐胺N-甲基转移酶启动子
<130>5051.530CN
<150>PCT/US01/47371
<151>2001-11-07
<150>US60/246488
<151>2000-11-07
<160>11
<170>PatentIn version 3.2
<210>1
<211>742
<212>DNA
<213>NtPMT启动子
<400>1
gtataccaaa aatcaattca acccccaaaa cataatacaa ccaatgttaa tgcaatatct 60
ctgctgctat cacgaagata attgtagctc acgaaagtag gatacattat gtaggttaca 120
tcacatagag gtaatctaaa gctcccaata ataagatgtg taatgttgat tatgtagaaa 180
tttgccaggt tatttagaat aaacaagaag aggagaaaaa aagtacaatt tacctgaact 240
cttgaatgta tcctacaaat aacctagact tcatggacgt cagttgtcag tttacttttg 300
ttttaatggt acatcatttg tcaaatactt tatttggata aaaacagttt tgcctaagga 360
gtaaacagat ccggagtaag aaagcagacg attaaagcaa tttttaaaaa aggagagaga 420
aattaatgag cacacacata tactagtgaa attagggtac taatttacta ataattgcac 480
cgagacaaac ttatatttta gttccaaaat gtcagtctaa ccctgcacgt tgtaataaat 540
ttttaactct attatattat atcgagttgc gccctccact cctcggtgtc caaattgtat 600
ttaaatgcat agatgtttaa tgggagtgta cagcaagctt tcggaaaata caaaccataa 660
tactttctct tcttcaattt gtttagttta attttgaaaa tggaagtcat atctaccaac 720
acaaatggct ctaccatctt ca 742
<210>2
<211>732
<212>DNA
<213>PMT启动子的缺失突变体
<400>2
aatcaattca acccccaaaa cataatacaa ccaatgttaa tgcaatatct ctgctgctat 60
cacgaagata attgtagctc acgaaagtag gatacattat gtaggttaca tcacatagag 120
gtaatctaaa gctcccaata ataagatgtg taatgttgat tatgtagaaa tttgccaggt 180
tatttagaat aaacaagaag aggagaaaaa aagtacaatt tacctgaact cttgaatgta 240
tcctacaaat aacctagact tcatggacgt cagttgtcag tttacttttg ttttaatggt 300
acatcatttg tcaaatactt tatttggata aaaacagttt tgcctaagga gtaaacagat 360
ccggagtaag aaagcagacg attaaagcaa tttttaaaaa aggagagaga aattaatgag 420
cacacacata tactagtgaa attagggtac taatttacta ataattgcac cgagacaaac 480
ttatatttta gttccaaaat gtcagtctaa ccctgcacgt tgtaataaat ttttaactct 540
attatattat atcgagttgc gccctccact cctcggtgtc caaattgtat ttaaatgcat 600
agatgtttaa tgggagtgta cagcaagctt tcggaaaata caaaccataa tactttctct 660
tcttcaattt gtttagttta attttgaaaa tggaagtcat atctaccaac acaaatggct 720
ctaccatctt ca 732
<210>3
<211>722
<212>DNA
<213>PMT启动子的缺失突变体
<400>3
acccccaaaa cataatacaa ccaatgttaa tgcaatatct ctgctgctat cacgaagata 60
attgtagctc acgaaagtag gatacattat gtaggttaca tcacatagag gtaatctaaa 120
gctcccaata ataagatgtg taatgttgat tatgtagaaa tttgccaggt tatttagaat 180
aaacaagaag aggagaaaaa aagtacaatt tacctgaact cttgaatgta tcctacaaat 240
aacctagact tcatggacgt cagttgtcag tttacttttg ttttaatggt acatcatttg 300
tcaaatactt tatttggata aaaacagttt tgcctaagga gtaaacagat ccggagtaag 360
aaagcagacg attaaagcaa tttttaaaaa aggagagaga aattaatgag cacacacata 420
tactagtgaa attagggtac taatttacta ataattgcac cgagacaaac ttatatttta 480
gttccaaaat gtcagtctaa ccctgcacgt tgtaataaat ttttaactct attatattat 540
atcgagttgc gccctccact cctcggtgtc caaattgtat ttaaatgcat agatgtttaa 600
tgggagtgta cagcaagctt tcggaaaata caaaccataa tactttctct tcttcaattt 660
gtttagttta attttgaaaa tggaagtcat atctaccaac acaaatggct ctaccatctt 720
ca 722
<210>4
<211>712
<212>DNA
<213>PMT启动子的缺失突变体
<400>4
cataatacaa ccaatgttaa tgcaatatct ctgctgctat cacgaagata attgtagctc 60
acgaaagtag gatacattat gtaggttaca tcacatagag gtaatctaaa gctcccaata 120
ataagatgtg taatgttgat tatgtagaaa tttgccaggt tatttagaat aaacaagaag 180
aggagaaaaa aagtacaatt tacctgaact cttgaatgta tcctacaaat aacctagact 240
tcatggacgt cagttgtcag tttacttttg ttttaatggt acatcatttg tcaaatactt 300
tatttggata aaaacagttt tgcctaagga gtaaacagat ccggagtaag aaagcagacg 360
attaaagcaa tttttaaaaa aggagagaga aattaatgag cacacacata tactagtgaa 420
attagggtac taatttacta ataattgcac cgagacaaac ttatatttta gttccaaaat 480
gtcagtctaa ccctgcacgt tgtaataaat ttttaactct attatattat atcgagttgc 540
gccctccact cctcggtgtc caaattgtat ttaaatgcat agatgtttaa tgggagtgta 600
cagcaagctt tcggaaaata caaaccataa tactttctct tcttcaattt gtttagttta 660
attttgaaaa tggaagtcat atctaccaac acaaatggct ctaccatctt ca 712
<210>5
<211>702
<212>DNA
<213>PMT启动子的缺失突变体
<400>5
ccaatgttaa tgcaatatct ctgctgctat cacgaagata attgtagctc acgaaagtag 60
gatacattat gtaggttaca tcacatagag gtaatctaaa gctcccaata ataagatgtg 120
taatgttgat tatgtagaaa tttgccaggt tatttagaat aaacaagaag aggagaaaaa 180
aagtacaatt tacctgaact cttgaatgta tcctacaaat aacctagact tcatggacgt 240
cagttgtcag tttacttttg ttttaatggt acatcatttg tcaaatactt tatttggata 300
aaaacagttt tgcctaagga gtaaacagat ccggagtaag aaagcagacg attaaagcaa 360
tttttaaaaa aggagagaga aattaatgag cacacacata tactagtgaa attagggtac 420
taatttacta ataattgcac cgagacaaac ttatatttta gttccaaaat gtcagtctaa 480
ccctgcacgt tgtaataaat ttttaactct attatattat atcgagttgc gccctccact 540
cctcggtgtc caaattgtat ttaaatgcat agatgtttaa tgggagtgta cagcaagctt 600
tcggaaaata caaaccataa tactttctct tcttcaattt gtttagttta attttgaaaa 660
tggaagtcat atctaccaac acaaatggct ctaccatctt ca 702
<210>6
<211>692
<212>DNA
<213>PMT启动子的缺失突变体
<400>6
tgcaatatct ctgctgctat cacgaagata attgtagctc acgaaagtag gatacattat 60
gtaggttaca tcacatagag gtaatctaaa gctcccaata ataagatgtg taatgttgat 120
tatgtagaaa tttgccaggt tatttagaat aaacaagaag aggagaaaaa aagtacaatt 180
tacctgaact cttgaatgta tcctacaaat aacctagact tcatggacgt cagttgtcag 240
tttacttttg ttttaatggt acatcatttg tcaaatactt tatttggata aaaacagttt 300
tgcctaagga gtaaacagat ccggagtaag aaagcagacg attaaagcaa tttttaaaaa 360
aggagagaga aattaatgag cacacacata tactagtgaa attagggtac taatttacta 420
ataattgcac cgagacaaac ttatatttta gttccaaaat gtcagtctaa ccctgcacgt 480
tgtaataaat ttttaactct attatattat atcgagttgc gccctccact cctcggtgtc 540
caaattgtat ttaaatgcat agatgtttaa tgggagtgta cagcaagctt tcggaaaata 600
caaaccataa tactttctct tcttcaattt gtttagttta attttgaaaa tggaagtcat 660
atctaccaac acaaatggct ctaccatctt ca 692
<210>7
<211>642
<212>DNA
<213>PMT启动子的缺失突变体
<400>7
gtataccaaa aatcaattca acccccaaaa cataatacaa ccaatgttaa tgcaatatct 60
ctgctgctat cacgaagata attgtagctc acgaaagtag gatacattat gtaggttaca 120
tcacatagag gtaatctaaa gctcccaata ataagatgtg taatgttgat tatgtagaaa 180
tttgccaggt tatttagaat aaacaagaag aggagaaaaa aagtacaatt tacctgaact 240
cttgaatgta tcctacaaat aacctagact tcatggacgt cagttgtcag tttacttttg 300
ttttaatggt acatcatttg tcaaatactt tatttggata aaaacagttt tgcctaagga 360
gtaaacagat ccggagtaag aaagcagacg attaaagcaa tttttaaaaa aggagagaga 420
aattaatgag cacacacata tactagtgaa attagggtac taatttacta ataattgcac 480
cgagacaaac ttatatttta gttccaaaat gtcagtctaa ccctgcacgt tgtaataaat 540
ttttaactct attatattat atcgagttgc gccctccact cctcggtgtc caaattgtat 600
ttaaatgcat agatgtttaa tgggagtgta cagcaagctt tc 642
<210>8
<211>630
<212>DNA
<213>PMT启动子的缺失突变体
<400>8
gtataccaaa aatcaattca acccccaaaa cataatacaa ccaatgttaa tgcaatatct 60
ctgctgctat cacgaagata attgtagctc acgaaagtag gatacattat gtaggttaca 120
tcacatagag gtaatctaaa gctcccaata ataagatgtg taatgttgat tatgtagaaa 180
tttgccaggt tatttagaat aaacaagaag aggagaaaaa aagtacaatt tacctgaact 240
cttgaatgta tcctacaaat aacctagact tcatggacgt cagttgtcag tttacttttg 300
ttttaatggt acatcatttg tcaaatactt tatttggata aaaacagttt tgcctaagga 360
gtaaacagat ccggagtaag aaagcagacg attaaagcaa tttttaaaaa aggagagaga 420
aattaatgag cacacacata tactagtgaa attagggtac taatttacta ataattgcac 480
cgagacaaac ttatatttta gttccaaaat gtcagtctaa ccctgcacgt tgtaataaat 540
ttttaactct attatattat atcgagttgc gccctccact cctcggtgtc caaattgtat 600
ttaaatgcat agatgtttaa tgggagtgta 630
<210>9
<211>620
<212>DNA
<213>PMT启动子的缺失突变体
<400>9
gtataccaaa aatcaattca acccccaaaa cataatacaa ccaatgttaa tgcaatatct 60
ctgctgctat cacgaagata attgtagctc acgaaagtag gatacattat gtaggttaca 120
tcacatagag gtaatctaaa gctcccaata ataagatgtg taatgttgat tatgtagaaa 180
tttgccaggt tatttagaat aaacaagaag aggagaaaaa aagtacaatt tacctgaact 240
cttgaatgta tcctacaaat aacctagact tcatggacgt cagttgtcag tttacttttg 300
ttttaatggt acatcatttg tcaaatactt tatttggata aaaacagttt tgcctaagga 360
gtaaacagat ccggagtaag aaagcagacg attaaagcaa tttttaaaaa aggagagaga 420
aattaatgag cacacacata tactagtgaa attagggtac taatttacta ataattgcac 480
cgagacaaac ttatatttta gttccaaaat gtcagtctaa ccctgcacgt tgtaataaat 540
ttttaactct attatattat atcgagttgc gccctccact cctcggtgtc caaattgtat 600
ttaaatgcat agatgtttaa 620
<210>10
<211>630
<212>DNA
<213>PMT启动子的缺失突变体
<400>10
aatcaattca acccccaaaa cataatacaa ccaatgttaa tgcaatatct ctgctgctat 60
cacgaagata attgtagctc acgaaagtag gatacattat gtaggttaca tcacatagag 120
gtaatctaaa gctcccaata ataagatgtg taatgttgat tatgtagaaa tttgccaggt 180
tatttagaat aaacaagaag aggagaaaaa aagtacaatt tacctgaact cttgaatgta 240
tcctacaaat aacctagact tcatggacgt cagttgtcag tttacttttg ttttaatggt 300
acatcatttg tcaaatactt tatttggata aaaacagttt tgcctaagga gtaaacagat 360
ccggagtaag aaagcagacg attaaagcaa tttttaaaaa aggagagaga aattaatgag 420
cacacacata tactagtgaa attagggtac taatttacta ataattgcac cgagacaaac 480
ttatatttta gttccaaaat gtcagtctaa ccctgcacgt tgtaataaat ttttaactct 540
attatattat atcgagttgc gccctccact cctcggtgtc caaattgtat ttaaatgcat 600
agatgtttaa tgggagtgta cagcaagctt 630
<210>11
<211>610
<212>DNA
<213>PMT启动子的缺失突变体
<400>11
acccccaaaa cataatacaa ccaatgttaa tgcaatatct ctgctgctat cacgaagata 60
attgtagctc acgaaagtag gatacattat gtaggttaca tcacatagag gtaatctaaa 120
gctcccaata ataagatgtg taatgttgat tatgtagaaa tttgccaggt tatttagaat 180
aaacaagaag aggagaaaaa aagtacaatt tacctgaact cttgaatgta tcctacaaat 240
aacctagact tcatggacgt cagttgtcag tttacttttg ttttaatggt acatcatttg 300
tcaaatactt tatttggata aaaacagttt tgcctaagga gtaaacagat ccggagtaag 360
aaagcagacg attaaagcaa tttttaaaaa aggagagaga aattaatgag cacacacata 420
tactagtgaa attagggtac taatttacta ataattgcac cgagacaaac ttatatttta 480
gttccaaaat gtcagtctaa ccctgcacgt tgtaataaat ttttaactct attatattat 540
atcgagttgc gccctccact cctcggtgtc caaattgtat ttaaatgcat agatgtttaa 600
tgggagtgta 610
Claims (24)
1.一种分离的DNA分子,所述DNA分子指导下游异源DNA区段在植物细胞中的根特异性转录,所述分离的DNA分子具有选自以下的一种序列:
(a)SEQ ID NO:1、SEQ ID NO:2、SEQID NO:3、SEQID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ IDNO:9、SEQ ID NO:10和SEQ ID NO:11,和
(b)与具有一种上述(a)序列的分离DNA或具有上述(a)序列的分离DNA的互补序列在洗涤严格性为0.3M NaCl、0.03M柠檬酸钠、0.1%SDS于60℃代表的条件下杂交并且指导下游异源DNA区段在植物细胞中的根特异性转录的DNA序列,其中所述DNA序列与具有一种上述(a)序列的DNA或其互补序列至少有75%同源性。
2.一种DNA构建体,所述DNA构建体以5′→3′方向包含根特异性启动子和位于所述启动子下游并且与其操作上连接的异源DNA区段,其中所述根特异性启动子具有选自以下的一种序列:
(a)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、SEQ ID NO:8、SEQID NO:9、SEQ ID NO:10和SEQ ID NO:11,和
(b)与具有一种上述(a)序列的分离DNA的互补序列在洗涤严格性为0.3M NaCl、0.03M柠檬酸钠、0.1%SDS于60℃代表的条件下杂交并且指导下游异源DNA区段在植物细胞中的根特异性转录的DNA序列,其中所述DNA序列与具有一种上述(a)序列的DNA或其互补序列至少有75%同源性。
3.一种权利要求2的DNA构建体,其中所述构建体是质粒。
4.一种权利要求2的DNA构建体,其中所述异源DNA区段是一种编码杀虫蛋白的基因。
5.一种权利要求3的DNA构建体,其中所述异源DNA区段是一种编码对昆虫有毒的苏云金芽孢杆菌(Bacillus thuringiensis)晶体蛋白的基因。
6.一种植物细胞,所述植物细胞已用一种按照权利要求2的DNA构建体转化。
7.一种产生转化植物的方法,所述方法包括从按照权利要求6的植物细胞再生植株。
8.一种细胞,所述细胞含有按照权利要求2的DNA构建体,并且其中所述DNA构建体还包含Ti质粒。
9.一种产生转化植物的方法,所述方法包括用按照权利要求8的构建体感染植物细胞,以产生转化植物细胞,然后从所述转化植物细胞再生植株。
10.一种携带权利要求2的DNA构建体的微粒,其中所述微粒适合于植物细胞的基因枪转化。
11.一种产生转化植物的方法,所述方法包括将按照权利要求10的微粒推进植物细胞中,以产生转化植物细胞,然后从所述转化植物细胞再生植株。
12.一种植物细胞原生质体,所述植物细胞原生质体包含一种按照权利要求2的DNA构建体。
13.一种产生转化植物的方法,所述方法包括从按照权利要求12的植物细胞原生质体再生植株。
14.一种转化植物细胞,所述转化植物细胞包含一种异源DNA构建体,所述构建体以5′→3′方向包含烟草PMT启动子和位于所述启动子下游且与其操作上连接的异源DNA区段,所述启动子指导所述异源DNA区段的根特异性转录。
15.一种权利要求14的转化植物细胞,其中所述启动子具有选自以下的一种序列:
(a)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、SEQ ID NO:8、SEQ IDNO:9、SEQ ID NO:10和SEQ ID NO:11,和
(b)与具有一种上述(a)的序列的分离DNA的互补序列在洗涤严格性为0.3M NaCl、0.03M柠檬酸钠、0.1%SDS于60℃代表的条件下杂交并且指导下游异源DNA区段在植物细胞中的特异性转录的DNA序列,其中所述DNA序列与具有一种上述(a)序列的DNA或其互补序列至少有75%同源性。
16.一种权利要求14的转化植物细胞,其中所述植物细胞是双子叶植物细胞。
17.一种权利要求14的转化植物细胞,其中所述植物细胞是单子叶植物细胞。
18.一种权利要求14的转化植物细胞,其中所述植物细胞是烟草植物细胞。
19.一种分离的DNA分子,所述DNA分子由指导下游异源DNA区段在植物细胞中的根特异性转录并且具有选自SEQ ID NO:1-11的一种序列的启动子组成。
20.一种包含表达盒的DNA构建体,所述构建体以5′→3′方向包含按照权利要求19的启动子和位于所述启动子下游并且与其操作上连接的异源DNA区段。
21.一种转化植物细胞,所述转化植物细胞含有一种按照权利要求20的DNA构建体。
22.一种分离的DNA分子,所述DNA分子包含SEQ ID NO:1核苷酸序列中的至少100个连续核苷酸。
23.一种分离的DNA分子,所述DNA分子包含SEQ ID NO:1核苷酸序列中的至少150个连续核苷酸。
24.一种分离的DNA分子,所述DNA分子包含SEQ ID NO:1核苷酸序列中的至少200个连续核苷酸。
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-
2001
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- 2001-11-07 DE DE60128149T patent/DE60128149D1/de not_active Expired - Lifetime
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ATE360701T1 (de) | 2007-05-15 |
CA2427825A1 (en) | 2002-05-16 |
EP1368467A2 (en) | 2003-12-10 |
WO2002038588A3 (en) | 2003-10-16 |
US7189570B2 (en) | 2007-03-13 |
AU2002230691A1 (en) | 2002-05-21 |
US20040031074A1 (en) | 2004-02-12 |
CN1484696A (zh) | 2004-03-24 |
EP1368467A4 (en) | 2005-09-07 |
JP2004533807A (ja) | 2004-11-11 |
DE60128149D1 (en) | 2007-06-06 |
WO2002038588A2 (en) | 2002-05-16 |
EP1368467B1 (en) | 2007-04-25 |
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