CN1152250A - 使用子房组织转录因子改良棉花 - Google Patents
使用子房组织转录因子改良棉花 Download PDFInfo
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Abstract
提供了新的方法,由此,在子房组织尤其在极早期果实发育中优选指导基因表达的编码序列可被用来表达棉花胚珠组织中植物生长修饰激素,该方法可带来棉铃及棉花植株性状的改进,并为改变纤维的尺度和强度等纤维品质提供了一种机制。
Description
技术领域
本发明涉及使用体外构建的DNA转录或表达盒指导棉花中所感兴趣的DNA序列的子房组织转录,产生表型改变的子房来源的细胞的方法。本发明已用子房组织启动子改变棉花棉铃产量的表型以及改变棉花纤维的质量等方法作了示范。本发明包括由此方法得到的棉花植株和棉花纤维。
背景
通过基因工程技术操纵控制棉花纤维质量的各种特性,可快速得到改良的棉花变种。棉花纤维质量习惯上根据强度,长度和细度(纤维精细程度的测量)来测定。
基因工程技术一般用以改变单个原核及真核细胞的表型,尤其是培养细胞的表型。植物细胞已证明比其它真核细胞较难改变,不仅因为缺乏合适的载体系统而且因为所涉及的目的不同。对许多应用而言,需要能够控制植株生长特定阶段或者植株特定部位的基因表达。为此,需要能在植物发育合适时间和/或合适细胞类型中提供所需转录起始并且对植物发育及产量无严重危害的调节序列。因此,分离在宿主植物生长周期中能提供植物细胞所需的转录调节作用的序列成为研究兴趣所在。
研究兴趣的其中一方面在于改变特定细胞类型如来源于子房组织的分化的上皮细胞的表型,以便得到成熟细胞类型的改变的或改良的特性的能力。为了达到所需的表型改变,使用了能在早期子房发育中起始转录的转录起始区。这些转录起始区在受粉开始前是活跃的,在果实胀大、组织成熟或其它类似事件发生前较少活跃或不活跃。
相关文献
调节西红柿果实中细胞激动素表达的方法及其组合物在美国专利号5,177,307中已有描述。美国专利号5,175,095描述了子房组织转录启动子,包括在胚珠珠被细胞中有活性的pZ7启动子。两项专利在此引为参考文献。但任一专利均未描述修饰棉花纤维质量特性的方法。
一类果实特异性的启动子可在果实发育过程中表达于花开期,表达至少延续到成熟开始。这类启动子在欧洲专利申请88.906296.4中已有讨论,在此引为参考文献。已分离了在棉花纤维中优先表达的cDNA克隆。分离的克隆之一对应于在晚期初级细胞壁和早期次级细胞壁合成阶段表达水平最高的mRNA和蛋白质(JohnCrow,Pro.Natl.Acad.Sci.(1992)89:5769-5773)。已对来自西红柿、在果实发育中显示差异表达的cDNA克隆作了分离和鉴定(Mansson et al.,Mol.Gen.Genet.(1985)200:356-361;Slater et al.,Plant Mol.Biol.(1985)5:137-147),这些研究主要侧重于果实成熟期中累积的mRNA。一种由成熟期特异性cDNA编码的蛋白质已被鉴定为多聚半乳糖醛酸酶(Slater et al.,Plant Mol.Biol.(1985)5:137-147)。一种编码西红柿多聚半乳糖醛酸酶的cDNA克隆已被测序(Grierson et al.,Nucleic Acids Research(1986)14:8395-8603)。西红柿果实贮存和通过转录调节多聚半乳糖醛酸酶基因的表达等多方面的进展已有报道(Sheehy et al.,Proc.Natl.Acad.Sci.(1988)85:8805-8809;Smithet al.,Nature(1988)334:724-726)。
编码psbA(光系统II的成分之一)的成熟质体mRNA在果实发育晚期达到最高水平,然而成熟开始之后,编码光系统I和II其它成分的质体mRNA在色质体中降至不能检测的水平(Piechulla et al.,Plant Mol.Biol.(1986)7:367-376)。最近,代表了与西红柿花粉(Mc Cormick et al.,Tomato Biotechnology(1987)Alan R.Liss,Inc.,NY)、雌蕊(Gasser et al.,Plant Cell(1989),1:15-20)相互作用明显相关基因的cDNA克隆已被分离和鉴定。
其他研究侧重于诱导性调节的基因,如编码丝氨酸蛋白酶抑制物的基因,它们在响应西红柿所受伤害的过程中表达。(Graham et al.,J.Biol.Chem.(1985)260:6555-6560;Graham et al.,J.Biol.Chem.(1985)260:6561-6554)。也有些研究侧重于与伤后成熟果实和叶中乙烯合成相关的mRNA.(Smithet al.,Planta(1986)168:94-100)。金属羧肽酶抑制剂蛋白的累积已在受伤土豆叶中有报道(Graham et al.,Biochem&Biophys.RRes.Comm(1981)228:281-286)。
在植物种子组织如胚胎或种皮中优先表达的基因也已有报道(参见欧洲专利申请87306739.1(于1988年2月3日公开,公开号0255378)和Kridl et al.,Seed Science R esearch(1991)1:209-219。
土壤杆菌介导的棉花转化已在Umbeck的美国专利号5,004,863和5,159,135中有述。棉花的粒子轰击转化在1992年9月17日公开的WO92/15675中已有报道。Brassica的转化已由Radke等人描述(Theor.Appl.Genet.(1988)75:685-694;Plant Cell Reports(1992)11:499-505)。
栽培西红柿的转化已由McCormick et al.,Plant Cell Research(1986)5:81-89和Fillatti et al.,Bio/Technology(1987)5:726-730)描述。
本发明概要
本发明一般性地包括一些DNA构建物的使用,这些构建物具有转录和翻译起始区域,它可在胚珠珠被细胞中起作用以在改变棉花植株和/或棉花纤维细胞的表型的方法中表达编码蛋白质的DNA序列,而所述蛋白质对于植物生长物质的产生有活性。植物生长物质一般指能引发植物生长、发育或代谢反应的化合物。这种物质不是代谢物,因为它们不是所控制途径的中间物或产物,而且它们在很低的浓度下具有活性。一些对促进生长或发育有活性,另一些却更多地起到同一过程的抑制物的作用。照此,植物生长物质包括植物生长素、赤霉素、细胞激动素、乙烯和脱落酸这类物质,也常指植物激素。
对于植物生长物质的产生有活性的蛋白质包括参与乙烯生物合成途径的酶。许多这种酶已被描述,包括ACC合成酶、乙烯形成酶(也称为pTOM13),SAM合成酶,ACC脱氨酶,SAM脱羧酶。
方法一般包括栽培转基因棉花以产生成熟的胚珠组织,其中成熟的胚珠组织细胞在其基因组中包括这种DNA构建物。这种构建物也具有转录终止区域作为其额外成分。至少一种成分对所说的至少一种另外的成分而言是外源性的,也即这些构建物成分并不天然地作为一个整体在一起出现。在此种情况下植物在成熟植物胚珠组织中表达对产生植物生长物质有活性的蛋白质。
目前已发现从这种构建物中表达对植物生长物质的产生有活性的蛋白质能被用来改变转基因棉花的棉铃生产效率。举例说明了在胚珠珠被细胞中表达细胞激动素可增加棉铃产量。
目前也已发现,转基因棉花的纤维质量也能用这种方法修饰。特别是棉花纤维的尺度特性如纤维长度,强度,细度的修饰已从pZ7转录和翻译起始区域表达细胞激动素作了示例。
附图的描述
图1表示cDNA克隆pZ130的DNA序列。相应于pZ7cDNA克隆的序列用下划线标明。
图2表示与pZ130cDNA克隆重叠的Calgene Lambda 140基因组克隆区(此区域已用下划线标明)的序列以及5′和3′区域到此区域的部分序列。pZ130基因转录本的起始点用2567位的有下划线的粗体“A”标明。基因序列中的内含子用2702位到2921位的小写序列标明。普通限制性酶位点也已标明。
序列中的符号有如下含义:
A=腺嘌呤核苷;C=胞嘧啶;G=鸟嘌呤;T=胸腺嘧啶或尿嘧啶;R=A或G;Y=C或T或U;M=C或A;K=T或U或G;W=T或U或A;S=C或G;N=C,T,A,G,U任选;B=非~D=非C;H=非G;V=非T非U。
图3表示Calgene Lambda 140的限制性内切酶图谱。B:BamH I;G:Bgl II;H:Hind III;R:EcoR I;S:Sal I。
图4表示cDNA克隆pZ70的完全DNA序列。相对于pZ8的cDNA克隆的序列已用下划线标明。pZ70基因编码的成熟蛋白质cDNA的起始与终止也已标明。
图5表示Calgene Lambda 116的限制性酶切图谱。B:BamH I;G:Bgl II;H:HindIII;R:EcoR I;S:Sal I;X:Xba I。
图6表示Northern印迹实验的结果,显示pZ7和pZ8 RNA积累的发展时序。UC82B果实发育(花和子房/果实)阶段描绘于上。数字1至21代表花开后的天数。
图7表示用于植物转化以表达黑色素合成的基因的二价载体。
图8表示接头区域位点图谱。
图9表示Northern印迹实验结果,显示pZ7和pZ8 RNA的棉花胚珠珠被细胞中累积的发展时序。“A”指开花期RNA,数字7,21和28代表花开后的天数。
本发明详述
根据本发明,提供了一种影响来自转基因棉花植株的棉花纤维质量的方法,同时也提供了一种能改变转基因棉花棉铃生产效率的方法。这些方法中使用的构建物依据其用途可包括几种形式。因此,构建物包括载体,转录盒,表达盒和质粒。转录和翻译起始区(有时也称为“启动子”)优选包括5′未翻译序列的转录起始调节区和翻译起始调节区,后者即“核糖体结合位点”-它负责将mRNA结合到核糖体和翻译起始。优选的是起始控制区的所有转录和翻译功能性元件来自同一基因。在某些实施方案中,启动子用加入序列如增强子或删除不必要的和/或不需要的序列来加以修饰。具有与天然启动子充分类似的DNA序列以为所感兴趣的DNA序列提供所需的转录特异性的启动子是我们想得到的。它包括天然的和合成的序列,以及合成与天然序列的结合。
典型的载体包括一个或多个核苷酸和一个与子房组织基因表达相关的转录起始调节区的核苷酸序列。一个用来在子房组织转录所感兴趣核苷酸序列的转录盒应在其转录的方向上包括,子房组织转录起始区和任选的翻译起始区,所感兴趣的DNA序列,在植物细胞中起作用的转录终止区和任选的翻译终止区。当上述盒能为感兴趣的DNA序列提供转录和翻译时,即被认为是表达盒。同时也会存在一个或多个内含子。
也存在其他序列,包括编码所需的转运肽的序列和分泌前导肽的序列。调节区能指导子房细胞花开期的转录,但在受粉和/或受精前后发生的初始变化之后,很少或不能指导表达;来自这些调节区的转录在花开期后大约三周即检测不到。而且,本发明的子房组织转录起始区通常不易于在其他植物组织中检测到。来自子房组织而又不是子房特异性的转录起始区有其特殊用途。尤其优选那些在果实发育各阶段不出现而只在花开期出现的转录起始区。能在其他植物组织和/或在子房发育其他阶段起始转录的转录起始区,除前述以外,还可以是这样的区域,它们能在处于感兴趣的特定时期的子房组织中提供明显的表达水平而且不会对整个植株有负面的影响,尤其不影响果实和/或果实相关部分的发育。子房组织启动子和/或能指导外果皮组织,内核心组织,表皮及类似物等特定子房组织转录的启动子也成为研究兴趣所在。
优选那些能在本发明感兴趣的子房组织时期-一般为植物繁殖周期的开花期以最大水平或接近最大水平表达的转录起始区。花开期前至少1到3天到花衰老这段时期尤其为研究兴趣点。转录水平应足以提供能产生修饰之果实的RNA量。此外“果实”指花的子房壁和其它任何紧密相关部分发育得到的成熟器官。参看Weirer,T.E.,1,ed.,Botany An Introduction to Plant Biology(6thed.)(John Wiley&Sons,1982);Tootill&Backmore,The Facts onFile Dictionary of Botany(Market Home Books Ltd.1984)。“经修饰的果实”意为相对同种未转化的植物有明显不同表型的果实。如那种在其基因组中没有讨论中的转录盒的果实。
特别感兴趣的是与子房组织基因表达相关的转录起始区,它们能在花开期前至少24小时至花凋亡这段时期指导转录。此处的“花开期”指与花张开和盛开相关的时期,“花凋亡”指与花死亡包括花瓣脱落相关的时期。例见AberCrombie,M.,et al.,A Dic tionary of Biology(6th ed)(Penguin Books,1973)。未开的花,或花芽被认为是“花前期”。花开期始于花瓣的张开,这代表植物繁殖周期的性接受部分。在受试的西红柿变种UCB82中开花通常持续大约一周。在如棉花的植物中,花开持续约2周,纤维从种皮组织发育而来。优选本发明的转录起始区在植物花出芽前不会长时间或明显起始转录。转录维持高水平达至少约1到3天并贯穿花开期前后是较理想的。
进一步的要求是,本发明的转录起始区在花开期开始后1-3天内显示出转录活性水平的降低,所述转录活性水平长时间内不会升高,优选为降低。西红柿胚囊的受精以产生形成胚胎植物的合子通常发生于花开后2-3天。这与本发明的转录起始区活性降低同时发生。这样就需要本发明的启动子转录活性在花开期开始后大约2天内明显降低。本发明的转录起始区将能指导子房组织在上述所优选时期内以高水平表达。
在一些实施方案中,需要在特定子房组织中选择性地调节转录。转录起始区与能在特定子房组织,如子房珠被(有时也称为“胚珠表皮细胞”)、核心或果皮组织等中起始翻译、表达的5′未翻译序列相联时,即能在特定组织中指导由感兴趣的DNA序列编码的所需的信息,以便更有效地影响所需的表型修饰。
至少能在子房外果皮组织中表达的转录起始区尤其是研究兴趣点。棉花中,子房结构类似物是棉铃的刺果。调节子房珠被和/或核心组织中的表达已产生有用的棉铃和棉花纤维修饰。棉花纤维是胚珠外珠被的分化的上皮单细胞,有四个不同的生长阶段即起始、延长(初级细胞壁合成)、次级细胞壁合成和成熟。纤维发育的起始似乎由激素引发。初级细胞壁在延长阶段初步形成,延续至花开期后25天(DPA)。次级细胞壁合成在延长阶段停止前开始,一直持续到大约40DPA,形成几乎为纯纤维素的壁。除子房组织启动子外,来自种子组织,特别是种皮组织所优先表达之基因的转录起始区,也被考虑用于修饰棉花纤维细胞。
在Brassica种皮细胞中高水平表达的一个基因例子是欧洲专利申请0255378描述的EA9基因。EA9基因cDNA的部分核酸序列在该申请中已提供,能用来取得相应序列。包括启动子区域。另一个在种胚和种皮细胞中表达的种子基因是Bce4Brassica基因。来自此基因的启动子区域在本发明中也具用途。此基因及相应的启动子区域在WO91/13980中有述,该文献公开于1991年9月19日。纤维特异性蛋白质受发育调节,这样,来自在纤维细胞中表达的蛋白质基因的转录起始区域也是研究兴趣所在。一个受发育调节的纤维细胞蛋白质的例子是E6(John andCrow.Proc.Natl.Acad.Sci.(1992)89:5769-5773)。尽管E6基因在叶,胚珠及花中仅有低水平转录,但在纤维中最为活跃。
为了取得特异衍生的转录起始区,可使用下列步骤。从所需发育阶段的组织分离信使RNA(mRNA),然后将该mRNA用来构建cDNA克隆,根据克隆的初级DNA序列和不同克隆在种群中的丰度,这种克隆对应于mRNA种群。mRNA也可从目的基因不应该表达的不同发育阶段的组织(互生组织)中分离。来自所需组织及互生组织的放射活性的cDNA可用来筛选cDNA克隆的复制拷贝。初步筛选可将cDNA克隆鉴定为对应于在两种组织均丰富的mRNA的cDNA克隆,也可鉴定为对应于在两种组织均不丰富的mRNA的cDNA克隆和那些在一种组织丰富而在另一组织相对不丰富的mRNA的cDNA克隆。随后相应于仅在所需组织丰度较高的mRNA的克隆被挑选出来,进一步鉴定。
因为上述的用于初步筛选的杂交探针是来自特定组织的总cDNA,它主要与最丰富的序列杂交。为了确定表达的实际水平、尤其是在mRNA不丰富的组织中的表达水平,使用克隆序列作为所需组织和各种不需要组织的总mRNA群体的杂交探针。这就是能给出特定组织中mRNA相对丰度和mRNA转录本大小之信息的Northern印迹试验最常见的步骤。
知道mRNA的丰度是否由单基因转录引起还是由多基因家族转录产物引起是极重要的,这一点可由基因组与cDNA克隆的Southern印迹反应测定。总基因组DNA用各种限制酶消化,然后与放射活性的cDNA克隆杂交。从杂交的类型和强度,可区分出mRNA由一个或两个或相关基因大家族编码的可能性。但难以决定是哪一个交叉杂交的基因编码了在所需组织中发现的大量表达的mRNA。例如,试验表明pZ130(见实施例4)是一个小的基因家族的成员,但pZ7探针能将pZ130从其余家族成员中区别出来。
按上述所得的cDNA可以被测序以测定开放阅读框架(可能的蛋白质编码区域cDNA)和转录方向,以便所需的目的DNA序列随后能在正确位置以正确方向插入转录盒。cDNA克隆的序列信息也使相应基因组的克隆,包括下文所述的图谱和亚克隆的定性研究变得容易。同时可从基因组文库中筛选含有包括位于转录序列侧翼的控制区的完全基因序列的克隆。基因组克隆一般含有DNA大片段(大约10-20kb),能用限制酶作出图谱,然后亚克隆并部分测序以决定哪一片段含有发育调节的基因。
使用限制酶切图谱和序列信息可以设计和构建质粒,它们有推定的子房基因或连接到基因的其他所需启动子区,所述基因可以在子房和/或其他所需组织尤其是子房来源组织中表达。这些杂合构建物在转化的再生植株中检验了其表达类型,以保证在启动子不再与天然开放阅读框架相连时仍能成功维持所需的计时表达和/或组织表达和/或整体表达水平。使用以上描述的方法已鉴别出几种转录调节区域。一个例子是西红柿来源的转录起始区,它可调节相应于pZ130 cDNA克隆的序列的表达。能与pZ130克隆杂交的序列如探针pZ7尤其在花开期的早期阶段显示出丰富的mRNA。信息在子房珠被和子房外果皮组织中表达,在其他组织或在果实发育其他阶段不表达或至少不易检测出表达。这样,一般认为pZ130转录起始区是子房专一性的,为本发明的目的所在。图1提供了cDNA克隆pZ130的DNA序列。由组成pZ130的结构基因编码的氨基酸序列与已有报道的硫蛋白的氨基酸序列同源(见Qing et al.,Mol.Gen.Genet.(1992)234:89-96)。尽管据报道硫蛋白在植物防御中有功能,但植物子房组织中的硫蛋白功能仍然未知。
在子房组织转录/翻译起始控制区的调节控制下的下游是所感兴趣的核苷酸序列,它提供如果实或纤维等成熟自子房组织的结构表型的修饰。核苷酸序列可以是编码所感兴趣的多肽如酶的任何开放阅读框架,或者是一个与基因组序列互补的序列,而基因组序列可以是一个开放的阅读框架,一个内含子,一个非编码的前导序列,或任何其互补序列可抑制转录,如剪接翻译等mRNA加工的其他序列。本发明的核苷酸序列可以合成,天然取得或者合成与天然取得相结合。依赖于所感兴趣的DNA序列的本质,需要用植物优选的密码子合成序列。植物优选的密码子可由所感兴趣的特定植物种类中大量表达的蛋白质中的最高频率密码子决定。表型修饰可通过调节内源性转录及翻译产物的量及相对分布等而取得,或者通过调节外源性转录或翻译产物,如在转基因宿主细胞或组织中提供一种新的功能或产物而取得。
特别感兴趣的是编码与植物细胞生长和发育的调节相关的表达产物的DNA序列,尤其是编码涉及参与植物果实发育调节的激素代谢的表达产物,如细胞激动素,生长素,乙烯,脱落酸,赤霉酸等的DNA序列。调节细胞激动素表达的方法和组合物和在美国专利号5,177,307中有述,在此被引为参考文献。
也可以使用来自包括其他真核或原核细胞来源的多种基因,如根瘤土壤杆菌T-DNA生长素和细胞激动素生物合成基因产物的基因和哺乳动物的干扰素基因。所感兴趣的乙烯途径中的基因包括编码ACC合成酶的DNA序列(WO92/04456)和pTOM13(WO91/01375)。实际上任何编码参与乙烯生物合成途径的酶的基因均有这种潜在的用途,如SAM合成酶。
另外,也可以引入和表达编码能降解植物细胞中乙烯前体的酶的DNA序列,以提供乙烯抗性细胞。这种酶的例子是ACCD(PCT/US91/02958)和SAM脱羧酶(WO91/09112)。每篇上面提到的文件均引为文献附后。
当需要转录反义序列时,棉花纤维强度、质地或尺度特性的修饰可利用转录盒。需要表达多肽时,可使用提供感兴趣的DNA序列的转录和翻译的表达盒。不同改变都是兴趣点,改变包括特定糖类、激素、酶或其它生物学参数的形成调节(增加或降低)。也包括修饰最终果实或纤维的组分,即改变水、固形物,纤维或糖的比例和/或量。其他感兴趣的用于修饰的表型特征包括对张力,生物体,除草剂,灌木、生长调节剂等的反应。这些结果可以通过提供一个或多个内源产物尤其是酶或辅因子的表达下降来获得,具体方式可以通过产生与天然基因转录产物互补(反义)的转录产物以抑制转录产物的成熟和/或表达,或者通过提供与植物果实发育相关联的内源或外源基因的表达来实现。
表达盒中所使用的终止区十分便利,因其有相对的可交换性。终止区可与转录起始区是同源的,也可与所感兴趣的DNA序列是同源的,也可衍生自另外的来源。终止区可以天然产生,或整体或部分合成。便利的终止区可取自根瘤土壤杆菌的Ti-质粒,如章鱼碱合成酶和胭脂氨酸合成酶的终止区。在一些实施方案中,有时需使用与在特定构建物中所用的子房组织转录起始区同源的3′终止区。
在一些情况下,构建物中包括额外的核苷酸序列以将特定基因产物定向于特定细胞器是有用的。如在使用了编码芳香族色素序列的构建物中,尤其是使用了编码以芳香族化合物如酪氨酸和吲哚为其底物的酶的序列的构建物中优选包含能提供将酶转移至质体功能的序列,如SSU转运肽序列。
例如,为产生黑色素,即需在棉花纤维细胞中提供酪氨酸酶和来自抗生链霉菌(Streptomyces antibioticus)的ORF438基因(Berman et al.,(1985)37:101-110),以用于从pZ130启动子表达。在链霉菌中,0RF438和酪氨酸酶从同一启动子区表达。为了从转基因植物基因组中的构建物表达,编码区应该处于不同的启动子区域调节控制之下。两个基因的启动子区可以是相同的或者不同的。另外,需要由单个植物启动子协同表达两个基因。从pZ130启动子表达酪氨酸酶和ORF438基因产物的构建物在以下实施例中有详述。也需要额外的启动子,如CaMV35S等植物病毒启动子能用来组成型表达所需的基因产物这一,同时别的基因产物受pZ130启动子影响在棉花纤维组织中表达。另外,也考虑了其他植物启动子在棉花纤维中用以表达基因的情况,例如上面讨论的Brassica种子启动子和E6基因启动子。同样,其他组成型启动子在某些应用中也具有价值,如美国专利号5,106,739和Comai et al.,Plant Mol.Biol.(1990)15:373-383)所述的mas,Mac或Double Mac启动子,当需要包含多个基因的植物时,可用两个构建物共转化得到植物,也可用各个构建物分别转化,然后用植物杂交方法得到表达两个所需基因的植物。
不同构建物通常连接到一个标记物上以便在植物细胞中选择。标记物常对抗生剂有抗性,尤其是对抗生素如卡那霉素,G418,博莱霉素,潮霉素,氯霉素等有抗性。所用特定标记物应能将转化细胞从未引入DNA的细胞中挑选出来。DNA构建物的成分,包括本发明的转录盒,可从宿主内部的(内源性的)或外部的(外源性的)的序列制得。“外部”即意味着序列在其即将转入的野生型宿主中找不到。异源构建物至少包含一个对于子房组织转录起始区域取自其中的基因是非同源的区域。
制备构建物中,不同DNA片段可被操作以提供正确方向的和正确表达阅读框架的DNA序列。衔接子和接头用以连接DNA片段,其他操作手段涉及提供方便的限制性位点,除去多余DNA,除去限制性位点等。体外诱变,引物修复,限制性酶切,退火,切除,连接等也可使用,其中将会涉及插入、缺失或取代如转换和颠换。载体或盒包括子房相关转录起始区下游的多克隆位点,以便构建物能有效用于不同序列。
在进行各步骤中,使用克隆以扩增DNA量进而分析DNA以确保操作方式正确。通过限制性酶切,切割或补平突出端以提供平端,接头的连接等合适操作可为连接提供片段的互补末端。有大量克隆载体可购得,这里的克隆载体包括在大肠杆菌中有功能的复制系统和筛选转化细胞的标记物,载体的例子包括pBR322,pUC系列,M13mp系列,pACYC184等。因此,序列可在合适限制性酶切位点插入载体,产生的质粒用以转化大肠杆菌,用恰当营养培养基培养大肠杆菌,收获并裂解细胞,最终回收质粒。分析将涉及序列分析,限制性酶切分析、电泳等。在每一操作后,最终构建物中使用的DNA序列应作限制性酶切并连接到下一个序列。每种部分构建物可克隆到相同或不同质粒。
各种将构建物引入植物细胞宿主的技术都可以使用并为本领域熟练技术人员所熟知。这些技术包括使用根瘤土壤杆菌或发根病土壤杆菌作转染剂的DNA转染、原生质体融合、注射、电穿孔、粒子加速等。为了用土壤杆菌转化,可在大肠杆菌中制备质粒,它含有与Ti质粒尤其是T-DNA同源的DNA。该种质粒可以或不能在土壤杆菌中复制即可能有或没有广谱原核复制系统,如pRK290就部分依赖于转录盒是整合入Ti质粒或仍被保留在独立的质粒上。土壤杆菌宿主将包含具有转移T-DNA至植物细胞所必需的vir基因的质粒,其T-DNA完全或不完全。Ti或Ri质粒的T-DNA的至少右边界,常常是左边界和右边界将作为侧翼区连接到转录构建物上。T-DNA转化植物细胞的运用已得到广泛研究,在欧洲专利申请流水号120,516,Hoekema,二元植物载体系统Offset-drukkerif,Kanters B.V.,Alblasserdam 1985,Chapter V.;Knallf等人.,土壤杆菌宿主范围表达的遗传学分析,细菌-植物相互作用的分子遗传学,Puhler.A.ed.,Springer-Verlag.NY,1983,P245.和An.et al.,EMBOJ.(1985)4:77-284中有充分描述。
为了进行感染,粒子加速和电穿孔,可将缺少发现于T-DNA区中的肿瘤基因的已解除防备的Ti质粒导入植物细胞。在辅助质粒帮助下,将构建物转移至根瘤土壤杆菌,产生的感染细菌用以感染植物细胞;将外植体与转化的根瘤土壤杆菌或发根病土壤杆菌一起培养,使得转录盒转移至植物细胞中。另外,为了加强对植物基因组的整合,转座子的末端重复可用作与转座酶连接的边界。这种情况下,转座酶的表达应是可诱导的,使得一旦转录构建物整合进入基因组,即可相对稳定地存在。然后将转基因植物细胞置于合适的选择性培养基中选择转基因细胞,并转移至生根培养基中培养产生愈伤组织、幼苗,并由幼苗长成小植株。
为确保转基因细胞和植物中转移基因的存在,可用本领域熟练人员熟知的方法作Southern印迹分析。根据产物的性质,转移基因的表达产物可用许多方法中的任一种检测,包括免疫分析,酶分析或者肉眼观察, 如检测在合适植物部位或细胞中色素形成。一旦已获得转基因植物,即可培养它们以产生具有所需表型的果实。收获果实或果实部分如棉花纤维和/或收集种子。种子可长出有所需特征的另一植株。转基因植物和转基因细胞包括衍生自转基因植物或转基因细胞的植物和细胞。
本文提供的各种序列可用作分离其他在本发明中有用之序列的分子探针,例如从相同或不同的植物来源获得相关的转录起始区域。得自本发明所提供序列的相关的转录起始区域至少有约60%的同源部分,进一步优选的区域与探针有更大的同源性百分比。取得有本文所述之时序和组织参数的相关转录起始控制区的能力极其重要。如使用探针pZ130鉴定了至少7个另外的克隆,但未作进一步鉴定。使用本申请所述技术及本领域中已知的其他技术(如Maniatis,et al.,分子克隆实验手册(Cold Spring Harbor,New York)1982),可以测定其他能如本发明所述指导子房组织转录的转录起始区域。构建物也能与植物再生系统联合使用,以取得植物细胞和植物;构建物也可用以修饰由此产生的果实的表型。
为了用于棉花,在所描述的方法中可使用不同变种和株系的棉花。载培的棉花种类包括Gossypium hirsutum和G.babadense(过长产品,或Pima棉花)等在New World中进化的棉花和Old World作物G.herbaceum和G.arboreum。
以下实施例用于说明而不是限制本发明。
实验
下列保藏物保存于美国典型培养物保藏中心(ATCC)(12301 Parklawn DriveRockyille,MD20852)。噬菌体Calgene Lambda 116和Calgene Lambda 140,每种含一个本发明的转录起始区域,它们于1989年7月13日被保藏,各自的入藏登记号为40632和40631。实施例1花前期西红柿子房cDNA文库的构建和子房特异性克隆的筛选cDNA文库的制备
西红柿植株(Lycopersicon esculentum cv UC82B)生长于温室条件下。Poly(A)+RNA用Mansson et al.,Mol.Gen.Genet.(1985)200:356-361所述的方法分离。使用BRL cDNA克隆试剂盒按制造商指导(BRL:Bethesda MD)从未开花的西红柿花(花前期)子房制备的Poly(A)+RNA合成cDNA。使用DNA Cloning Vol.1:A Practical Approach,Glover,ed.,(IRt Press,Oxford 1985)第2章所述程序,将限制性内切酶EcoR I接头(1078,New England Biolabs;Beverly,MA)加到所形成的双链cDNA.将cDNA克隆到噬菌体Lambda ZAP(Stratagene;La Jolla.CA)的EcoR I位点并包装所产生的重组噬菌体(使用Giga Pack Gold,Stratagene),均按各自的商品程序中的描述完成。
从相同的花前期阶段mRNA按前述方法制备两个cDNA文库。对于比第一个文库含明显较长的cDNA的第二个文库,在进行克隆之前,遵照生产商指导用RNA旋转柱(Boehringer Mannheim Biochemicals;Ind ianapolis,IN)过滤Poly(A)+RNA样品。
cDNA文库筛选
使用自花前期mRNA,叶mRNA和幼苗mRNA制备的32P标记的cDNA探针通过鉴别杂交筛选第一个cDNA文库。根据仅对花前期mRNA有杂交性筛选克隆。从噬菌体载体上切割相应于所选Lambda ZAP(Stratagene)克隆的cDNA并作为质粒繁殖(按生产商指导)。
从最初筛选的1000个cDNA中,分离了30个选定的克隆,根据其cDNA插入物的序列,将它们分为5个类型。选择两个克隆即克隆pZ7和pZ8作进一步研究。pZ7和pZ8的DNA序列分别用图1和图4中下划线部分表示。
用放射性标记的DNA探针的混合物通过噬菌斑杂交(Statagene Cloning试剂盒指导手册有述),筛选来自第二个cDNA文库的几千个重组克隆。用pZ7和pZ8 DNA探针筛选大约3千个来自第二个文库的重组克隆,筛选到14个有强烈杂交信号的克隆。将筛选出的克隆从噬菌体载体上切下来作为质粒增殖。从每个克隆中分离DNA,用限制性内切酶EcoR I切割,然后通过0.7%琼脂糖凝胶电泳。复制印迹杂交按Maniatis et al.Molecular Cloning:A laboratory Manual(ColdSpring H arbor,New York,1982)所述方法用代表所感兴趣的基因(pZ7和pZ8)的经放射性标记的探针进行。已挑选了7个与pZ7杂交的克隆,3个与pZ8杂交的克隆。进一步鉴定了对每个探针而言最长的克隆,如pZ130(与pZ7杂交)和pZ70(与pZ8杂交)并在其他试验中使用了这些克隆。实施例2cDNA克隆分析
Northern分析
cDNA克隆的组织特异性被阐明如下:使用经下列修饰的Ecker和Davis方法(Proc.Natl.Acad.Sci,(1987)84:503),从开花期后1,2,3,4,5,6,7,10,14,17和21天,花开期和花前期阶段的西红柿子房,西红柿叶和未组织化的西红柿愈伤组织中分离RNA。初次沉降核酸后,沉淀物在冰上重悬浮于2ml经焦碳酸二乙酯(DEP)处理的水中。溶液中加入MgCl2至终浓度1mM,并加入1/4体积的8MLiCl。样品充分混合并在4℃贮藏过夜。然后,样品在8000转4℃离心20分钟。干燥沉淀物,按前述重新在冰上悬浮于DEP处理过的水中,再用乙醇沉淀一次。根据Fourney等,Focus(1988)10:5-7所述的方法用甲醛/琼脂糖凝胶电泳RNA,固定在Nytran膜上(Schleicher&Schuell;Keene,NH),并与32P标记的探针杂交。
根据32P-标记的pZ7的EcoR I插入DNA或pZ8的EcoR I插入DNA的Northern分析,可明显看出两个基因在西红柿变种UC83B花开期中得到最高度的表达,花开前及花开一天后表达降低。图6表明西红柿在发育的不同阶段的花,在发育的同一阶段紧接花的下部剖分出一个代表性的子房。如图6所见,花开期后两天,两个基因的表达急剧下降。与pZ7探针杂交的mRNA大小约800nt,与pZ8探针杂交的mRNA大约500nt。
从花开期后两天起,pZ8 RNA的积累明显维持在相对低的水平,而pZ7 RNA的积累持续稳定下降,直到花开期后三周,此分析检测不到。pZ8 RNA累积在分离自比待熟果实不成熟的绿色期老的西红柿果实的RNA样品中用本方法检测不到。愈伤组织中对pZ7或pZ8没有RNA杂交;在叶组织中对pZ7没有杂交,长时间曝光条件下,pZ8杂交信号在叶RNA中刚刚可检测到。
表达水平
通过比较已知量的从克隆体外合成的RNA(在Ribopro bd系统(Promege)中使用T7或T3 RNA聚合酶)与从花开期和3周龄西红柿子房得到的RNA的杂交强度可测定对应于cDNA探针的信使丰度。此分析表明pZ7和pZ8 cDNA代表了花开期西红柿子房中丰富的RNA类型,各自约占信使RNA的5%和2%。
细胞特异性
cDNA探针的细胞特异性用原位杂交技术证明。花前期UC82B西红柿子房用4%多聚甲醛、磷酸盐缓冲液(PBS),5mM MgCl2溶液,pH7.4(PBS是10mM磷酸盐缓冲液,pH7.4,150mM NaCl)固定过夜(Singer,et al.,Biotechniques(1986)4:230-250)。固定后,将组织通过以50%醇开始的梯度叔丁醇(TBA)系列,用Paraplast浸润,并置于石蜡块中以便切片(Berlyn and Miksche,BotanicalMicrotechnique and Cytochemistry(1976)Iowa)。包埋的子房在ReichertHistostat旋转切片机上横切成8mm厚的切片。容纳5-7个子房切片的石蜡带固定到有色明胶明矾浸泡的载片上(Berlyn and Miksche(1976)文献同上)并于无灰尘盒子里存放直至进行原位杂交。准备杂交的载片在二甲苯中脱去石蜡,并通过如同Singer et al.,文献同上(1986)所述的水合乙醇系列以重新水合。
2X的杂交混合物包括100μl 20X SSC,20μl 10%BSA,100μl 750mMDTT,200μl 50%的葡聚糖硫酸酯,50μl RNasin,30μl无菌水。根据制造商的方案用T3和T7RNA聚合酶进行体外转录反应(Riboprobe Promega Biotec或Stratagene)从感兴趣的cDNA产生有义和反义35S-RNA探针。2.5μltRNA(20mg/ml),2.5μl鲑精DNA(10mg/ml)和4X 106cpm/探针一起用冻干机干燥。此混合物然后用含25μl 2X的杂交混合物/载片的25μl 90%甲酰胺重新悬浮。将40μl的杂交混合物置于每个载片上。将盖玻片置于切片上,边缘用胶泥密封。载玻片置于玻璃载片盒内的载片架上,盖一盖置37℃干燥过夜以杂交。杂交后处理在Singer等的上述文献中(1986)有述。
使用液体乳剂NTB-3,按KODAK Materials for Light Microscope(KODAK(1986);Rochester,NY)中所述方法进行放射自显影。载片在避光盒中曝光约两周。在冲洗了放射自显影载片后,切片在0.05%甲苯胺蓝中染色,然后通过梯度醇系列脱水,二甲苯∶100%乙醇,1∶1,然后两次更换为100%二甲苯,在每种溶液中脱水5分钟。盖片用Cytoseal(VWR,San Francisco,CA)固定,放在载片干燥器中直到干燥(45-50℃,1-2天)。放射自显影的载片然后可用作显微观察。
当花前期西红柿子房与有义和反义35S-pZ7 RNA杂交时反义转录本特异性杂交到子房外果皮区域和胚珠的外围区域(珠被)。有义转录本(阴性对照)未显示杂交。当花前期西红柿子房与35S-pZ8有义和反义RNA杂交时,反义转录本与子房内核区的胚珠外围区域特异性杂交。有义转录本未显示杂交。
总之,由相应于pZ7和pZ8的基因编码的mRNA转录本在西红柿果实发育的很特定的阶段,主要在花开期和花开前后一天大量表达。转录本还在发育的这一阶段在西红柿子房细胞类型的特定子集特别是珠被(pZ7和pZ8)及子房外果皮(pZ7)和内核区(pZ8)中表达。实施例3 pZ130和pZ70 cDNA克隆的测序
cDNA pZ130和pZ70克隆的完整的DNA序列用Sanger等(1971)的双脱氧技术测定。pZ130和pZ70的DNA的序列在3个阅读框架中被翻译。这些序列,包括每种序列的最长的开放阅读框架示于图1(pZ130)和图4(pZ70)。实施例4 基因家族分析
Southern分析按Maniatis等文献同上(1982)中的方法进行。来自载培品种UC82B的西红柿总DNA用Bam H I、EcoR I和Hind III消化,用琼脂糖凝胶电泳分离并转移到硝酸纤维素膜上。使用32P标记的由pZ130或pZ70随机引发产生的探针来进行Southern杂交。简单的杂交模式表明编码pZ130和pZ70的基因在西红柿基因组中仅有几个或可能仅有一个拷贝。
使用pZ130杂交探针杂交用限制性内切酶Bg II消化的西红柿基因组DNA的另外的分析,表明这个基因实际是一个小的基因家族(约5-7千成员)的成员之一。然而,最初含有局限于较长的pZ130克隆的3′未翻译区序列的pZ7 cDNA克隆,在使用经Bgl II消化的西红柿基因组DNA的Southern分析中仅对一条带强烈杂交,对第二条带微弱地杂交。实施例5 基因组克隆pZ130和pZ70的制备
用如下方法取得两个基因组克隆,一个代表pZ130 cDNA克隆,另一个代表pZ70 cDNA克隆。将从西红柿培养品种UC82B的DNA构建的基因组文库用限制性酶Sau3A部分消化,按生产厂商指导(Stratagene;La Jalla,CA)将之建立于Lambda噬菌体载体Lambda-FIX中。该文库用32P标记的pZ130和pZ70作探针来筛选,分离了一个含大约14.5kb的来自西红柿基因组的序列并与pZ70杂交的基因组克隆。与pZ70探针杂交的区域位于Calgene Lambda 116(见图5)的大约2kb的Xba I-Hind III的限制片段内。第二个基因克隆也被分离出来,它含有来自西红柿基因组的13kb序列并能与pZ130(和pZ7)杂交,与pZ130探针杂交的区域位于CalgeneLambda 140(见图3)的较大的EcoR I-Hind III限制性片段内。pCGN2015的制备
用Xba I消化pCGN565,用绿豆核酸酶钝化末端将所得片段插入经EcoRV消化的Bluescript KSM13-(Stratagene)载体产生pCGN2008。pCGN2008用EcoR I和Hind III消化,用Klenow补平,分离出1156bp的氯霉素片段。Bluescript KSM 13+(Stratagene)用Dra I消化,分离出2273bp的片段并连接到pCGN2008的氯霉素片段,从而产生pCGN2015。
pCGN2901/pCGN2902的制备
pCGN2901包含pZ130基因组克隆中与pZ7杂交的区域的邻近区域,包括5′方向大约1.8kb和3′方向大约4kb。为制备pCGN2901,用Sal I消化Calgene Lambda140,将含pZ7杂交区域的所得片段在pCGN2015的唯一Sal I位点插入pCGN2015,产生pCGN2901。
pCGN2902包含pZ130基因组的其他Sal I片段(不与pZ7杂交),所述片段来自Calgene Lambda 140的Sal I消化产物并被置于pCGN2015构建物中。实施例6 pZ130表达构建物的制备
用Nco I完全消化分离自pCGN2901的质粒DNA,然后用分离自绿豆的外切核酸酶(Promega,Madison,WI)处理以消除包括组成Nco I识别序列之ATG序列的单链DNA序列。样品然后用Sac I完全消化。产生的1.8kb(大约)的5′Sac I至Nco I片段被插入pUC衍生的氨苄青霉素抗性质粒,pCGP261(下述),该质粒如下制备。用Xba I完全消化pCGP261,单链DNA序列用DNA聚合酶I的Klenow片段处理以补平,然后用Sac I再次消化pCGP261 DNA产生的表达构建物命名为pCGN2903,在5′至3′转录方向包括衍生自Lambda 140的子房组织启动子,tmr基因和tmr的3′转录终止区。
pCGP261质粒从8762位至9836位含有来自根瘤土壤杆菌章鱼碱Ti质粒pTiLS955的序列(同Barker等的测序,Plant Mol.Biol.(1983)2:335-350)。该区域含有命名为tmr的遗传基因座的整个编码区,tmr编码异戊烯转移酶(Akiyoshi,et al.,Pro.Natl.Acad.Sci.(1984)81:4776-4780),翻译起始密码子ATG 5′端8个bp及翻译终止密码子TAG 3′端341bp的序列。
用下列方法得到质粒pCGP261,质粒pCGN1278(美国专利号5,177,307,1990年7月127日提交,全文引为文献)用Xba I和EcoR I消化,所产生的单链DNA序列用DNA聚合酶I的Klenow片段处理以补平,然后将含tmr基因的Xba I到EcoR I片段连接到载体ml3 Bluescript-(Stratagene Inc.,La Jolla,CA)的Sma I位点,产生质粒pCGp259。用BSpMI和BstXI消化pCGP259可消除AT翻译起始密码子上游的所有区域和一些tmr基因编码区域,产生的编码区域和最初在第一个ATG密码子上游发现的8bp序列被再次引入质粒,并且Xba I位点通过如下合成的寡核苷酸引入质粒,所述合成寡核苷酸含有序列5′AATTAGATGCAGGTCCATAAGTTTTTTCTAGACGCG 3′。所产生的质粒是pCGP261。
包含pZ130基因5′端、tmr基因编码区及3′端区域的pCGN2903的Xba I-Kpn I片段插入二元盒pCGN1557中,命名为pCGN2905。制备转基因植物。(见美国专利号5,177,307,上述)。实施例7 pZ130启动子盒的制备
pZ130 5′端含有转录起始位点1.8kb(pCGN2909)或5kb(pCGN2928)的DNA,3′端含有pZ130基因的区域(从TAA终止密码子到下游1.2kb位点)。pZ130盒如下构建。
转录起始区域
分离自PCGN2901质粒(见美国专利No.5,177,307,上述)的质粒DNA用NCOI完全消化,然后用分离自绿豆的外切核酸酶(Promega,Madison,WI)处理,以消除包括组成Nco I部分识别序列的ATG序列的单链DNA序列。样品用Sac I完全消化,产生的5′端Sac I至Nco I的1.8kb片段然后插入pCGN2015(上述)以产生pCGN2904。
为了消除多余的限制性酶位点,使随后的克隆更容易,将得自质粒pCGN2904的质粒DNA用Sal I和EcoR I完全消化,产生的1.8kb片段,含pZ130 5′端序列,将其插入pBluescript II(Stratagene;La Jolla,CA)以产生pCGN2907。
转录和翻译终止区域
用EcoR I的BamH I完全消化分离自pCGN2901的质粒DNA。产生的0.72kb EcoRI至BamH I片段位于pZ130编码区的下游(3′端),将此片段插入pCGN2907,产生PCGN2908。
按下述方法完成0.5kb(大约)DNA序列的插入,该序列包括pZ130基因TAA终止密码子和位于终止密码子和下游(3′端)EcoR I位点之间的序列以及加入独特的限制性位点以使外源基因容易插入。
合成包括序列5′GTTCCTGCAGCATGCCCGGGATCGATAATAATTAAGTGAGGC-3′的多接头/“引物”以产生具有下列位点:Pst I-Sph I-Sma I-Cia I,并包括pZ130基因TAA终止密码子和下列pZ130基因3′端区域序列的(3′)13个bp的多接头。合成包括序列5′-CAAGAATICATAATATTATATATAC-3′的另一个寡核苷酸以产生具有EcoR I限制性位点和紧邻EcoR I位点之pZ130基因3′端区域的16个碱基对,所述EcoR I位点距pZ130基因TAA终止密码子3′端约0.5kb。
这些合成的寡核苷酸可用于聚合酶链反应中(PCR),其中按制造商的教导,分离自pCGN2901的质粒DNA被用作热循环仪(Perkin-Elmer/cetus,Norwalk,CT)的底物。用Pst I和EcoR I完全消化所得的0.5kb DNA产物,产生的0.5kbDNA片段插入pCGN2908产生pCGN2909。从Pst I位点到EcoRI位点的0.5kb区域完整的DNA序列用Sanger等(1971)的双脱氧技术测定以证实PCR反应过程中在寡核苷酸引物间序列未曾出错。
pZ130盒pCGN2909,包括从808位的Sal I位点到2636位的5′端pZ130 DNA序列(见图2),可方便插入基因的单个Pst I、Sph I和Sma I位点,以及从3173位TAA终止密码子(图2)到4380位的BamH I位点间的3′端pZ130 DNA序列。
pZ130盒pCGN2928可通过将pCGN2059的3.2kb的从Kpn I到Sal I片段插入到pCGN2909的Kpn I和Sal I位点来制备。将pCGN2902的3.2kb Sal I至Bgl II片段插入M13mp19可制备pCGN2059。pCGN2928与pCGN2909一致,除了前者包括一个额外的约3.2kb的808位Sal I位点上游的pZ130 DNA序列(见图2)。实施例8检测构建物的制备和分析
β-葡糖苷酸酶(GUS)报道基因用以评价pZ130-GUS构建物的表达和组织特异性。GUS可产生高稳定性的酶,在植物组织中背景(内源)酶活性很小或没有,而且该酶易于利用荧光或分光光度底物分析,所以在植物系统中GUS是一种有用的报道基因(例见Jefferson,Plant Mol.Rep.(1987)5:387-405)。GUS酶活性的组织化学染色也可用以分析转基因植物中酶累积的模式,Jefferson(1987)文献同上。
检测构建物pCGN2917和pCGN2918的制备
这些构建物含1.8kb的pZ130 5′序列,GUS基因编码区域和1.2kb的pZ130 3′序列。pCGN2917和pCGN2918仅在与二元载体质粒其他元件如来自CaMV的35S启动子有关的pZ130/GUS构建的方向上有所不同。
这些构建物通过将pRAJ250(Jefferson(1987)文献同上)的Pst I片段或具有含GUS编码区的Pst I片段的任何其他质粒构建物插入pCGN2909的Pst I位点而制得。所产生的质粒,在与pZ130基因启动区有关的有义方向上有GUS基因,命名为pCGN2914。pZ130/GUS构建物作为Xba I至Kpn I片段被切割下来,并克隆到二元载体pCGN1557和pCGN1558,分别得到pCGN2917和pCGN2918。pCGN1557和pCGN1558在McBrid和Gummerfelt,Plant Mol.Biol.(1990)14:269-296中有述。
检测构建物pCGN2926的制备
该构建物含5kb的pZ130 5′端序列,GUS基因编码区域和1.2kb的pZ1303′端序列。它通过将pCGN2059的3.2kb的Kpn I片段插入至pCGN2914的Kpn I和Sal I位点而制得,产生的质粒命名为pCGN2923。pZ130/GUS/pZ130构建物然后从pCGN2923上作为Xba I-Kpn I片段切割下来,克隆到二元载体pCGN1557,产生pCGN2926。
GUS酶活性分析
转化子的β-葡糖苷酸酶活性用4-甲基散形基(umbelliferyl)葡糖苷酸作底物来测定,Jefferson(1987)上述文献中有简要说明。GUS酶活性在转化植物的子房中易于检测,相对未转化西红柿植物子房和转化植物叶中的背景活性要高得多。比较pCGN2917和pCGN2981转化子,十分有趣地发现,接近35S CaMV增强子区域(pCGN1558)能降低或消除子房组织特异性。实施例9 pZ7棉花转化
外植体制备
将Coker315种子置于50%CloroxR(2.5%次氯酸钠溶液)中20分钟作表面消毒,在灭菌的蒸馏水中清洗3次。表面消毒后将种子在25×150含25ml 1/2×MS盐,1/2×B5维生素,1.5%葡萄糖,0.3%gelrite的灭菌管中萌发。幼苗在28℃的黑暗中萌发7天,在第七天,将幼苗置于28+2℃的光中。
混合培养和植物再生
包含二元质粒pCGN2917和pCGN2926的根瘤土壤杆菌菌株2760的单菌落转移到5ml的肉汤培养基中,在30℃过夜培养。混合培养前细菌培养物稀释到1×108个细胞/ml。从八天龄幼苗上切下下胚轴,切成0.5-0.7cm的切片,放置到烟草饲养平皿上(Horsch et al.,(1985))。饲养平皿在使用前一天通过将1.0ml烟草悬浮培养物铺在培养皿上制得,该培养皿含不加抗生素的Callus InitiationMedium(CIM)(MS盐,维生素B5,3%葡萄糖,0.1mg/L 2,4-D;0.1mg/L细胞分裂索,0.3%gelrite,高压灭菌前pH值调整到5.8)。使用前将灭菌滤纸盘(Whatman#1)置于饲养小室顶部。所有切片准备完毕后,每个切片浸入根瘤土壤杆菌培养物中,在无菌滤纸上沾一下,送回烟草饲养培养皿中。
在饲养皿中混合培养两天后,将下胚轴切片置于新鲜的含75mg/L卡那霉素和500mg/L羧苄青霉素的CIM上。在28+2℃30uE 16:8光:暗期孵育组织4周,将整个外植株转移到含抗生素的新鲜CIM培养基中。第二次转移两周后,从外植体除去愈伤组织,并在CIM和再生培养基(MS盐;40mM KNO3;10Mm NH4Cl,维生素B5;3%葡萄糖;0.3%gelrite;400mg/L羧苄青霉素;75mg/L卡那霉素)之间分裂。
胚发生愈伤组织在起始后2-6月鉴定,在新鲜的再生培养基上传代培养。选择胚胎用以萌发并放入静态液体胚胎脉冲培养基(Stewart and Hsu medium,0.01mg/L NAA;0.01mg/L细胞分裂素,0.2mg/L GA3)上,在30℃过夜孵育。胚胎在纸巾上沾后即放入含40ml用Gelrite TM固化的Stewart和Hsu培养基的Magenta盒子内。萌发的胚胎保持在28±2℃,50uEm-2S-1,16:8光周期。有根小植株在土壤中建立后转移到温室。
生长室内棉花生长条件如下:16小时光周期,约80-85°F的温度,光强度约500μEinsteins。温室内棉花生长条件如下:14-16小时光周期,光强度至少400μEinsteins,白昼温度90-95°F,夜间温度70-75°F,相对湿度约80%。
植株分析
在花开期将来自温室生长条件下的T1植株的花贴上标鉴。从不同发育阶段的植株收获棉花芽、花、棉铃等并分析GUS活性。GUS荧光及组织化学分析在手工切片上进行,Jefferson(1987)文献同上中有述。
来自每一构建物(pCGN2917和pCGN2926)的至少10个转基因植物送入生长室/温室培养。大约80%(9/11)的2917号植株,100%(12/12)的2926号植株表达GUS,其水平通过荧光或组织化学试验均检测得到。用组织化学分析检测了来自几株被pCGN2917和pCGN2926转染植株的棉花芽的GUS表达,其中表达GUS的细胞被染成蓝色。初步分析表明所有植物在发育的花部位均表达GUS。胚株和花药染色极深,苞叶和子囊壁有时也呈蓝色。来自这些植物第5、9及12DPA(花开期后的天数)的棉铃的纤维也表达GUS。
几种GUS分析在从棉花出芽到花开期后53天各个棉铃发育阶段进行,GUS活性在棉花芽及花中极高,棉铃中GUS活性各植株有所不同。从2926植株中的两个在花开期后43天及53天的纤维中检测到GUS活性存在。
β-葡糖苷酸酶相当稳定,因此,GUS活性的存在与基因表达可能不存在时间上的直接联系,但在组织和/或来自子房珠被的结构中表达的特异性明显。GUS裂解的不同及表达的不同可以解释表达模式的多样性。
棉花和西红柿GUS表达比较
最初的MUG检测在来自被pCGN2917转染的西红柿及棉花植株组织中进行。西红柿根、茎、叶及分生组织、开花部位都能发现GUS活性。不同植株活性的量各不相同。棉花中,开花部位活性最高但在相同植株的根、茎部也可检测得到。
棉花转化
用pCGN2926转化的棉花植株中,其纤维细胞的嵌合型pZ130/GUS基因表达的时序模式可以使用经如下改变的Hall等的方法通过分离自植株2926-13的花开期后7,17-21及28天的纤维中的RNA来检测,(Proc.Natl.Acad.Sci(1978)75:3196)。在用2M LiCl第二次清洗后,沉淀溶于1/10原体积的10mM Tris(pH7.5)中,调整醋酸钾pH6.5到35mM,并慢慢加入1/2体积的乙醇。混合物置于冰上15分钟,然后在20000g,4℃,离心15分钟。醋酸钾浓度调整至0.2M,加入2.5体积的乙醇,RNA在-20℃放置数小时。沉淀物在12000g,4℃离心30分钟,沉淀重悬于焦碳酸二乙酯处理过的水中。
使用作如下改进的实施例2所述方法,从植株2926-13的花开期胚珠中分离RNA。应小心倾倒以避免在最后的乙醇沉淀中存在明显的沉淀物或在离心前将沉淀物从乙醇可溶性物质中分离出来。纤维和胚珠RNA然后用上述实施例2的方法作Northern分析。
根据32P-标记的GUS编码区探针的Northern分析,清楚看出嵌合pZ130 GUS基因在植株2926-13花开期胚珠中表达,这些组织中GUS mRNA的积累即为证据。图8提供了花开期阶段RNA与来自花开期后7、21和28天纤维的RNA比较。如图8所示,到花开期后第7天,基因的表达在分离的纤维中急剧降低到本方法不能检测的水平。表达模式与西红柿子房中内源性pZ130(硫蛋白)基因中观察到的模式极相似(见图6)。A道是花开期阶段的胚珠;B道是7天龄的纤维;C道是21天龄的纤维;D道是28天龄的纤维。实施例10转基因的黑色素合成基因的表达
一个用于植物转化以表达黑色素合成基因的二元构建物按如下方式制备。抗生链霉菌的mel操纵子(Bernan et al.(1985)34:101-110)作为Bcl I片段亚克隆到Bluescript载体中。通过突变,在紧邻(包括)ORF438的起始密码子ATG 5′端处插入Nco I和BamH I位点,所产生的质粒是pCGN4229。通过在紧接ORF438终止密码子后插入Pst I位点和在AroA基因座起始密码子处加入Nco I和BamH I位点,进一步诱变pCGN4229,这样,提供了诱变的mel操纵子。来自质粒载体的Pst I位点紧接tyrA编码区的3′端。
诱变pZ130盒pCGN2909以重新插入包括pZ130编码序列起始MET的ATG密码子在内的Nco I位点,产生pCGN4228。诱变pCGN4228以除去pZ130转录终止区域3′端的BamH I位点,并在其位置上插入Asc I接头片段,产生pCGN4235。诱变其他质粒以除去BamH I位点但无Asc I接头在此位置取代,它们被命名为pCG;N4236。然后诱变pCGN4236以插入Asc I接头到pZ130转录起始区域的5′端(在Xho I/Sal I处消化,Klenow处理),产生pCGN4241。
链霉菌ORF438区域通过用Nco I和Pst I消化诱变的mel操纵子构建物取得,并插入到经Nco I/Pst I消化的pCGN4235。tyrA区域作为来自诱变的mel操纵子构建物的Nco I/Pst I片段,克隆到经Nco I/Pst I消化的pCGN4241。
编码转移肽和成熟蛋白的12个氨基酸的烟草核酮糖二磷酸羧化酶小亚单位基因片段作为Nco I/BamH I片段插入到具有ORF438编码序列的阅读框架。这个片段同样插入tyrA编码的序列之前。产生的构建物含转运肽/ORF438和转运肽/tryA融合蛋白,定位于pZ1305′和3′端调节区以供表达。
用于插入ORF438和tyrA构建物的二元载体(见图7)从pCGN1578制备(McBrideet al.,文献同上)。上述制备过程是通过用含下列酶切位点Asp718/Asc/Pac/Xba I/BamH I/Swa/Sse/Hind III的接头区替换pCGN1578的接头区来实现的(见图8),结果产生了pCGN1578PASS,Asc,Pac,Swa和Sse是在8碱基识别位点切割的限制性酶。Asc,Pac购自New England Biolabs,Swa购自Boehringer Manheim,Sse购自Takara(日本)。
ORF438 pZ130构建物作为Asp/Asc片段插入pCGN1578PASS。tyrA pZ130构建物作为Asc/Xba片段插入ORF438 pZ130构建物的邻近区。实施例11用pZ130表达构建物pCGN2905和pCGN2925转化之植物的制备和分析
pCGN2925的制备
一个表达构建物(实施例6中有述)在其转录的5′至3′方向含有来源于Lambda140的1.8kb子房组织启动子,及tmr编码区和tmr3′端转录终止区(命名为pCGN2903),这个构建物用如下方法改进以产生pCGN2925。来自pCGN2903的2.8kbEcoR I片段被插入pCGN2015的EcoR I位点以产生pCGN2910。pCGN2059的3.2kb大小的Kpn I至Sal I片段被插入到pCGN2910的Kpn I和Sal I位点以产生pCGN2922。来自pCGN2922的6kb的Kpn I至Xba I片段被插入二元盒pCGN1557的Kpn I和Xba I位点产生pCGN2925。pCGN2925与pCGN2905相同,除前者包括一个额外的约3.2kb的pZ130 DNA序列,该序列位于图2的808位Sal I位点上游外(pCGN2905含有插入本申请实施例6中所述二元盒pCGN1557的源自pCGN2903的从Xba I到KpnI片段)
转基因植物的制备
转基因棉花植株Coker 130变种用pCGN2905和pCGN2925转化,所用方法在实施例9中有述。近亲交配的转基因西红柿株系UC82B用pCGN2905转化,所用方法在美国专利号5,177,307的实施例7中有述。
转基因植物的农业性状分析
已获得用pCGN2905和pCGN2925转化的棉花植株,通过基因表达已证明获得了对抗生素卡那霉素的抗性。由几个初始转化子的分离后代所做温室试验,支持了棉花子房中细胞激动素水平的增加会增加果实调节/果实保留能力的结论。5个非转化对照植株中花开期花数在30和53朵之间变动;来自转化子2925-2的两个后代有83朵和96朵花开期花。对照组收获期棉铃的数目变动于20和38之间;两个2925-2植株收获时有66个和58个棉铃,来自2905-2的后代收获时有46个棉铃,来自2905-3的后代收获时有45个棉铃。已取得转化的西红柿植株,通过Southern分析证明对来自pCGN2905的嵌合基因pZ130/tmr/tmr是纯合的。果实重量数据的检查结合重复棉田分析中得到的植株产量信息表明,子房中增加的细胞激动素水平在受检的至少两株转基因株系中增加了果实调节。一块地中2905-9和2905-18品系每米植株的果实数目比非转基因对照组高许多(大约分别为207和192对162)。
转基因棉铃和纤维性状的分析
经pCgN2905和pCGN2925转化的棉花植株的纤维样品分析表明纤维品质特征桐对非转化的对照组植物的纤维已经改变。如对照植株中纤维长度在1.12和1.15英寸之间变化,植株2905-2A的纤维长度是1.17英寸,植株2905-3B是1.19英寸。对照样品中纤维强度在24.3和25.7grams/tex之间变化;而在植株2905-2B中纤维强度是26.8 grams/tex,2905-3B植株中是27.6 grams/tex。
对照植株中细度测量在4.4与4.8之间变化。受检的3个2905-2后代中的细度测量为4.0,3.5和4.0,在2905-3B中细度测量为4.2(细度的描述,见Munro,Cotton,2d ed(1987)Longman Scientific&Technical,Essex,England)。与对照相比,两个2925-2后代的其他纤维品质测量也有明显改变。2925-2A和2925-2B纤维样品的长度和强度分别为1.09和20.6,1.06和19.3。
经pCGN2905转化的西红柿植株的果实样品分析表明,与非转化的对照植株相比果实质量已改变。例如果实总的固形物水平在受检的7个独立的转基因西红柿品系中的6个中均明显增加(对照组5.79对6个转化子中6.27-6.72)。可溶性果实固形物水平在受检的7个独立的转基因西红柿品系中的5个中均明显增加(对照中5.18对5个转化子中5.68-5.96)。果实样品的糖酸比率在7个独立品系中的6个中也明显增加(对照组9.3对6个转化子中的10.5-12.0)。实施例12表达盒构建物pCGN2937和pCGN2939的制备
pCGN2931和pCG2932的制备
质粒pCGN2931含有来自根瘤土壤杆菌章鱼碱Ti质粒pTi15955的3288位至5810位的序列(已由Baker等测序,Plant Mol.Biol(1983)2:335-350)。该区域含有tms-2(或iaaH)遗传基因座的整个编码区域,所述编码区域编码吲哚乙酰胺水解酶(Schroder et al.,Eur.J.BioChem(1983)138:387-391;Thomashow et al.,Pro.Natl.Acad.Sci(1984)81:5071-075),翻译起始ATG密码子5′端347bp和翻译终止TAG密码子3′端772bp序列。pCGN2931按如下方式产生。来自pTi15955,或任何其他含有来自pTi15955 T-DNA区域的iaaH基因座的质粒的DNA,在标准聚合酶链反应(PCR)中用作模板,该反应使用引物5′-CGCGGGTCGACTGCAGTGTTAGAAAAGATTCG-3′和5′-CGGACTCTAGAGATGTGAGGTGTG-3′。
分离产生的约2.5kb的片段,用限制酶Sal I和Xba I切割并插入质粒pBluescript KS II-(Stratagene)的Sal I和Xba I位点,产生质粒pCGN2931。来自pCGN2931的2.5kb的Pst I片段插入二元盒pCGN1557得到pCGN2932。
pCGN2930的制备
质粒pCGN2930含有来自根瘤土壤杆菌章鱼碱Ti质粒pTi15955的5809位至8076位的序列(Barker等已测序,Plant Mol.Biol.(1983)2:335-350)。该区域含tms-1(或iaaM)遗传基因座的整个编码序列,所述编码序列编码色氨酸单加氧酶(Thomashow et al.Science(198 6)231:616-618)。pCGN2930用如下方式得到。来自pTi15955或任何其他含来自pTi15955T-DNA区iaaM基因座的质粒的DNA,在标准PCR中作为模板,用5′-CGAATCTGCAGATGTCAGCTTCACC-3′和5′-CGGGGCTGCAGCTAATTTCTAGTGC-3′作引物。分离所得到的约2.3kb片段,用限制性酶Pst I消化并插入到质粒pBluescript KS II-(stratagene)的Pst I位点,产生pCGN2930。
pCGN2934和pCGN2936的制备
pCGN2934和pCGN2936各自含pZ130基因的5kb和1.8kb的翻译起始位点5′端DNA,约2.3kb的iaaH编码区域和pZ130基因的3′区域(以TAA终止密码子到下游1.2kb处的位点)。这些质粒用如下方式得到。
来自pCGN2930的2.3kb Pst I片段被插入pCGN2928的Pst I位点。所产生的质粒命名为pCGN2934,该质粒在与pZ130基因启动子区相关的有义方向含有iaaM。
来自pCGN2930的2.3kb的PstI片段被插入pBCKS II-(stratagene)的Pst I位点,得到pCGN2935。然后将pCGN2935的2.3kb的Pst I片段插入pCGN2909的Pst I位点,产生的质粒命名为pCGN2936,该质粒在与pZ130基因启动子区有关的有义方向有iaaM。
pCGN2937和pCGN2939的制备
pZ130/iaaM/pZ130构建物作为Xba I-Kpn I片段(分别为11kb和7.8kb)从pCGN2934和pCGN2936上切下,并克隆到二元载体质粒pGN2932(已含iaaH)。产生的质粒命名为pCGN2937(1.8kbpZ130启动子)和pCGN2939(5.0kb pZ1306启动子)。
如以上结果表明,感兴趣的基因表达能在子房来源的细胞如西红柿果实和棉花纤维中得到。
本文说明书中提到的所有出版物和专利申请均引为文献,如同每个独立的出版物或专利申请被特别和单独指明已引入作为参考文献。
尽管本发明已通过以弄清和理解为目的的阐述和实施例详细描述,但对本领域普通技术人员而言,一定的改变和改进也是显而易见的,而与所附权利要求的精神及范围并不背离。
Claims (22)
1.一种改进转基因棉花棉铃产率的方法,所述方法包括如下步骤:
培养转基因棉花以产生成熟的胚珠组织,其中所述成熟胚珠组织细胞在其基因组中含有一个或多个DNA构建物,所述构建物在其转录方向上含有可操作连接的成分,所述成分包括在植物胚珠珠被细胞中有功能的转录和翻译起始区、编码对植物生长物质的产生有活性的蛋白质的DNA序列和转录终止区,其中至少一种所述成分对至少一种其他所述成分而言是外源的,
其中所述植物在成熟植株胚珠组织中表达对植物生长物质的产生有活性的所述蛋白质从而在所述转基因棉花中改进棉铃产率。
2.根据权利要求1的方法,其中所述改进是转基因棉花中产生的棉铃数目的增加。
3.根据权利要求2的方法,其中所述的转基因棉花细胞是Gossypi umNirsutum L.植株。
4.根据权利要求3的方法,其中所述的转录和翻译起始区来自pz130基因,所述的植物生长物质是细胞激动素。
5.根据权利要求4的方法,其中所述植物生长物质选自由生长素、赤霉素、细胞激动素、乙烯和脱落酸组成的一组。
6.根据权利要求5的方法,其中所述的对植物生长物质的产生有活性的蛋白质是与乙烯生物合成途径相关的酶。
7.根据权利要求6的方法,其中所述的乙烯生物合成的酶选自由ACC合成酶,pTOM13,SAM合成酶,ACC脱氨酶和SAM脱羧酶组成的一组。
8.根据权利要求1的方法产生的转基因棉花胚珠珠被细胞。
9.含有权利要求8的棉花胚珠珠被细胞的转基因棉花植株。
10.权利要求9的植株的种子。
11.根据权利要求10的种子萌发的植物。
12.一种改进转基因棉花的纤维质量的方法,所述方法包括如下步骤:
培育转基因棉花以产生成熟的胚珠组织,其中所述的成熟的胚珠组织细胞在其基因组中含有一个或多个DNA构建物,所述构建物在其转录方向含有可操作连接的成分,所述成分包括在植物胚株株被细胞中有功能的转录和翻译起始区、编码对植物生长物质的产生有活性的蛋白质的DNA序列和转录终止区,其中至少一种所述成分对至少一种其他所述成分而言是外源的,
其中所述植物在成熟植株胚珠组织中表达对植物生长物质的产生有活性的蛋白质,从而所述转基因棉花的纤维的尺寸量度及强度质量特性得以改进。
13.根据权利要求12的方法,其中所述的质量改进是指对纤维细度的改进。
14.根据权利要求12的方法,其中所述的质量改进是指对纤维强度的改进。
15.根据权利要求12的方法,其中所述质量改进是指对纤维长度的改进。
16.根据权利要求12的方法,其中所述棉花胚珠珠被细胞是棉花纤维细胞。
17.根据权利要求16的方法,其中所述棉花纤维细胞是Gossypium hirsuturrL.细胞。
18.根据权利要求12的方法,其中所述棉花生长物质选自由生长素,赤霉素,细胞激动素,乙烯和脱落酸组成的一组。
19.根据权利要求18的方法,其中所述的转录和翻译起始区来自pZ130基因,所述的植物生长物质是细胞激动素。
20.根据权利要求18的方法,其中所述的对植物生长物质的产生有活性的蛋白质是与乙烯生物合成途径相关的酶。
21.根据权利要求20的方法,其中所述的乙烯生物合成的酶选自由ACC合成酶,pTOM13,SAM合成酶,ACC脱氨酶和SAM脱羧酶组成的一组。
22.根据权利要求12的方法产生的经改进的棉花纤维。
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WO2021084325A1 (en) | 2019-11-01 | 2021-05-06 | Purecircle Usa, Inc. | Stevia cultivar '18136109' |
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US11617335B2 (en) | 2021-02-26 | 2023-04-04 | Monsanto Technology Llc | Cotton variety 19R249B3XF |
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NZ221259A (en) * | 1986-07-31 | 1990-05-28 | Calgene Inc | Seed specific transcriptional regulation |
US5004863B2 (en) | 1986-12-03 | 2000-10-17 | Agracetus | Genetic engineering of cotton plants and lines |
US5177307A (en) | 1987-05-26 | 1993-01-05 | Calgene, Inc. | Compositions and methods for modulation of endogenous cytokinin levels |
US5495070A (en) * | 1988-10-04 | 1996-02-27 | Agracetus, Inc. | Genetically engineering cotton plants for altered fiber |
US5175095A (en) | 1989-07-19 | 1992-12-29 | Calgene, Inc. | Ovary-tissue transcriptional factors |
WO1991001323A1 (en) * | 1989-07-19 | 1991-02-07 | Calgene, Inc. | Compositions and methods for modulation of endogenous cytokinin levels |
ES2163392T3 (es) * | 1990-03-16 | 2002-02-01 | Calgene Llc | Nuevas secuencias expresadas preferentemente en el desarrollo de semillas precoces, y procedimientos relacionados con las mismas. |
US5512466A (en) | 1990-12-26 | 1996-04-30 | Monsanto Company | Control of fruit ripening and senescence in plants |
AU1663392A (en) | 1991-03-06 | 1992-10-06 | Agracetus, Inc. | Particle mediated transformation of cotton |
WO1993007272A1 (en) | 1991-10-03 | 1993-04-15 | Calgene Pacific Pty. Ltd. | Transgenic plants |
WO1994012014A1 (en) | 1992-11-20 | 1994-06-09 | Agracetus, Inc. | Transgenic cotton plants producing heterologous bioplastic |
JPH08509871A (ja) | 1993-09-30 | 1996-10-22 | アグラシータス インコーポレイテッド | 異種ペルオキシダーゼを生産するトランスジェニック綿植物 |
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1995
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1996
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- 1996-02-28 CN CN96190406.2A patent/CN1152250A/zh active Pending
- 1996-02-28 EP EP96910441A patent/EP0758841A4/en not_active Withdrawn
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN101663400B (zh) * | 2007-03-09 | 2013-04-17 | 孟山都技术公司 | 分生组织切除和转化方法 |
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WO1996026639A1 (en) | 1996-09-06 |
US6329570B1 (en) | 2001-12-11 |
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EP0758841A4 (en) | 1999-02-03 |
AU713836B2 (en) | 1999-12-09 |
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