CN102105591A - 提高水稻光合固碳的方法 - Google Patents
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Abstract
本发明涉及刺激植物生长和/或改善植物生物质产量和/或提高植物固碳的方法,包括将一种或多种核酸引入水稻植物细胞、水稻植物组织或水稻植物中,引入的一种或多种核酸可导致叶绿体从头表达具有乙醇酸脱氢酶活性的一种或多种多肽。
Description
水稻(Oriza sativa)是全球种植的最重要谷物,是全世界约一半人口的主食。由于世界人口的增长和全球可耕地需求压力的增大,需要不断提高土地的作物产量。因此,继续需要新的提高作物生产力的解决办法,而作为最重要的谷物的水稻(rice)是此类解决办法的一个主要目标作物。
作物的产量受多种因素的影响,一方面是影响植物产生生物质能力(光合作用、营养物和水摄取)的因素,另一方面是影响植物对抗某些应激状态,例如生命应激(昆虫、真菌、病毒等)或非生命应激(干旱、含盐度)能力的因素。
影响生物质产量的一个重要因素是光合作用。光合作用的机制是植物通过它捕获大气二氧化碳将其转化成糖,然后掺入植物组织中产生生物质。
大多数植物具有光合作用机制,其中叶绿体酶RuBisCo(核酮糖-1,5-二磷酸羧化酶/加氧酶)是捕获二氧化碳将其转化成糖的主要酶。那些植物称为C3植物,水稻就是C3植物。已知C3植物光合作用机制中的一个问题是固碳效率在某些环境条件下不是最优,此时所固碳的一部分因RuBisCo的另一活性(称为氧合作用)而损失。
RuBisCO能催化核酮糖-1,5-二磷酸的羧化和氧合。这两种活性之间的平衡主要取决于叶片中的CO2/O2比率,植物对某些环境条件反应后该比率可能改变。每次羧化反应产生两分子的磷酸甘油酸进入卡尔文循环,最终形成淀粉和糖并产生核酮糖-1,5-二磷酸。氧合反应产生一分子磷酸甘油酸和磷酸乙醇酸。后者通过光呼吸作用再循环产生磷酸甘油酸(Leegood R.C.等,1995)。每产生两分子磷酸乙醇酸放出一分子CO2,导致净损失一个固定碳,最终减少了产生的糖和生物质量。该反应中还损失了氨,需要叶绿体通过耗能反应再固定。
已有报道将克服光呼吸作用作为提升光合作用最大效率和提高生产力的目标(Zhu等.,2008),迄今已描述了为减少植物碳损失从而提高糖和生物质产量所作的多种尝试。一些植物种类已获得一些有希望的结果,但迄今对于水稻尚无正面结果报道。
Kebeish等报道了可在叶绿体中引入细菌的光呼吸作用底物乙醇酸盐分解代谢途径来缓解拟南芥(Arabidopsis thaliana)的光呼吸损失(WO03/100066;Kebeish R.等,2007)。该作者先将大肠杆菌(Escherichia coli)乙醇酸脱氢酶的3个亚基靶向(引入)拟南芥叶绿体,然后引入大肠杆菌乙醛酸聚醛酶和大肠杆菌羟基丙二酸半醛还原酶(tartronic semialdehyde reductase)从而完成了与内源性光呼吸途径相平行的将乙醇酸转化成甘油酸的途径。这种利用5种大肠杆菌基因的逐步核转化可导致拟南芥植物叶绿体乙醇酸直接转变为甘油酸。这些转基因植物生长较快,产生更多的嫩枝和根生物质,含有更多的可溶性糖。在仅过表达乙醇酸脱氢酶此3个亚基的拟南芥植物中可见此作用但程度较轻。
另一策略是将C4-或C4-样途径或该途径的组分转移给C3植物。
1996年,Gehlen J.等报道在最佳温度时,能表达谷氨酸棒状杆菌(C.glutamicum)的PEPC(磷酸烯醇丙酮酸羧化酶)基因的转基因马铃薯其光合特征有所改变。
1999年,Ku等利用玉米PEPC将该方法应用于水稻。然而,在转基因水稻植物中,CO2的同化速度无显著改变,对植物生理学和生长性能仅有微弱影响,虽然检测到PEPC活性水平升高达100-倍(Matsuoka等.,2001;也参见EP-A 0 874 056)。
另一项研究报道,过表达靶向水稻叶绿体的类黍尾稃草(Urochloapanicoides)磷酸烯醇丙酮酸羧基激酶(PCK)可导致诱生单个细胞内的内源性PEPC和建立C4-样循环。然而,未观察到生长参数升高(Suzuki等.,2000;也参见WO 98/35030)。最近(2008),Y.Taniguchi等将轮叶黑藻(Hydrillaverticillata)的C4-样途径引入水稻植物的叶肉细胞。产生了独自过表达或组合过表达4种C4酶,即磷酸烯醇丙酮酸羧化酶、正磷酸双激酶(orthophosphatedikinase)、NADP-苹果酸酶和NADP-苹果酸脱氢酶的不同转基因水稻植物。发现所有4种酶组合过表达略微改善了光合作用,但同时导致转基因植物生长发生轻微但可重现的发育障碍。
因此,仍需要提高水稻固碳的有效方法,以刺激植物生长和/或提高生物质产量和/或种子产量。
本发明涉及提高水稻植物生物质产量和/或种子产量和/或固碳的方法,包括将编码具有乙醇酸脱氢酶活性的一种或多种多肽的一种或多种核酸引入水稻植物细胞的基因组,引入的所述一种或多种核酸可导致从头表达一种或多种具有乙醇酸脱氢酶活性的多肽,而所述的一种或多种多肽定位于所产生的水稻植物的叶绿体中。
就本发明而言,生物质产量是个体植物或种植植物地表面积所产生的物质量。可检测几种参数以测定生物质产量是否提高。此类参数的例子有,植物高度、叶片面积、枝条干重、根干重、种子数、种子重量、种子大小等。就该方面而言,种子产量或种子产率是生物质产量的一种特定指标。可测定每个植物或种植植物土地单位表面积的种子产量或种子产率。通常在土壤中生长确定时期后或在生长的特定阶段检测这些参数,例如在营养期结束时,在用一种或多种本发明核酸转化的植物与未用一种或多种核酸转化的植物之间作比较。
可通过检测气体交换和叶绿素荧光参数来测定植物固碳的提高。一种方便的方法是利用LI-6400***(Li-Cor)和生产商提供的软件,可参见通过引用纳入本文的R.Kebeish等,2007的描述。
本发明方法所涉及的核酸编码具有乙醇酸脱氢酶活性的一种或多种多肽。
可利用有机辅因子氧化乙醇酸形成乙醛酸来测定乙醇酸脱氢酶的活性,而且,例如,植物过氧化物酶体中存在的乙醇酸氧化酶能利用分子氧作为辅因子并释放过氧化氢。
根据辅因子性质的(不同),乙醇酸脱氢酶和乙醇酸氧化酶之间如此明显的差异并非总是这样,例如以前曾将gcl操纵子编码的大肠杆菌乙醇酸脱氢酶称为乙醇酸氧化酶(Bari等.,2004)。
可采用本申请实施例4描述的技术,按照Lord J.M.1972检测乙醇酸脱氢酶的活性。
或者,可对缺乏形成活性内源性乙醇酸脱氢酶3个亚基的大肠杆菌突变株进行互补分析。这些大肠杆菌突变株不能利用乙醇酸作为唯一碳源生长。当这些缺陷型突变株的某种酶过表达使该菌在含有乙醇酸作为唯一碳源的培养基上恢复生长时,则意味着该酶编码大肠杆菌乙醇酸脱氢酶的功能等价物。互补分析的方法和工具可参见通过引用纳入本文的Bari等,2004中的描述。
已鉴定到各种来源,包括细菌、藻类和植物的具有乙醇酸脱氢酶活性的多肽和编码它们的核酸。
表1:已知乙醇酸脱氢酶的例子
可从,例如任何来源,包括细菌、哺乳动物、藻类、真菌和植物来源的基因组DNA或cDNA文库中,分离得到编码具有乙醇酸脱氢酶活性的一种或多种多肽的核酸分子。或者,可借助重组DNA技术(例如,PCR)或化学合成产生它们。可采用已知的乙醇酸脱氢酶核酸分子的序列或那些序列的一部分,或视情况采用这些分子的反向互补链(例如,通过标准方法杂交,参见,例如Sambrook等,1989)来鉴定和分离此类核酸分子。
对于本发明目的,所述乙醇酸脱氢酶可以是任何天然产生的乙醇酸脱氢酶或其任何活性片段或其任何变体,所述片段或变体中的一些氨基酸(优选1-20个,更优选1-10个,甚至更优选1-5个氨基酸)可被替代、添加或删除,而该酶仍保留了乙醇酸脱氢酶活性。
根据本发明,乙醇酸脱氢酶可以是嵌合型乙醇酸脱氢酶。术语“嵌合型乙醇酸脱氢酶”指通过组合各种来源的酶的一部分,例如第一种酶的N-末端部分与第二种酶的C-末端部分获得的乙醇酸脱氢酶,从而得到新的功能性嵌合型乙醇酸脱氢酶,其中各部分根据其特定特性而选择。例如,可制备功能性嵌合型乙醇酸脱氢酶以组合第一种乙醇酸脱氢酶的有效活性位点与第二种乙醇酸脱氢酶提供的在水稻中的良好稳定性。
根据本发明,“核酸”或“核酸分子”应理解为是多聚核苷酸分子,可以是DNA或RNA类型,优选DNA类型,特别是双链核酸。其可以是天然或合成来源。可在体外产生合成核酸。此类合成核酸的例子是其中编码具有乙醇酸脱氢酶活性的一种或多种多肽的密码子按照表达宿主生物作了优化(例如,与原始宿主相比,利用此类宿主生物的密码子选择表中或此类宿主生物所属种群中更偏爱或最偏爱的那些密码子作密码子替换)的那些核酸。技术人员熟知密码子优化的方法。
可采用一种或多种多肽获得本发明方法涉及的乙醇酸脱氢酶活性。所述活性获自一种以上多肽时,可用一个质粒构建物或几个独立构建物将编码所述多肽的核酸转移入植物细胞内。
具有乙醇酸脱氢酶活性的优选多肽是大肠杆菌glc操纵子(gi/1141710/gb/L43490.1/ECOGLCC)编码的那些多肽。最优选包含SEQ IDNO:2(Glc D)、4(Glc E)和6(Glc F)所示氨基酸序列的多肽。因此,可采用包含SEQ ID NO:1、3和5所示多核苷酸序列的核酸来实施本发明。
或者,可采用具有乙醇酸脱氢酶活性衍生自拟南芥或其它高等植物来源的一种或多种多肽。优选的拟南芥多肽包含SEQ ID NO:8所示氨基酸序列,由包含SEQ ID NO:7所示多核苷酸序列的核酸编码。可采用包含SEQ ID NO:7所示多核苷酸序列的核酸来实施本发明。
或者,可采用具有乙醇酸脱氢酶活性衍生自藻类,特别是衣藻属(Chlamydomonas)或集胞蓝细菌(Synechocystis)的一种或多种多肽(Eisenhut等.,2006)。优选的衣藻属多肽包含SEQ ID NO 12所示氨基酸序列,由包含SEQ ID NO 11所示多核苷酸序列的核酸编码。因此,可采用包含SEQ ID NO:11所示多核苷酸序列的核酸来实施本发明。优选的集胞蓝细菌多肽包含SEQID NO 16所示氨基酸序列,由包含SEQ ID NO 15所示多核苷酸序列的核酸编码。因此,可采用包含SEQ ID NO:15所示多核苷酸序列的核酸来实施本发明。
在本发明的另一实施方式中,可采用保留了乙醇酸脱氢酶活性的截短多肽。优选的衣藻属多肽包含SEQ ID NO 14所示氨基酸序列,由包含SEQ IDNO 13所示多核苷酸序列的核酸编码。因此,可采用包含SEQ ID NO:13所示多核苷酸序列的核酸来实施本发明。
由于氨基酸序列中可能发生一些改变而不实质性改变乙醇酸脱氢酶的酶活性,可采用包含与SEQ ID NO:2、4和6,或SEQ ID NO:8或SEQ ID NO:10或SEQ ID NO:12或SEQ ID NO:14或SEQ ID NO:16基本上相似的氨基酸序列的任何蛋白质来实施本发明,其中低于20个,优选低于10个、更优选1-5个氨基酸可被其它氨基酸替换而不实质性影响乙醇酸脱氢酶活性。
本发明方法包括将编码具有乙醇酸脱氢酶活性的一种或多种多肽的一种或多种核酸引入水稻植物细胞的基因组中,在氨基酸序列水平上所述的一种或多种多肽包含与SEQ ID NO:2、4和6,或SEQ ID NO:8或SEQ ID NO:10或SEQ ID NO:12或SEQ ID NO:14或SEQ ID NO:16至少有60、70、80或90%,特别是至少有95%、97%、98%或至少有99%序列相同性的序列,引入的所述一种或多种核酸可导致从头表达具有乙醇酸脱氢酶活性的至少一种多肽,所述活性位于叶绿体内。
本发明方法还包括将编码具有乙醇酸脱氢酶活性的一种或多种多肽的一种或多种核酸引入水稻植物细胞的基因组中,所述的一种或多种核酸包含与SEQ ID NO:1、3和5,或SEQ ID NO:7或SEQ ID NO:9或SEQ ID NO:11或SEQ ID NO:13或SEQ ID NO:15至少有60、70、80或90%,特别是至少有95%、97%、98%或至少有99%序列相同性的核酸序列,引入的所述一种或多种核酸可导致从头表达具有乙醇酸脱氢酶活性的至少一种多肽,所述活性位于叶绿体内。
对于本发明的目的,以百分数表示的两条相关核苷酸或氨基酸序列的“序列相同性”指两条最佳比对序列中具有相同残基的位置数(x100)除以所比较的位置数。空位,即某残基在一条序列中存在而在另一条中不存在的比对位置视作具有不相同残基的位置。可通过EMBOSS(Rice等.,2000)中的Needleman和Wunsch算法(Needleman和Wunsch 1970),利用默认设置(空位开放罚分10,空位延伸罚分0.5)进行两条序列的比对以在序列的全长上找出最佳比对。
一旦已知外源DNA的序列,可借助分子生物学技术开发能特异性识别核酸(DNA或RNA)样品中这些序列的引物和探针。例如,可开发PCR方法来鉴定生物学样品(例如,植物、植物材料或包含植物材料的产品)中可用于本发明方法的基因(gdh基因)。此类PCR依据至少两种特异性“引物”,例如二者均能识别本发明所用gdh编码区(例如,SEQ ID No.1、3、5、7、9、11、13或15所示编码区)中的序列,或者一种识别gdh编码区内的序列,另一种识别相关转运肽序列内或调控区内的序列,例如包含本发明所用DNA的嵌合型基因的启动子或3’末端。所述引物优选含有15-35个核苷酸的序列,在最优PCR条件下能特异性识别本发明所用gdh嵌合基因内的序列,从而扩增含有本发明所用gdh基因的核酸样品中的特异性片段(“整合片段”或差别扩增子)。这意味在最优PCR条件下只扩增植物基因组或外源DNA中的靶向整合片段,而不扩增其它序列。
本发明方法还包括将编码具有乙醇酸脱氢酶活性的一种或多种多肽的一种或多种核酸引入水稻植物细胞的基因组中,所述的一种或多种核酸包含能在严谨条件下与选自下组的核苷酸序列杂交的一种或多种核酸:SEQ ID NO 1、3和5、SEQ ID NO 7、SEQ ID NO 9、SEQ ID NO 11、SEQ ID NO 13和SEQID NO 15,引入所述的一种或多种核酸可导致从头表达至少一种具有乙醇酸脱氢酶活性的多肽,所述活性位于叶绿体内。本文所用的严谨性杂交条件具体指以下条件:将相关DNA序列固定在滤膜上,42℃将该滤膜在50%甲酰胺、5%SSPE、2x Denhardt试剂和0.1%SDS中,或68℃在6x SSC、2xDenhardt试剂和0.1%SDS中预杂交1-2小时。然后将变性的地高辛-或放射性标记探针直接加入预杂交液体,在上述合适温度下培育16-24小时。培育后,室温用2x SSC,0.1%SDS洗涤膜30分钟,然后68℃用0.5x SSC和0.1%SDS进行各30分钟的两次洗涤。-70℃,用增感屏使膜暴露于X-射线胶片(Kodak XAR-2或等效物)24-48小时。该方法当然可采用等价条件和参数,而仍能维持所需的严谨性杂交条件。
该本文中通篇使用的术语“包含”某序列X的DNA或蛋白质,指至少包含或含有序列X的DNA或蛋白质,而其它核苷酸或氨基酸序列,例如编码可选择标记蛋白的核苷酸序列、编码转运肽的核苷酸序列、和/或5′前导序列或3′尾随序列可包含在5’(或N-末端)和/或3’(或C-末端)端。类似地,本申请文本和权利要求书中通篇使用的术语“包含”或“含有”应理解为暗示包括所述整数或步骤或整数或步骤的组合,但不排除任何其它整数或步骤或整数或步骤的组合。
本发明方法包括在叶绿体内产生乙醇酸脱氢酶活性。这可通过以下方式实现:将编码乙醇酸脱氢酶活性的一种或多种核酸引入植物细胞的核基因组,然后将该蛋白的一种或多种编码序列与编码叶绿体转运肽的核酸融合。或者,可用编码相应酶的一种或多种核酸直接转化叶绿体基因组而在叶绿体中产生乙醇酸脱氢酶活性。
本领域熟知转化植物细胞或植物组织,特别是水稻植物细胞的通用技术。一系列方法包括用附着有DNA序列的颗粒轰击细胞、原生质体或组织。另一系列方法包括利用***根癌土壤杆菌(Agrobacterium tumefaciens)Ti质粒或发根土壤杆菌(Agrobacterium rhizogenes)Ri质粒中的嵌合基因作为转运入植物的工具。可采用其它方法,例如显微注射或电穿孔或直接用PEG沉淀。技术人员可选择任何合适的方法和工具转化植物细胞或植物,特别是水稻植物细胞或植物。对于水稻,优选实施土壤杆菌介导的转化(Hiei等.,1994和Hiei等.,1997,通过引用纳入本文)、电穿孔(美国专利5,641,664和美国专利5,679,558,通过引用纳入本文)或轰击(Christou等.,1991,通过引用纳入本文)。转化单子叶植物,特别是水稻的合适技术可参见通过引用纳入本文的WO 92/09696中的描述。
为在植物细胞中表达编码具有本发明所需酶活性的一种或多种多肽的一种或多种核酸,可采用任何方便的调控序列。这种调控序列将提供转录和翻译起始以及终止区,所述转录启动可以是组成型或诱导型启动。将编码区操作性连接于此类调控序列。合适的调控序列的代表是组成型35S启动子。或者,可采用组成型泛素启动子,特别是玉米泛素启动子(GenBank:gi19700915)。诱导型启动子的例子有RUBISCO小亚基的光诱导型启动子和“光捕获叶绿素复合体结合蛋白(lhcb)”的启动子。优选采用水稻gos2基因的启动子区,包括GOS2基因的5’UTR和内含子(de Pater等.,1992)、水稻核酮糖-1,5-二磷酸羧化酶小亚基基因的启动子区(Kyozuka J.等.,1993)或水稻肌动蛋白1基因的启动子区(McElroy D.等.,1990)。
根据本发明,该启动子还可联用位于启动子与编码序列之间的其它调控序列,例如转录激活子(“增强子”),例如专利申请WO 87/07644所述的烟草花叶病毒(TMV)或Carrington和Freed 1990所述的烟草蚀纹病毒(TEV)的翻译激活子,或内含子,例如玉米的adh1内含子或水稻肌动蛋白的内含子1。
作为调控终止子或聚腺苷酸化序列,可采用细菌来源的任何相应序列,例如根癌土壤杆菌的nos终止子,病毒来源的任何相应序列,例如CaMV 35S启动子,或植物来源的任何相应序列,例如组蛋白终止子,见专利申请EP 0 633317所述。
在优选转化核基因组的本发明的一个具体实施方式中,将编码叶绿体转运蛋白的核酸与编码乙醇酸脱氢酶的核酸序列5’端(融合),该转运肽序列位于启动子区域与编码乙醇酸脱氢酶的核酸之间,从而允许转运肽/乙醇酸脱氢酶融合蛋白表达。该转运肽能将乙醇酸脱氢酶引入质体中,更具体地说叶绿体中,当乙醇酸脱氢酶进入质体时,该融合蛋白在转运肽与乙醇酸脱氢酶之间被切断。转运肽可以是一种肽,例如EPSPS转运肽(描述见美国专利5,188,642)或植物核酮糖双羧化酶/加氧酶小亚基(RuBisCO ssu)的转运肽,例如衍生自马铃薯(Solanum tuberosum)的核酮糖-1,5-二磷酸羧化酶基因的叶绿体转运肽(GenBank:G68077,氨基酸1-58),如果需要,可包括成熟RuBisCO ssu的N-末端部分几个氨基酸(EP 189707),或者马铃薯rbcS1基因的叶绿体靶向肽(gi21562)。转运肽可以是完全天然产生(野生型)的转运肽,其功能片段、其功能突变体。也可以是嵌合型转运肽,其中至少两种转运肽以功能方式彼此连接或者不同转运肽的一部分以功能方式彼此连接。此类嵌合型转运肽的一个例子包括与玉米RuBisCO ssu的N-末端部分融合,与玉米RuBisCO ssu的转运肽融合的葵花籽RuBisCO ssu的转运肽,如专利EP 508909所述。
本领域技术人员能构建适合实施本发明的核酸,其包含编码成熟(即,不含转运肽)乙醇酸羟化酶的核酸,为在水稻中表达作了优化或未作优化,其中可缺失或不缺失第一个ATG密码子(如果有的话),将其操作性连接于叶绿体转运肽。适合实施本发明的此类核酸的一个例子是为在水稻中表达而优化的操作性连接于编码嵌合型叶绿体转运肽序列的拟南芥乙醇酸脱氢酶DNA序列,如SEQ ID NO 9所述。
或者,可采用转化叶绿体基因组在叶绿体中直接表达所述多肽。将感兴趣核酸整合入叶绿体基因组中的方法是本领域熟知的,特别是基于同源重组机制的方法。本领域技术人员知道合适的载体和选择***。可将多肽的编码序列转移入单个载体中或一个构建物中,可将各开放读框融合于一个或几个聚顺反子RNA,其中在各个开放读框之前加入核糖体结合位点以便独立释放。可用于此类整合入叶绿体基因组的工具和方法的例子见,例如WO 06/108830,其内容通过应用纳入本文。
当将核酸直接整合入叶绿体基因组时,不需要转运肽序列。在该情况中,可将(Met)翻译起始密码子加入到编码成熟蛋白质的序列以确保启动翻译。
本发明的主题还有包含一种或多种核酸的水稻植物细胞、水稻植物组织或水稻植物,所述一种或多种核酸在叶绿体内可表达具有乙醇酸脱氢酶活性的一种或多种多肽。
将核酸引入水稻植物细胞、水稻植物组织或水稻植物的优选实施方式如上所述。
按照本发明,水稻植物细胞应理解成源自或在水稻植物中发现的任何细胞,其能形成未分化组织如胼胝体,分化组织如胚芽、植物的各部分、植物或种子,或是它们的一部分。
本发明还涉及含有转化细胞的水稻植物,特别是转化细胞再生的植物。可通过任何合适的方法获得再生。可引用以下专利和专利申请,特别是关于转化植物细胞和再生植物的方法:US 4,459,355、US 4,536,475、US 5,464,763、US 5,177,010、US 5,187,073、EP 267,159、EP 604662、EP 672752、US4,945,050、US 5,036,006、US 5,100,792、US 5,371,014、US 5,478,744、US5,179,022、US 5,565,346、US 5,484,956、US 5,508,468、US 5,538,877、US5,554,798、US 5,489,520、US 5,510,318、US 5,204,253、US 5,405,765、EP442174、EP 486233、EP 486234、EP 539563、EP 674725、WO 91/02071和WO 95/06128。
本发明还涉及通过培养和/或杂交上述再生植物产生的转化植物或其一部分,还涉及转化植物的种子,其特征是它们含有本发明的转化植物细胞。
本发明还涉及通过加工本发明的植物、其各部分或种子所获得的任何产田,例粗粉。例如,本发明包括加工本发明水稻种子获得的水稻谷粒,还包括进一步加工水稻种子或水稻谷粒获得的粗粉以及从所述粗粉获得的任何食品。
序列表:
SEQ ID NO 1:大肠杆菌gcl D的DNA序列
SEQ ID NO 2:SEQ ID NO 1编码的氨基酸序列
SEQ ID NO 3:大肠杆菌gcl E的DNA序列
SEQ ID NO 4:SEQ ID NO 3编码的氨基酸序列
SEQ ID NO 5:大肠杆菌gcl F的DNA序列
SEQ ID NO 6:SEQ ID NO 5编码的氨基酸序列
SEQ ID NO 7:编码成熟(即,不含转运肽)拟南芥乙醇酸脱氢酶的DNA序列,为在水稻中表达作了优化
SEQ ID NO 8:SEQ ID NO 7编码的氨基酸序列
SEQ ID NO 9:优化的拟南芥乙醇酸脱氢酶DNA序列,操作性连接于编码优化叶绿体转运肽序列
SEQ ID NO 10:SEQ ID NO 9编码的氨基酸序列
SEQ ID NO 11:编码成熟(即,不含转运肽)衣藻属乙醇酸脱氢酶的DNA序列
SEQ ID NO 12:SEQ ID NO 11编码的氨基酸序列
SEQ ID NO 13:编码截断的衣藻属乙醇酸脱氢酶的DNA序列
SEQ ID NO 14:SEQ ID NO 13编码的氨基酸序列
SEQ ID NO 15:编码集胞蓝细菌乙醇酸脱氢酶的DNA序列
SEQ ID NO 16:SEQ ID NO 15编码的氨基酸序列
实施例
实施例1:构建编码大肠杆菌GDH的植物表达载体
通过化学DNA合成获得大肠杆菌乙醇酸脱氢酶的glcD、glcE和glcF(gi/1141710/gb/L43490.1/ECOGLCC)亚基的编码序列。质粒pTTS84含有3个表达盒,编码这3个大肠杆菌GDH亚基。如de Pater等.(1992)所述,glcE由水稻gos2基因的启动子区域驱动,包括GOS2基因的5’UTR与内含子;如Kyozuka等.(1993)所述,glcF由水稻核酮糖-1,5-二磷酸羧化酶小亚基基因的启动子区域驱动;glcD由水稻肌动蛋白1基因的启动子区域驱动(McElroy等.,1990)。如EP 0508909所述,这三个大肠杆菌GDH亚基基因各含有优化的编码转运肽(OTP)叶绿体靶向序列的序列。质粒pTTS84包含含有p35S启动子和3’nos终止区的bar表达盒。
实施例2:构建编码拟南芥GDH的植物表达载体
通过化学DNA合成获得拟南芥(At5g06580)GDH编码区的编码序列。设计合成基因时,排除了推定线粒体靶向序列的编码序列,以OTP叶绿体靶向序列的编码序列替换。利用该合成基因制备了不同载体。在质粒pTTS86中,该基因由p35S启动子驱动,而在质粒pTTS87中,如Kyozuka等.(1993)所述,采用水稻核酮糖-1,5-二磷酸羧化酶小亚基基因的启动子区域,。这两种质粒均包含含有p35S启动子和3’nos终止区的bar表达盒。
实施例3:植物转化和再生
受者土壤杆菌菌株ACH5C3(pGV4000)携带有删除了T-区域的不致癌(无害的)Ti质粒。该Ti质粒携带有使中间克隆载体的T-DNA区域转移至植物基因组所必需的功能vir基因。
在大肠杆菌中构建中间克隆载体(例如,pTTS84、pTTS86、pTTS87)。通过热激将其转移至受者根癌土壤杆菌中。此中间克隆载体经土壤杆菌介导的基因转移可导致T-DNA边界重复序列之间的DNA片段转移至植物基因组。
作为转化的靶组织,必须采用PCT专利公布号WO 92/09696中所述技术从切割成小片的日本和印度水稻品种获得不成熟胚芽或胚衍生胼胝体。将土壤杆菌与该水稻组织共同培养一定天数,然后用合适的抗生素除去土壤杆菌。将草铵膦(glufosinate ammonium)(含5mg/L草铵膦(phosphinothricin))加入水稻组织培养基,选择转化的水稻细胞。
将生长在含草铵膦培养基上的胼胝体转移至再生培养基。发育成含根和枝条的植株时,将它们转移至土壤中置于温室内。
实施例4:叶绿体的分离和酶试验
采用Kleffmann等.,2007所述方法分离得到完整的叶绿体。这些制品不含污染的过氧化氢酶和活性延胡索酸酶(>95%纯度)。如Lord J.M.1972所述检测乙醇酸脱氢酶活性。将100μg叶绿体蛋白质提取物加入100μmol磷酸钾(pH 8.0)、0.2μmol DCIP、0.1ml 1%(w/v)PMS和10μmol乙醇酸钾液中,终体积2.4ml。在固定的时间间隔加入0.1ml 12M HCl以终止各试验。静置10分钟后,加入0.5ml 0.1M苯肼-HCl。将该混合物再静置10分钟,然后检测因乙醛酸苯腙形成导致的324nm消光。
实施例5:叶绿体提取物中标记乙醇酸的CO2释放
在紧闭的15-ml反应试管中,将1μCi[1,2-14C]-乙醇酸盐(哈特曼分析公司(Hartmann Analytics))加入50μg叶绿体蛋白提取物中。释放的CO2被与15-ml试管内壁相连的500-μl反应管内的0.5M NaOH吸收。培育样品5小时,用注射器不时混合反应管中的气相。
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Claims (30)
1.一种提高水稻植物生物质产量和/或种子产量和/或固碳的方法,包括将编码具有乙醇酸脱氢酶活性的一种或多种多肽的一种或多种核酸引入水稻植物细胞中,引入的所述一种或多种核酸可导致从头表达具有乙醇酸脱氢酶活性的一种或多种多肽,所述的一种或多种多肽定位于所产生的植物的叶绿体中。
2.如权利要求1所述的方法,其特征在于,所述的一种或多种核酸被引入到水稻植物细胞的核基因组中,所述的一种或多种核酸编码一种或多种多肽,这种多肽含有使所述多肽靶向叶绿体的氨基酸片段。
3.如权利要求1或2所述的方法,其特征在于,具有乙醇酸脱氢酶活性的所述一种或多种多肽衍生自大肠杆菌(E.coli)glc操纵子。
4.如权利要求3所述的方法,其特征在于,所述多肽包含与SEQ ID NO:2、4或6所示序列至少有60%序列相同性的氨基酸序列。
5.如权利要求3所述的方法,其特征在于,所述核酸包含与SEQ ID NO:1、3或5所示多核苷酸序列至少有60%序列相同性的多核苷酸序列。
6.如权利要求1或2所述的方法,其特征在于,具有乙醇酸脱氢酶活性的所述多肽是植物乙醇酸脱氢酶。
7.如权利要求6所述的方法,其特征在于,具有乙醇酸脱氢酶活性的所述多肽是拟南芥乙醇酸脱氢酶。
8.如权利要求7所述的方法,其特征在于,所述多肽包含与SEQ ID NO:8所示序列至少有60%序列相同性的氨基酸序列。
9.如权利要求7所述的方法,其特征在于,所述一种或多种核酸序列包含与SEQ ID NO:7所示多核苷酸序列至少有60%序列相同性的多核苷酸序列。
10.如权利要求1所述的方法,其特征在于,具有乙醇酸脱氢酶活性的所述一种或多种多肽是藻类乙醇酸脱氢酶。
11.如权利要求10所述的方法,其特征在于,具有乙醇酸脱氢酶活性的所述一种或多种多肽是衣藻属(Chlamydomonas)或集胞蓝细菌(Synechocystis)乙醇酸脱氢酶。
12.如权利要求11所述的方法,其特征在于,所述一种或多种多肽包含与选自下组的序列有至少60%序列相同性的氨基酸序列:SEQ ID NO:12、SEQ ID NO:14和SEQ ID NO:16。
13.如权利要求11所述的方法,其特征在于,所述一种或多种核酸序列包含与选自下组的多核苷酸序列有至少60%序列相同性的多核苷酸序列:SEQ ID NO:11、SEQ ID NO:13和SEQ ID NO:15。
14.一种转基因水稻植物,其包含编码具有乙醇酸脱氢酶活性的一种或多种多肽的一种或多种核酸,其中所述一种或多种多肽定位于所述水稻植物的叶绿体中。
15.如权利要求14所述的水稻植物,其特征在于,所述的一种或多种多肽各自含有使所述多肽靶向叶绿体的氨基酸序列。
16.如权利要求14或15所述的水稻植物,其特征在于,具有乙醇酸脱氢酶活性的所述多肽衍生自大肠杆菌glc操纵子。
17.如权利要求16所述的水稻植物,其特征在于,所述多肽包含与SEQ IDNO:2、4或6所示序列至少有60%序列相同性的氨基酸序列。
18.如权利要求16所述的水稻植物,其特征在于,所述一种或多种核酸序列包含与SEQ ID NO:1、3或5所示多核苷酸序列至少有60%序列相同性的多核苷酸序列。
19.如权利要求14所述的水稻植物,其特征在于,具有乙醇酸脱氢酶活性的所述一种或多种多肽是植物乙醇酸脱氢酶。
20.如权利要求19所述的水稻植物,其特征在于,具有乙醇酸脱氢酶活性的所述一种或多种多肽是拟南芥乙醇酸脱氢酶。
21.如权利要求20所述的水稻植物,其特征在于,所述一种或多种多肽包含与SEQ ID NO:8所示序列至少有60%序列相同性的氨基酸序列。
22.如权利要求20所述的水稻植物,其特征在于,所述一种或多种核酸序列包含与SEQ ID NO:7所示DNA序列至少有60%序列相同性的多核苷酸序列。
23.如权利要求14所述的水稻植物,其特征在于,具有乙醇酸脱氢酶活性的所述一种或多种多肽是藻类乙醇酸脱氢酶。
24.如权利要求23所述的水稻植物,其特征在于,具有乙醇酸脱氢酶活性的所述一种或多种多肽是衣藻属或集胞蓝细菌乙醇酸脱氢酶。
25.如权利要求24所述的水稻植物,其特征在于,所述一种或多种多肽包含与选自下组的序列有至少60%序列相同性的氨基酸序列:SEQ ID NO:12、SEQ ID NO:14和SEQ ID NO:16。
26.如权利要求24所述的水稻植物,其特征在于,所述一种或多种核酸序列包含与选自下组的DNA序列有至少60%序列相同性的多核苷酸序列:SEQ ID NO:11、SEQ ID NO:13和SEQ ID NO:1。
27.水稻种子,其特征在于,该种子获自权利要求书14-26中任一项所述的转化植物。
28.通过加工权利要求27所述水稻种子获得的水稻谷物。
29.通过加工权利要求27所述水稻种子或权利要求28所述水稻谷物获得的粗粉。
30.从权利要求29所述粗粉获得的食品。
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CN110628810B (zh) * | 2019-08-13 | 2022-06-28 | 浙江大学 | 一种提高植物光合效率的方法 |
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