CN101146439A - 耐旱植物 - Google Patents
耐旱植物 Download PDFInfo
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- CN101146439A CN101146439A CNA2006800090389A CN200680009038A CN101146439A CN 101146439 A CN101146439 A CN 101146439A CN A2006800090389 A CNA2006800090389 A CN A2006800090389A CN 200680009038 A CN200680009038 A CN 200680009038A CN 101146439 A CN101146439 A CN 101146439A
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Abstract
本发明涉及耐旱植物的开发。本发明涉及在衰老诱导型启动子的控制下,表达参与细胞分裂素合成的蛋白质的转基因植物的制备。
Description
相关申请的交叉引用
本申请要求US申请60/664,035的优先权,其内容纳入本文作为参考。
在联邦资助下进行研究和开发的发明权利的声明
无
参考“序列表”,在磁盘上提交表格或计算机程序列表附件
无
发明背景
生理学和基因研究表明,衰老是一种受到高度调节的过程(Nooden,Senescence and Aging in Plants(植物的衰老与老化),(L.D.Nooden和A.C.Leopold编),第391-439页,Academic Press,San Diego,Calif.,1988;Thomas等,Ann.Rev.Plant Physiol.31:83-111,1980)。分子研究提示,基因表达的改变与衰老进程有关。例如,衰老期间编码参与光合作用的蛋白质的mRNA水平降低(Bate等,J.Exp.Bot.42:801-811,1991;Hensel等,Plant Cell 5:553-564,1993;Jiang等,Plant Physiol.101:105-112,1993),而编码认为参与衰老进程的蛋白质的基因mRNA水平升高(Graham等,Plant Cell4:349-357,1992,Hensel等,Plant Cell 5:553-564,1993;Kamachi等,Plant Physiol.93:1323-1329,1992;Taylor等,Proc.Natl.Acad.Sci.USA90:5118-5122,1993)。
已提示,可利用衰老特异性启动子来驱动所选衰老基因的表达。例如,美国专利5,689,042采用的一种基因构建物包含衰老特异性启动子SAG12,其可操作地连接于天然不与该启动子序列相连的编码土壤杆菌属异戊基转移酶(IPT)的DNA序列。含此构建物的转基因植物通过SAG12启动子所驱使的IPT表达能保持绿叶更长时间。已知IPT能提高细胞分裂素水平,细胞分裂素是一种衰老期间其浓度减低的植物激素,因而在控制叶片衰老中可能起作用。
类似地,Gan和Amasino表明,通过自身调节细胞分裂素产生的可抑制叶片衰老(Gao等,Science270:1986-1988,1995)。还鉴定了其它衰老诱导型启动子。例如,肾豆(Phaseolus vulgaris)的SARK启动子在WO99/29159和Hajouj等,Plant Physiol.124:1305-1314(2000)中描述。
一个内在调节感兴趣基因表达的有用而理想的方面是,能够只调节经历衰老的那些细胞中细胞分裂素的表达,从而保持正常细胞不受影响并避免细胞分裂素过度产生的可能导致的不良作用。
虽然已证明利用SAG12来控制IPT表达能够控制叶片衰老,但是,对这类植物的其它表型尚未很好地了解。本发明解决了这些和其它需要。
发明概述
本发明涉及耐旱植物的开发。本发明方法提供了耐旱性能提高和具有其它有益特征如产量提高的植物。此外,本发明的植物也具有更高的的水分利用效率。本发明涉及能在衰老诱导型启动子控制下表达参与细胞分裂素合成的蛋白质的转基因植物的制备。
本发明方法包括:(a)将包含SARK启动子的重组表达盒引入植物群中,该SARK启动子可操作地连接于编码参与细胞分裂素合成的蛋白质的核酸序列;和(b)选择能耐受干旱应激的植物。引入表达盒的步骤可采用任何已知方法进行。例如,通过有性杂交(sexual cross)或采用土壤杆菌属引入该表达盒。
SARK启动子可从肾豆方便地制备,它的序列与SEQ ID NO:1至少95%相同。在一些实施方式中,参与细胞分裂素合成的蛋白质是土壤杆菌属的异戊烯基转移酶(IPT)。一种示例性序列(IPT)与SEQ ID NO:3至少95%相同。
可将该序列引入能用重组表达构建物转染的任何植物中。本文阐述其在烟草中的表达。本发明常规使用的其它植物包括草坪植物。
附图说明
图1表明,野生(WT)烟草植物在无水干旱应激5和7天内出现进行性叶片萎蔫,而两个独立的转基因株不显示萎蔫症状。
图2A-2L示出了4月龄烟草植株遭受干旱应激后复水(rewatering)。野生型(图2A)和转基因植株(图2B和2C)在干旱7天后都出现了叶片萎蔫症状。干旱18天后,WT(图2D)和两个转基因株(图2E和2F)的叶片萎蔫症状都变得更加明显。植株复水7天对萎蔫的WT植物几乎没有作用(图2G),但诱导了转基因株的部分恢复(图2H和2I),转基因株T4-24(图2I)显示比转基因株T2-36(图2H)恢复得更好。植株复水14天不能使WT植株恢复(图2J),却使两个转基因株完全恢复(图2K和2L)。
图3显示了经14天复水后图2所示植物的鲜重。数值是平均值±SD(n=40)。
图4显示了经干旱应激和复水5天后的WT鼠耳芥属植株和T1转基因植株(pSARK:IPT)。
发明详述
定义
如本文所用,术语“耐旱”或“抗旱”表示植物从经历干旱应激(即几天时间没有或几乎没有水)期后恢复的能力。通常,干旱应激至少为5天,可长达18-20天。
术语“水分利用效率”表示植物在低于正常水分含量(通常约一半)的延长的时间范围内生长基本上不受到妨碍的能力。
术语“衰老”(也称为程序性细胞死亡)表示植物细胞和组织丧失对组织和功能的遗传控制的主动过程。
术语“衰老相关基因”表示参与衰老的基因。衰老过程可诱导(或改变)该基因的表达。
如本文所用,术语“启动子”包括所有能驱使编码序列在植物细胞中转录的序列。因此,本发明构建物中所用的启动子包括顺式作用的转录控制元件以及与基因转录时机和/或转录速率的调节或调整有关的调节序列。例如,启动子可以是顺式作用的转录控制元件,包括增强子、启动子、转录终止子、复制起点、染色体整合序列、5’和3’非翻译区、或内含子序列,它们与转录调节有关。这些顺式作用的序列通常与蛋白质或其它生物分子相互作用以进行(开/关、调节、调整等)转录。
“可诱导成熟的启动子”是赋予可操作连接的编码序列暂时特异性、从而在成熟完成和/或衰老过程期间发生表达的启动子。
“衰老诱导型启动子”是能赋予可操作连接的编码序列暂时特异性、从而在衰老过程期间驱使表达的启动子。
术语“植物”包括全植物、枝条营养器官/结构(例如,叶、茎和块茎)、根、花和花样器官/结构(例如,苞片、萼片、花瓣、雄蕊、心皮、花药和胚珠)、种子(包括胚胎、胚乳和种皮)和果实(成熟子房)、植物组织(例如,维管组织、地面组织等)和细胞(例如,保卫细胞、卵细胞、毛状体等)、以及它们的后代。本发明方法中可使用的植物类型通常广泛地包括适应转化技术的低等和高等植物类型,包括被子植物(单子叶和双子叶植物)、裸子植物、厥类植物和多细胞藻类。其包括各种多倍体水平的植物,例如非整倍体、多倍体、二倍体、单倍体和半合子。
如下所述,当排列对比两条核酸序列或多肽的最大相应时,如果两条序列中的核苷酸或氨基酸残基序列分别相同,则认为这两条核酸序列或多肽“相同”。术语“互补”在这里用于表示该序列与所有或一部分参比多核苷酸序列互补。
可用以下算法进行比较序列的最佳排列对比:Smith和Waterman Add.APL.Math.2:482(1981)的局部同源性算法,Needle man和Wunsch J.MoI.Biol.48:443(1970)的同源性排列对比算法,Pearson和Lipman Proc.Natl.Acad.Sci.(U.S.A.)85:2444(1988)的相似性搜索方法,这些算法的计算机实现(GAP、BESTFIT、BLAST、FASTA和TFASTA,Wisconsin Genetics软件包,GeneticsComputer Group(GCG),575Science Dr.,Madison,WI),或通过检查。
通过在比较窗口内比较两条最佳排列对齐序列来确定“序列相同百分比”,其中,与参比序列(不包括插入或缺失)相比,比较窗口中的多核苷酸序列部分可包括插入或缺失(即缺口)以实现两个序列的最佳排列对齐。通过以下方法计算百分比:确定两条序列中核酸碱基或氨基酸残基相同的位置数得到匹配位置数,将匹配位置数除以该比较窗口的总位置数,并将结果乘以100,得到序列相同百分比。
术语多核苷酸序列“基本相同”表示采用本文所述程序,优选用标准参数的BLAST,与参比序列相比,多核苷酸包含至少70%序列相同、至少80%序列相同、至少85%、90%、93%、95%或97%相同,如下所述。技术人员将明白,考虑到密码子简并、氨基酸相似性、读码框定位等,可适当调节这些数值以确定两条核苷酸序列编码蛋白质的对应相同性。为此目的氨基酸序列基本相同通常指用本文所述程序,与参比序列相比,至少40%、60%、70%、80%、90%、95%或97%序列相同。“基本相似”的多肽如上所述具有共同的序列,只是不相同的残基位置因保持性氨基酸(取代)变化而不同。保守氨基酸取代指,具有相似侧链残基的互换性。例如,具有脂肪族侧链的一组氨基酸有甘氨酸、丙氨酸、缬氨酸、亮氨酸和异亮氨酸;具有脂肪族-羟基侧链的一组氨基酸有丝氨酸和苏氨酸;具有含酰胺侧链的一组氨基酸有天冬酰胺和谷氨酰胺;具有芳香族侧链的一组氨基酸有苯丙氨酸、酪氨酸和色氨酸;具有碱基侧链的一组氨基酸有赖氨酸、精氨酸和组氨酸;具有含硫侧链的一组氨基酸有半胱氨酸和蛋氨酸。优选的保守氨基酸取代分组是:缬氨酸-亮氨酸-异亮氨酸、苯丙氨酸-酪氨酸、赖氨酸-精氨酸、丙氨酸-缬氨酸、天冬氨酸-谷氨酸以及天冬酰胺-谷氨酰胺。
核苷酸序列基本相同的另一个指标是,在严谨条件下,两个分子是否能相互杂交、或与第三核酸杂交。严谨条件是序列依赖性的,在不同状况下条件不同。通常,在确定的离子强度和pH下,严谨条件比特定序列的热解链温度点(Tm)低约5℃。Tm是50%的目标序列与完美匹配探针杂交的温度(在确定的离子强度和pH下)。严谨条件一般为pH7、盐浓度约0.02摩尔,温度至少约60℃的条件。
在本说明书中,杂交的严谨条件,包括63℃用0.2X SSC至少洗涤20分钟一次,或等同的条件。适度严谨条件包括温度至少约50℃,通常约55℃,用0.2X SSC至少洗涤一次(通常2次)20分钟,或等同的条件。
术语“表达盒”指能在任何细胞中,包括例如植物细胞、原核细胞、酵母、真菌、昆虫或哺乳动物细胞中,体外或体内结构性地或可诱导地表达本发明核酸序列的任何重组表达系统。该术语包括线形或环形表达系统。该术语包括所有载体。表达盒可保持游离或整合到宿主细胞基因组中。表达盒可具有自身复制的能力或没有,只驱使在细胞中短暂表达。该术语包括只包含重组核酸转录所需最少元件的重组表达盒。
表达盒的制备
本发明表达盒包含衰老诱导型启动子。下面示例性地描述了肾豆的SARK启动子。该启动子在WO99/29159和Hajouj等的Plant Physiol.124:1305-1314(2000)中描述。其它合适的启动子包括鼠耳芥属SAG12启动子,如Gan等,Science,270:1986-8(1995)所述。技术人员将明白本发明构建物中使用的具体启动子,只要是衰老可诱导表达的。因此,例如,可将其它物种的SARK或SAG12基因同类物方便地用于本发明表达盒中。
可利用启动子来驱动编码能抑制或减缓衰老过程的蛋白质的表达。在一些优选的实施方式中,该基因编码参与细胞分裂素合成的蛋白质。例如,异戊二烯转移酶(IPT)能催化细胞分裂素的合成。IPT序列的例子参见:Crespi等,EMBOJ.11:795-804(1992);Goldberg等,Nucleic Acids.Res.12:4665-4677(1984);Heide Kamp等,Nucleic Acids Res.,11:6211-6223(1983);Strabala等,Mol.Gen.Genet.216:388-394(1989)GenBank登录号:NC_003308,以及X14410(见SEQID NO:2和3)。
转基因植物的制备
可通过多种常规技术将本发明DNA构建物引入所需植物宿主的基因组中。例如,可采用诸如电穿孔和显微注射植物细胞原生质体的技术将DNA构建物直接引入植物细胞基因组DNA中,或者可采用诸如DNA粒子轰击的冲击方法将DNA构建物直接引入植物组织。或者,可将DNA构建物可与合适的T-DNA侧翼区组合并引入常规根癌土壤杆菌宿主载体中。当该细菌感染细胞时,根癌土壤杆菌宿主的毒力功能将直接介导构建物和毗邻的标记物插入植物细胞DNA中。
显微注射技术是本领域已知的,在科学文献和专利文献中已很好地描述。采用聚乙二醇沉淀法引入DNA构建物参见Paszkowski等,Embo J.3:2717-2722(1984)。电穿孔技术在Fromm等,Proc.Natl.Acad.Sci.USA82:5824(1985)中描述。冲击转化技术在Klein等,Nature327:70-73(1987)中描述。
根癌士壤杆菌介导的转化技术,包括消除和使用二元载体已在科学文献中很好地描述。例如,参见Horsch等,Science 233:496-498(1984)和Fraley等,Proc.Natl.Acad.Sci.USA80:4803(1983)。
可培养由任何上述转化技术得到的转化的植物细胞,再生具有转化基因型、因而具有所需表型如无核性的全植物。这种再生技术依赖于操纵组织培养生长培养基中的某些植物激素,通常依赖于与所需核苷酸序列一起引入的杀微生物剂和/或除草剂标记物。由培养的原生质体再生植物在Evans等,原生质体的分离与培养(Protoplasts Isolation and Culture),《植物细胞培养手册》(Handbookof Plant Cell Culture),第124-176页,MacMillilan Publishing Company,纽约,1983;和植物结合与再生(Binding,Regeneration ofPlants),《植物原生质体》(Plant Protoplasts),第21-73页,CRC Press,Boca Raton,1985中描述。再生物也可由植物愈伤组织、外植体、器官或其部分得到。这种再生技术大致在Klee等,Ann.Rev.of Plant Phys.38:467-486(1987)中描述。
技术人员将明白,表达盒稳定结合入转基因植物并证实可操作之后,可通过有性杂交引入其它植物。根据待交叉的物种,可采用许多标准育种技术。
可利用本发明的表达盒赋予基本上任何植物耐旱性能。因此,本发明可应用于许多植物,包括天门冬属、颠茄属、燕麦属、芸苔属、柑桔属、西瓜属、辣椒属、黄瓜属、南瓜属、胡罗卜属、草莓属、大豆属、棉属、向日葵属、Heterocallis、大麦属、天仙子属、莴苣属、Linurn、Loliutn、番茄属、苹果属、木薯属、马郁兰属(Majorana)、苜蓿属、烟草属、稻属、Panieum、Pannesetum、鳄梨属、豌豆属(Pisum)、犁属、樱桃属、萝卜属、黑麦属、千里光属、芥子属、茄属(Solarium)、高梁属、胡芦巴属、小麦属、葡萄属、豇豆属和玉蜀黍属的植物。
在一些实施方式中,可使用本发明方法赋予草坪植物耐旱性能。许多草坪植物是本领域技术人员已知的。例如,可使用牛毛草、羊茅属(例如高羊茅,紫羊茅,硬羊茅和细叶羊茅)。其它草坪植物包括草地早熟禾(Kentucky bluegrassPoa pratensis)和匍匐剪股颖(creeping bentgrass Agrostis palustris)。
技术人员将明白,可使用许多植物物种作为预测转基因表达在其它植物中的表型效应的模型。例如,众所周知烟草(Nicotiana)和鼠耳芥属(Arabidopsis)植物是有用的转基因表达模型,尤其是在其它双子叶植物中。
可根据许多公知技术评价耐旱性能。例如,可使植物在低于最佳水分供应的条件下生长。通过许多标准方法中的任一种来确定耐旱性能,例如膨压、生长、产量等。在一些实施方式中,可常规使用实施例部分所述方法。
本说明书的所有出版物和专利申请纳入本文作为参考,如同各个出版物或专利申请具体且单独纳入参考那样。虽然为了清楚理解的目的,上述说明以阐述和实例的方式进行了详细描述,但本领域技术人员借鉴本发明的内容不难明白,可进行某些改变和改进而不背离所附权利要求的精神的范围。
实施例
SARK(衰老相关受体激酶)基因的鉴定
通过Hajouj等,(2000)所述的差异显示技术分离肾豆SARK基因的cDNA。SARK的全长cDNA序列揭示,其编码丝氨酸/苏氨酸蛋白激酶。观察到疏水跨膜区域,提示SARK基因编码受体激酶(Hajouj等,2000)。RNA印迹(Northernblot)分析揭示,叶片衰老早期SARK基因上调。SARK基因表达开始于附着的豆叶衰老出现任何视觉征兆(变黄)之前。在暗处培养时,叶盘加速变黄。
与完整的附着叶片相似,在看见叶片变黄之前衰老过程开始时,SARK基因的转录水平的升高(Hajouj et al(2000))。因此,我们将SARK基因视作衰老相关基因(SAG)。而且,在附着或脱落叶片的衰老非常早期已出现SARK转录物,提示SARK对衰老过程有调节作用。制备针对SARK蛋白的抗体,用于蛋白质印迹(western blot)分析。SARK蛋白水平的瞬时表达模式与其RNA(产生)模式相似,进一步支持以下观点:SARK蛋白与脱落和附着叶片的衰老过程有关。
分离SARK启动子
用Maniatis等(Molecular cloning,实验室手册第二版)所述的反向PCR技术分离得到SARK基因的5’-末端上游区域。根据生产商的说明书,用植物DNA提取试剂盒(Scotlab)分离得到肾豆基因组DNA。用限制性内切酶Xbal消化该DNA重连接(relegation)后进行再循环(扩增)。采用以下引物进行PCR反应。
1)5’ACGTCCAACCAAAGACC3’
2)5’TCTGCAGCTAGTGCGATATCC3’
在下面的条件下进行PCR反应:94℃30秒,55℃30秒,72℃2分钟,进行40个循环,然后72℃10分钟。
扩增1.4kb的DNA片段。该片段的DNA序列分析揭示,其包含340bp的SARK DNA5’末端。该序列揭示,SARK基因5’末端附近有一个内含子存在。
为了分离SARK基因5’末端上游的较长片段,进行热不对称交错(TAIL)PCR技术,如Liu等(Plant J.8:457-463)所述。采用以下三种PCR引物:
1)5’TCTGCAGCTAGTGCGATATCC3’
2)5’TTGGTGGATGAATAATGGAG3’
3)5’ACTGTAACTCACAAATTAGA3’
进行三种PCR反应以扩增目标序列。
对该PCR产物进行序列分析。鉴定到约800bp的cDNA5’末端,见SEQ IDNO:1。将该PCR片段克隆入pUC57中。
产生携带pSARK:IPT构建物的转基因植物
使土壤杆菌属ipt(异戊二烯基转移酶)融合于SARK启动子,所述ipt是能催化细胞分裂素生物合成中限速步骤的酶。Gan和Amasino(Science270;1996(1995)已证明,鼠耳芥属SAG12基因(衰老相关基因)连接于ipt基因时,可诱导细胞分裂素合成而延迟叶片衰老进程。将土壤杆菌属IPT可操作地连接于830个核苷酸长度的SARK基因启动子并以HinIII/XbaI片段的形式引入pBI101(ClonTech),形成pBI p-SARK:IPT。电穿孔进行土壤杆菌转化。
烟草转化
通过土壤杆菌属介导的转化方法转化植物。在SARK启动子调节下表达土壤杆菌属异戊基转移酶(IPT)基因使得烟草叶片延迟衰老。该包含p-SARK-IPT的转基因烟草显示各个叶片和全植物水平上常规衰老的显著延迟。通常发芽后3-3.5个月WT植物开花并在4个月后开始显示最初叶片(在底部)变黄。然而,转基因植物显示显著延迟衰老,发芽后一直到10个月没有出现任何最初叶片变黄。
在黑暗条件下培养时,转基因烟草的脱落叶片也可显著延迟变黄。通常,脱落烟草叶片在黑暗中培养5-6天后显示初期变黄,10-12天后完全变黄。然而,转基因植物的脱落叶片黑暗中培养20天、甚至30天后不显示任何变黄征兆,它们仍然为绿色,虽然也可观察到初期变黄。这些结果表明,除附着叶片之外,转基因植物脱落叶片中细胞分裂素合成的自身调节机制也起作用。
鼠耳芥属转化
采用以下引物,通过Pfu turbo DNA聚合酶反应仪(Stratagene)进行pSARK:IPT的PCR扩增。
SARKIPF5’T T C C T T A G A T G C T G T C A C A A T C A3’
SARKIPTR5’G A A C A T C T T A T C C A G A T G A A G A C A G3’
PCR扩增模板是含pSARK:IPT的转基因烟草DNA。
根据生产商的说明书(Invitrogen),用TOPO克隆试剂盒将PCR产物(pSARK:IPT)克隆到Topo感受态细胞(DH5α-T1)中。
用Qiaprep试剂盒(Qiagen)进行DNA质粒小量制备。
用BgIII和EcoRI消化质粒,并与Cambia1380载体(CAMBIA,CanberraAustralia)连接。
使携带pSARK:IPT的Cambia载体电穿孔进入(DH5a)感受态细胞。用Qiaprep试剂盒(Qiagen)进行转染DH5α克隆的DNA质粒小量制备。使包含pSARK:IPT的Cambia载体电穿孔引入土壤杆菌属,用于植物转化。
通过真空渗透技术,用包含pSARK:IPT和潮霉素抗性基因(hptIIgene)的根癌士壤杆菌转化拟南芥植物,进行植物选择。
SARK基因启动子调节下异戊基转移酶(IPT)在烟草植物中的表达赋予了耐旱性能。
将携带pSARK:IPT转基因烟草植物在温室中培养生长2-3个月。在最初的3-4个月期间,转基因和WT植物之间没有观察到形态差异。
开始开花后,使3月龄烟草植株遭受干旱应激(罐子中不加水),持续5-16天。WT植株显示进行性叶片萎蔫(图1)。然而,转基因植株(两个独立株)在无水的干旱应激5和7天内不显示萎蔫症状(图1)。16天长期脱水导致WT植株严重不可逆的萎蔫,而携带pSARK:IPT的植株显示严重程度较低、可逆的萎蔫。复水(脱水植物的加水)使转基因pSARK:IPT植株恢复,而WT植株不能从干旱应激恢复(图1)。
野生型植株(WT)和两种携带pSARK-IPT的转基因烟草植株(T2-36和T4-24)在温室中生长5个月。最佳条件下生长的最初3-4个月期间,转基因和野生型植株之间没有观察到形态差异。开始开花后,使4月龄烟草植株遭受干旱应激(罐子中不加水),连续持续18天(图2,A-F)。野生型(图2A)和转基因植株(图2B和2C)在干旱7天后都显示叶片萎蔫。18天干旱后,WT(图2D)和两个转基因株(图2E和2F)中都出现了更加明显的叶片萎蔫症状。植株复水7天对萎蔫的WT植物几乎没有作用(图2G),却可诱导转基因株部分恢复(图2H和2I),转基因株T4-24(图2I)比转基因株T2-36(图2H)恢复得更好。植株复水14天不能使WT植物恢复(图2J),却使转基因株完全恢复(图2K和2L)。复水阶段结束时测定野生型和转基因植物的鲜重显示,转基因株的鲜重约为250克/植物,而野生型保持干燥,重量不超过转基因株的20%(图2)。图3示出了14天复水后图2所示植株的鲜重。值是平均值±SD(n=40)。
SARK基因启动子调节下IPT基因的表达赋予转基因鼠耳芥属株耐早性能。
拟南芥植物在23℃、长时间日照方案(16/8h)下生长。正常条件下生长的WT和转基因(pSARK:IPT)植株之间没有观察到形态和发育上的差异。但是,当两月龄植株(提前开花期)经受干旱应激(罐中不加入水)时,它们显示不同的应激耐受性。WT植物脱水12天后经历严重不可逆的萎蔫和叶片变黄,而10个独立的T1转基因植株(pSARK:IPT)株显示轻度萎蔫,并且复水5天后可从干旱应激恢复(图4)。
SEQ ID NO:1
TTCTTCCTTAGATGCTGTCACAATCATTTTCATTATTTTTATATTTGGTTTTACTGC
ACAAGTGACATAATGAGTGCTGAATTGTGGTATTGTGGGAACCTTAAGCAATAGT
TTCATTAGACCACTTGTGCAGGTTTTTGGGGTGGTAGAAGGAATGCTCGTTGTCT
CTGAATGAGTTCTATTTTCATCTTTAGAAACTAGTAATTTAGTTAGTTTTGGGTCT
CGTGGTTCTACAGAGGGTTGAGATACTTTTGAAGTATCTCTCTTTTATTATATTAT
ACTTTTTGCTGATAAAAAAAGGTAGGTAGTTTTTTTTGGAATATTTTGTAGGATTT
TGTGGAGGTGTTTGGTATAAGGATTGAAATATTTCAAAAATATTTCCATTTAATTT
ACTTTTTCTTATAAAAAAAATCCTCCATGAAACAAGATCATCTTCTAGAAACAAC
AAGTAATATATTAGAATCTCTTTCTGAATTTTCTCATTTGTGAGTTATAGTACTTT
TTTTCCAATAATAATTATAAGTGGTAAGATGTGTGGTTGTGGAAGTTGGAAGGAA
AGAAGGAAAGAAAGGTTAGTTTTTGTTTTGTATTTGAAAGTAAGTCAAGGTCATT
GGCTTAGGGTTCTACCACTGCAACTATTCCACATTGGCTTCTACCACTGCAATTAT
TCCACATTGGCTTG TACTGTAAGGACAAACCTTGGCATGTCAAATACTTTTCATC
ACATATAACCATATTATAAACTACTTTCCATCTCCATTATTCATCCACCAAAATCT
AGAGTCACTGAGAGTGCAGATAACACAATTCTCTAATATAAAAATCAGTTTGTAT
TCAATATACTGCAAAAAACTTATGGACCTGCATCTAATTTTCGGTCCAACTTGCA
CAGGAAAGACGACGACCGCGATAGCTCTTGCCCAGCAGACAGGGCTTCCAGTCC
TTTCGCTTGATCGGGTCCAATGCTGTCCTCAACTATCAACCGGAAGCGGACGACC
AACAGTGGAAGAACTGAAAGGAACGACGCGTCTCTACCTTGATGATCGGCCTCT
GGTGGAGGGTATCATCGCAGCCAAGCAAGCTCATCATAGGCTGATCGAGGAGGT
GTATAATCATGAGGCCAACGGCGGGCTTATTCTTGAGGGAGGATCCACCTCGTTG
CTCAACTGCATGGCGCGAAACAGCTATTGGAGTGCAGATTTTCGTTGGCATATTA
TTCGCCACAAGTTACCCGACCAAGAGACCTTCATGAAAGCGGCCAAGGCCAGAG
TTAAGCAGATGTTGCACCCCGCTGCAGGCCATTCTATTATTCAAGAGTTGGTTTA
TCTTTGGAATGAACCTCGGCTGAGGCCCATTCTGAAAGAGATCGATGGATATCGA
TATGCCATGTTGTTTGCTAGCCAGAACCAGATCACGGCAGATATGCTATTGCAGC
TTGACGCAAATATGGAAGGTAAGTTGATTAATGGGATCGCTCAGGAGTATTTCAT
CCATGCGCGCCAACAGGAACAGAAATTCCCCCAAGTTAACGCAGCCGCTTTCGA
CGGATTCGAAGGTCATCCGTTCGGAATGTATTAGGTTACGCCAGCCCTGCGTCGC
ACCTGTCTTCATCTGGATAAGATGTTCAGATC
SEQ ID NO:2
1 ggatcccgtt acaagtattg cacgttttgt aaattgcata ttaatgcaat ctggatgttt
61 aataacgaat gtaatggcgt agaaatatgt attttattgt atttatcttt cactatgttg
121 aagtttgcaa taatatgcta atgtaaaatt aaaaaattat gtactgccgc atttgttcaa
181 atggcgccgt tatttcaaaa atatctttga ttttgttacg aggacaacga ctgcaggaag
241 taaataaaag acgctgttgt taagaaattg ctatcatatg tgcccagcta tagggccatt
301 taagttcaat tgtgaaatag ccgcccttat tttgacgtct catcaaatca aatattaaaa
361 aatatctcac tctgtcgcca gcaatgatgt aataaccgca gaaaagtgag agtaaatcgc
421 ggaaaaacgt cgccgagtgg catgaatagc ggcctccgta ttgctgattt agtcagctt
481 atttgactta agggtgccct cgttagtgac aaattgcttt caaggagaca gccatgcccc
541 acactttgtt gaaaaacaag ttgccttttg ggaagaacct aaagccactt gctcttcaag
601 gaggaatatc gaggaagaga atataacagc ctctggtaca gacttctctt gtgcaaaaat
661 caatttgtat tcaacatatc gcaagaccga tggatctacg tctaattttc ggtccaactt
721 gcacaggaaa gacatcgact gcgatagctc ttgcccagca gactggcctc ccagtcctct
781 cgctcgatcg cgtccaatgc tgtcctcaac tatcaaccgg aagcgggcga ccaacagtgg
841 aagaactgaa aggaacgact cgtctgtacc ttgatgatcg ccctttggta aagggtatca
901 ttacagccaa gcaagctcat gaacggctca ttgcggaggt gcacaatcac gaggccaaag
961 gcgggcttat tcttgaggga ggatctatct cgttgctcag gtgcatggcg caaagtcgtt
1021 attggaacgc ggattttcgt tggcatatta ttcgcaacga gttagcagac gaggagagct
1081 tcatgagcgt ggccaagacc agagttaagc agatgttacg cccctctgca ggtctttcta
1141 ttatccaaga gttggttcaa ctttggaggg agcctcggct gaggcccata ctggaaggga
1201 tcgatggata tcgatatgcc ctgctatttg ctacccagaa ccagatcacg cccgatatgc
1261 tattgcagct cgacgcagat atggagaata aattgattca cggtatcgct caggagtttc
1321 taatccatgc gcgtcgacag gaacagaaat tccctttggt gggcgcgaca gctgtcgaag
1381 cgtttgaagg accaccattt cgaatgtgat agattgcacc agttttgttt cagacttgtc
1441 gctatttgaa taagatgttc gttctttgtt gtgttggtgt gttgtgatag aggcaagtgg
1501 tttgaaactt gtttttactg gtttattttc agtctcttgg acgatgtttt acaaatataa
1561 tattgtgaaa attgtggttt tatattcgta gaacgaaata aatggtaagt atagccgtta
1621 tcaaaattta gcaaaaattg ttaaaggttc ttttatgcgg tgaggttgtc gacttttcat
1681 cattgtcgcg taaggagtta cggatatcca taactgtaaa aacgccgcag aatttacggg
1741 tggtgcattt agtttgccgt tcaacatgat tttggcaata gttggtaacc aagcactagc
1801 caaccgttcg ataatcactt aatcgatgga accgttcagc tttccttcgt gaggctgctc
1861 ttgatgatga gctgccgtct agtttttata acgccgggtt acgcattata gacaagctt
SEQ ID NO:3
MDLRLIFFGPTCTGKTSTAIALAQQTGLPVLSLDRVQCCPQLSTGSGRPTVEELKGTTRLYLDDRPLVKGIITAKQ
AHERLIAEVHNHEAKGGLILEGGSISLLRCMAQSRYWNADFRWHIIRNELADEESFMSVAKTRVKQMLRPSAGLS
IIQELVQLWREPRLRPILEGIDGYRYALLFATQNQITTPDMLLQLDADMENKLIHGIAQEFLIHARRQEQKFPLVG
ATAVEAFEGPPFRM
Claims (10)
1.一种制备能耐受干旱应激的植物的方法,该方法包括:
(a)将包含SARK启动子的重组表达盒引入植物群中,所述SARK启动子可操作地连接于编码参与细胞分裂素合成的蛋白质的核酸序列;和
(b)选择能耐受干旱应激的植物。
2.如权利要求1所述的方法,其特征在于,所述引入步骤通过有性杂交实现。
3.如权利要求1所述的方法,其特征在于,所述引入步骤采用土壤杆菌属实现。
4.如权利要求1所述的方法,其特征在于,所述SARK启动子来自肾豆。
5.如权利要求3所述的方法,其特征在于,所述SARK启动子与SEQ ID NO:1至少95%相同。
6.如权利要求1所述的方法,其特征在于,所述参与细胞分裂素合成的蛋白质是异戊二烯转移酶。
7.如权利要求6所述的方法,其特征在于,所述编码异戊二烯转移酶的核酸序列来自土壤杆菌属。
8.如权利要求7所述的方法,其特征在于,所述异戊二烯转移酶与SEQ IDNO:3至少95%相同。
9.如权利要求1所述的方法,其特征在于,所述植物是双子叶植物。
10.如权利要求9所述的方法,其特征在于,所述植物是烟草。
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CN104884619A (zh) * | 2012-12-31 | 2015-09-02 | 创世纪种业有限公司 | 一种棉花异戊烯基转移酶ipt2及其编码基因与应用 |
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US20110065158A1 (en) * | 2006-12-08 | 2011-03-17 | The University Of York | Regulation of plant metabolism |
US10126760B2 (en) | 2011-02-25 | 2018-11-13 | Mks Instruments, Inc. | System for and method of fast pulse gas delivery |
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WO2017083757A1 (en) * | 2015-11-13 | 2017-05-18 | Board Of Regents Of The Nevada System Of Higher Education On Behalf Of The University Of Nevada, Reno | Methods of engineered tissue succulence in plants |
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US5689042A (en) | 1995-03-29 | 1997-11-18 | Wisconsin Alumni Research Foundation | Transgenic plants with altered senescence characteristics |
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US20050086718A1 (en) | 1999-03-23 | 2005-04-21 | Mendel Biotechnology, Inc. | Plant transcriptional regulators of abiotic stress |
AU2001259754A1 (en) * | 2000-05-10 | 2001-11-20 | Phycogen, Inc. | Transgenic plants incorporating_genes of zostera marina |
WO2003050287A2 (en) | 2001-12-10 | 2003-06-19 | Thomas Schmulling | Method for modifying plant morphology, biochemistry and physiology comprising expression of plant cytokinin oxidase |
AUPQ994600A0 (en) | 2000-09-06 | 2000-09-28 | Agriculture Victoria Services Pty Ltd | Manipulation of plant senescene |
MXPA04009778A (es) * | 2002-04-08 | 2004-12-13 | Pionner Hi Bred International | Exsercion de fibra sedosa mejorada bajo estres. |
US7138566B2 (en) | 2002-06-21 | 2006-11-21 | Regents Of The University Of California | Methods of modulating cytokinin related processes in a plant using B3 domain proteins |
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CN103108955A (zh) * | 2010-07-15 | 2013-05-15 | 工业研究与发展基金会有限公司 | 用于提高植物中非生物胁迫耐受性的核酸构建体 |
CN103108955B (zh) * | 2010-07-15 | 2015-11-25 | 工业研究与发展基金会有限公司 | 用于提高植物中非生物胁迫耐受性的核酸构建体 |
US10106812B2 (en) | 2010-07-15 | 2018-10-23 | Technion Research & Development Foundation Limited | Nucleic acid construct for increasing abiotic stress tolerance in plants |
CN104884619A (zh) * | 2012-12-31 | 2015-09-02 | 创世纪种业有限公司 | 一种棉花异戊烯基转移酶ipt2及其编码基因与应用 |
CN104884619B (zh) * | 2012-12-31 | 2017-09-08 | 创世纪种业有限公司 | 一种棉花异戊烯基转移酶ipt2及其编码基因与应用 |
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EP1863334B1 (en) | 2011-11-02 |
BRPI0607732B1 (pt) | 2017-11-07 |
AU2006226863A1 (en) | 2006-09-28 |
ES2376924T3 (es) | 2012-03-20 |
US9624503B2 (en) | 2017-04-18 |
ZA200707905B (en) | 2008-12-31 |
EP1863334A4 (en) | 2008-11-26 |
BRPI0607732A2 (pt) | 2010-03-16 |
CA2601605C (en) | 2014-09-09 |
AU2011202138A1 (en) | 2011-05-26 |
US20080282365A1 (en) | 2008-11-13 |
WO2006102559A2 (en) | 2006-09-28 |
EP1863334A2 (en) | 2007-12-12 |
EP2324703A1 (en) | 2011-05-25 |
PL1863334T3 (pl) | 2012-04-30 |
MX2007011612A (es) | 2007-10-18 |
PL2324703T3 (pl) | 2015-11-30 |
CN101146439B (zh) | 2013-03-27 |
EP2324703B1 (en) | 2015-05-06 |
ATE531807T1 (de) | 2011-11-15 |
WO2006102559A3 (en) | 2007-02-22 |
ES2540588T3 (es) | 2015-07-10 |
CA2601605A1 (en) | 2006-09-28 |
AU2006226863B2 (en) | 2011-02-10 |
AU2011202138B2 (en) | 2011-11-03 |
HUE025741T2 (en) | 2016-04-28 |
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