CN101389212B - 具有增加的游离赖氨酸的转基因玉米种子 - Google Patents
具有增加的游离赖氨酸的转基因玉米种子 Download PDFInfo
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Abstract
含有高水平游离赖氨酸的转基因玉米种子,由重组DNA构建体所致,该构建体具有与抑制内源赖氨酸分解代谢产物产生的基因抑制元件可操作性连接的胚乳特异性启动子和胚特异性启动子。
Description
相关申请的引用[用于美国]
本申请是2005年12月19提交的美国专利申请Nos.11/311,892和2006年3月31日提交的美国专利申请Nos.11/394,567的部分继续申请,在此通过援引并入本文。
序列表的合并
CD-R上的序列表计算机可读形式(CRF),含有以38-21(53489)D_SeqListing.txt命名的文本文件,其为37KB大小(用MS-WINDOWS测定),创建于2006年10月18日,在此通过援引并入本文。
技术领域
在此公开的是由转基因玉米细胞中的重组DNA所导致的含有提高的游离赖氨酸水平的转基因玉米种子,以及生产和使用该种子的方法。
背景技术
Dizigen等在US 1005/0132437A1中公开了高赖氨酸玉米组合物,含有表达二氢吡啶甲酸合酶的重组DNA,其作为营养增强的动物饲料是有用的。
发明概述
本发明提供的转基因玉米种子具有至少1300ppm的游离赖氨酸,例如在1300和4000ppm的游离赖氨酸之间,由转基因玉米细胞中重组DNA的表达所导致,该重组DNA包含与抑制内源LKR-SDH蛋白产生的DNA可操作性连接的胚特异性启动子和与抑制内源LKR-SDH蛋白产生的DNA可操作性连接的胚乳特异性启动子。在本发明的一个实施方式中,转基因玉米种子进一步包含编码在赖氨酸生物合成中有活性的蛋白的DNA,该DNA与胚乳特异性启动子可操作性连接。本发明的几个方面是玉米细胞,其具有的重组DNA包含pMON99142的T-DNA边界间的DNA或者pMON99143的T-DNA边界间的DNA。
本发明的另一方面提供了重组DNA构建体,该构建体对于提供游离 赖氨酸水平提高的转基因玉米种子是有效的。该重组DNA构建体包含与抑制内源LKR-SDH蛋白产生的DNA可操作性连接的胚特异性启动子和与抑制内源LKR-SDH蛋白产生的DNA可操作性连接的胚乳特异性启动子。该重组DNA的实施方式进一步包含编码在赖氨酸生物合成中有活性的蛋白的DNA,该DNA与胚乳特异性启动子可操作性连接。该重组DNA的几个方面由pMON99142或pMON99143的T-DNA边界间的DNA说明。
本发明也提供了生产游离赖氨酸水平提高的玉米籽粒的方法,通过繁殖具有转基因细胞的玉米植物进行,所述转基因细胞是用本发明的重组DNA构建体转化的玉米细胞的后代。
附图简述
图1和图2阐述了质粒图。
图3是重组DNA构建体的示意图。
发明详述
在此使用的LKR-SDH意指赖氨酸分解代谢蛋白赖氨酸酮戊二酸还原酶(lysine catabolic protein lysine ketoglutarate reductase)/酵母氨酸脱氢酶(saccharopine dehydrogenase)。针对LKR结构域或者SDH结构域设计用于本发明的有效基因抑制元件。
本领域普通技术人员能够利用可商购的材料和公知的、已公开的方法容易地制备重组DNA构建体。用于基因抑制的重组DNA构建体可根据US2004-0029283A1所阐述和公开的来制备。制备用于转化的重组DNA构建体和载体的实用技术是GATEWAYTM克隆技术(可获自Invitrogen Life Technologies,Carlsbad,California),利用来自噬菌体λ载体构建的整合酶att系统的位点特异性重组酶LR克隆反应,而不是限制性内切酶和连接酶。该LR克隆反应公开于美国专利5,888,732和6,277,608以及美国专利公开US 2001-283529 A1、US2001-282319 A1、US 2002-0007051 A1和US 2004-0115642 A1中。Invitrogen也提供GATEWAYTM克隆技术使用手册,提供了用于将任何目的DNA克隆入含有可操作性植物表达元件的载体的常规方法的简要说明。
另一种载体的制备方法采用Aslandis C.等,Nucleic Acids Res.,18,6069-6074,1990和Rashtchian,A.等,Biochem.,206,91-97,1992公开的不依赖连接的克隆(ligation-independent cloning)方法,其中具有单链5’和3’端的DNA 片段接入目的载体内,其然后能够在体内扩增。将DNA分子按预定次序组装入DNA构建体内的方法也公开于公布的美国专利申请US 2006-__(系列号11/298,234)中。
在植物细胞中具有活性的许多启动子已经在文献中描述。可用于本发明的转基因种子中的有用启动子包括从种子基因中获取的启动子,如napin(美国专利5,420,034)、cornL3油质蛋白(cornL3 oleosin)(美国专利6,433,252)、玉米蛋白Z27(zein Z27)(Russell等(1997)Transgenic Res.6(2):157-166)、球蛋白1(globulin 1)(Belanger等(1991)Genetics 129:863-872)、谷蛋白1(glutelin1)(Russell(1997)同上)、过氧化物氧化还原酶的抗氧化剂(Perl)(peroxiredoxinantioxidant)(Stacy等(1996)Plant Mol Biol.31(6):1205-1216)、以及B32(Hartings等(1990)Plant Mol Biol,14:1031-1040)。
实践中,在任一转化试验中,DNA仅导入很小百分比的靶细胞中。标记基因用于提供鉴定已稳定转化细胞的有效系统,所述细胞已经接收了转基因DNA构建体并且已将其整合入它们的基因组。优选的标记基因提供了选择性标记,其赋予对选择剂的抗性,如抗生素或除草剂抗性。对于选择性标记,本发明的植物可能所抗的任何除草剂都是有用的试剂。将可能转化的细胞暴露在该选择剂中。幸存下来的细胞群将是这样的一些细胞,其通常整合了赋予抗性的基因并且以足以允许细胞存活的水平表达。细胞可以被进一步检验,以证实已稳定整合了外源DNA。通常使用的选择性标记基因包括那些赋予抗生素抗性的基因,如卡那霉素(npt II)、潮霉素B(aph IV)和庆大霉素(aac3和aacC4),或者赋予除草剂抗性的基因,如草铵膦(bar或pat)以及草甘膦(EPSPS)。这样的选择性标记的实例在美国专利5,550,318、5,633,435、5,780,708和6,118,047中有述。还可以使用筛选标记,其提供了肉眼鉴定转化体的能力,例如表达有色蛋白或荧光蛋白如荧光素酶或绿色荧光蛋白(GFP)的基因,或者表达β-葡萄糖醛酸酶的基因,或者uidA基因(GUS),其不同的显色底物是已知的。
本发明提供了转基因种子,在它的基因组内具有重组DNA构建体,该构建体包含(a)可操作性连接至少一个第一基因抑制元件的植物胚乳特异性启动子,所述基因抑制元件抑制赖氨酸分解代谢蛋白例如LKR-SDH的产生,(b)与所述植物胚乳特异性启动子方向相反的植物胚特异性启动子,位于所述的至少一个第一基因抑制元件的3′端。该植物胚特异性启动子可操作性连接第 二基因抑制元件,以抑制赖氨酸分解代谢蛋白LKR-SDH的产生。第二基因抑制元件可以包含与第一个基因抑制元件相同的或不同的DNA,例如靶向LKR-SDH基因不同结构域。基因抑制元件能够以例如与邻近的启动子(头接头)或与邻近的终止子(尾部接尾部)相同的或相反的方向组装入重组DNA构建体内。
在转基因种子的一些实施方式中,重组DNA构建体进一步包含编码赖氨酸生物合成酶的DNA,该DNA与胚乳特异性启动子连接。在一些实施方式中,基因抑制元件和表达蛋白的表达元件与一个启动子可操作性连接,该基因抑制元件可以被嵌入到内含子中,在许多实施方式中所述内含子优选为转录增强内含子,例如,″增强子″诸如水稻肌动蛋白1基因、水稻肌动蛋白2基因、玉米乙醇脱氢酶基因、玉米热激蛋白70基因和玉米皱缩1基因(com shrunken 1gene)的5′端内含子。编码赖氨酸生物合成酶的有用DNA来自于外源赖氨酸不敏感的二氢吡啶甲酸(dihydrodipicolinic acid)合酶(DHDPS)基因,例如公开于美国专利5,773,691和6,459,019中的棒杆菌(Corynebacterium)DHDPS基因。当表达编码DHDPS酶的DNA时,将它与编码转运肽的DNA连接是有用的,如玉米DHDPS转运肽基因。
本发明提供的转基因植物细胞具有本发明的重组DNA构建体,该构建体已稳定整合入它们基因组内,以致于该重组DNA遗传给后代植物和种子。这样的转基因植物细胞重的某些实施方式中,组DNA是纯合的。
本发明进一步提供了用于转化植物细胞的重组DNA构建体、它们的使用方法,以及含有这样构建体的稳定转基因植物细胞。这样的重组DNA构建体可以在质粒pMON99142和pMON99143上找到,以下有更详细地描述。
用重组DNA转化植物细胞的许多方法在本领域是已知的,并且可用来制备转基因玉米。通常使用的两个植物转化方法是土壤农杆菌(Agrobacterium)介导的转化和微粒轰击。用于生产转基因玉米细胞的微粒轰击方法描述于美国专利5,550,318、5,538,880、6,160,208和6,399,861中,用于生产转基因玉米细胞的土壤农杆菌介导的转化方法描述于美国专利5,591,616中。基于根癌土壤农杆菌(Agrobacterium tumefaciens)的植物转化系统,转化构建体上存在的其它元件包括根癌土壤农杆菌T-DNA的左和/或右边界序列(通常包括左右两个边界序列,但是优选的是至少一个边界序列,例如至少右边界序列), 以利于重组多核苷酸整合入植物基因组中。
玉米的转化优选在受控环境下的培养基上在组织培养中进行。“培养基”是指用于细胞体外生长的多种养分混合物,体外也就是指完整活生物体以外。受体细胞靶包括,但不局限于,分生组织细胞、愈伤组织、未成熟的胚,以及配子细胞如小孢子(microspores)、花粉、精子和卵细胞。考虑到可由其再生出,可育植物的任何细胞均可用作受体细胞。愈伤组织可以源自于组织来源(tissue sources),包括但不限于,未成熟的胚、幼苗顶端分生组织、小孢子等等。能够增殖成为愈伤组织的细胞也是用于遗传转化的受体细胞。制备本发明的转基因植物的实用的转化方法和材料,例如不同的培养基和受体靶细胞,未成熟胚的转化和可育转基因植物的随后再生,已公开于美国专利6,194,636和6,232,526和美国专利申请公开US 2004-0216189 A1中。
转基因植物的种子可以从可育转基因植物收获,并且用于生长本发明的转化植物的后代,包括含表达基因抑制元件的重组DNA构建体的杂交植物品系。
本发明进一步提供了生产转基因玉米种子的不同方法,该转基因玉米种子具有提高的赖氨酸水平,即至少1300ppm的游离赖氨酸或更多,例如至少1500ppm或更高,至少2000ppm或300ppm,高达4000ppm的游离赖氨酸。这样的方法包括转化玉米植物品系,并将本发明的重组DNA从细胞内含有该重组DNA的玉米植物渗入(introgressing)到另一玉米植物品系。这样方法的一个方面包括(a)选择第一转基因玉米植物,它的细胞内包含本发明的重组DNA构建体;(b)将重组DNA构建体渗入到第二玉米植物;(c)种植第二玉米植物的种子,产生后代玉米植物群体;(d)筛选后代玉米植物群体,获得与非转基因玉米植物相比其产生的玉米种子中具有提高的赖氨酸水平的后代玉米植物;(e)从所述群体中选择与非转基因玉米植物相比,其产生的玉米种子具有提高的赖氨酸水平的一种或多种后代玉米植物;(f)验证重组DNA构建体已稳定地整合入中选的后代玉米植物中;(g)验证中选的后代玉米植物与缺少重组DNA构建体的玉米植物相比,其体内的LKR-SDH赖氨酸分解代谢基因(lysine catabolism gene)已被沉默;(h)从中选的后代玉米植物中收集转基因玉米种子。
实施例1
本实施例详述重组DNA构建体的制备,该构建体用于制备含有本发明的转基因细胞的转基因玉米植物和种子。参考图1和SEQ ID NO:1(其是质粒pMON99142上的DNA的一条链的核苷酸序列),将两个基因抑制重组DNA构建体插入根癌土壤农杆菌的左和右T-DNA边界之间来制备植物转化质粒。T-DNA的边界之间是(a)LKR基因抑制重组DNA构建体,是将玉米L3启动子和前导序列(即美国专利6,433,252公开的来自于玉米L3油质蛋白基因的玉米胚特异性启动子和前导序列)的DNA可操作性与″LKR抑制元件″(稳定的反义构建体,其包含来自玉米LKR-SDH基因LKR结构域的DNA的大约947碱基对作为反义向的片段排列)的DNA连接,随后连接有义向的片段并且以小麦hspl7终止子(即小麦(Triticum aestivum)热激蛋白17基因的多聚腺苷酸化位点(polyadenylation site)和信号)的DNA终止来制备的。(b)SDH基因抑制重组DNA构建体,是将玉米B32启动子和前导序列(即玉米B32基因的玉米胚乳特异性启动子和前导序列,该基因为GenBank登录号X70153公开的848-1259间的核苷酸)的DNA可操作性与″SDH抑制元件″(稳定的反义构建体,包含玉米LKR-SDH基因SDH结构域DNA的大约1254碱基对作为反义向排列的片段,并随后连接有义向的片段)的DNA连接,并且以玉米Glbl终止子(即玉米球蛋白1基因(Zea mays globulin 1 gene)的多聚腺苷酸化位点(polyadenylation site)和信号)的DNA终止来制备的。该质粒还含有T-DNA边界外的DNA,用作草甘膦抗性选择性标记、土壤农杆菌复制起点、大肠杆菌抑制蛋白、大肠杆菌复制起点ColE1和杀菌性抗生素选择性标记(用于大观霉素(spectromycin)/链霉素)。用于草甘膦抗性标记的DNA包含水稻肌动蛋白1的启动子、前导序列和内含子,可操作性连接编码叶绿体转运肽和EPSPS基因的嵌合DNA、和具有根癌土壤农杆菌胭脂碱合酶的聚腺苷酸化位点和信号的终止子元件DNA。表1显示了在SEQ ID NO:1中该质粒关键元件的位置。SDH基因抑制重组DNA构建体和LKR基因抑制重组DNA构建体是尾部对尾部连接。这样,在下文的表1中,SEQ ID NO:1中针对LKR基因抑制重组DNA构建体元件鉴定的DNA,包含那些元件编码链的反向互补序列中的核苷酸。 表1
元件 | pMON99142上的相配物(Coordinates) SEQ ID NO:1 |
土壤农杆菌T-DNA右边界 | 1-331 |
玉米L3的启动子和前导序列 | 345-1383 |
LKR抑制元件 | 1396-2343 |
小麦hspl7终止子 | 2349-2558 |
玉米Glbl终止子 | 2589-3582 |
SDH抑制元件 | 3588-4742 |
玉米B32启动子和前导序列 | 4747-5179 |
土壤农杆菌T-DNA左边界 | 5235-5676 |
草甘膦抗性选择性标记 | 5683-8954 |
土壤农杆菌复制起点 | 9006-9402 |
大肠杆菌ColE1抑制蛋白 | 10911-11102 |
大肠杆菌复制起点,ColE1 | 11530-12118 |
细菌抗生素选择性标记 | 12649-13537 |
实施例2
实施列2
本实施例阐述了重组DNA构建体的制备,其用于制备具有本发明的转基因细胞的转基因玉米植物和种子。参考实施例1、图2和3、SEQ ID NO:2(其是质粒pMON99143上的DNA的一条链的核苷酸序列),将两个基因抑制重组DNA构建体插入根癌土壤农杆菌的左和右T-DNA边界间制备植物转化质粒。T-DNA边界之间是(a)SDH基因抑制重组DNA构建体,是将玉米B32胚乳特异性启动子和前导序列可操作性与其中嵌入″SDH抑制元件″的玉米Hsp70内含子(即来自于玉米热激蛋白70基因(Zea mays heat shock protein 70 gene))顺序连接,该内含子与编码玉米DHDPS转运肽的DNA(即来自于玉米的二氢吡啶甲酸转运肽基因)连接,编码玉米DHDPS转运肽的DNA与编码棒杆菌(Corynebacterium)DHDPS的DNA(即来自于赖氨酸不敏感的棒杆菌DHDPS基因)连接,并以小麦HSP17 终止子DNA终止来制备的;和(b)LKR基因抑制重组DNA构建体,是将玉米L3胚特异性启动子和前导序列可操作性地与″LKR抑制元件″顺序连接,并以玉米GIb 1终止子DNA终止来制备的。该质粒还含有T-DNA边界外的DNA,用作草甘膦抗性选择性标记、土壤农杆菌复制起点、大肠杆菌抑制蛋白、大肠杆菌复制起点ColE1和杀菌性抗生素选择性标记(用于大观霉素/链霉素)。表2显示了在SEQ ID NO:2中,该质粒各元件的位置。SDH基因抑制重组DNA构建体和LKR基因抑制重组DNA构建体是尾部对尾部连接。这样,在下文的表2中,SEQ ID NO:2中针对LKR基因抑制重组DNA构建体元件鉴定的DNA,包含那些元件编码链的反向互补序列中的核苷酸。表2
元件 | pMON99143上的相配物(Coordinates) SEQ ID NO:2 |
土壤农杆菌T-DNA的右边界 | 1-357 |
玉米L3的启动子和前导序列 | 374-806 |
玉米Hsp70内含子-5’端 | 812-1256 |
SDH抑制元件 | 1270-2424 |
玉米Hsp70内含子-3’端 | 2429-2778 |
玉米DHDPS转运肽 | 2783-2953 |
棒杆菌(Corynebacterium)DHDPS | 2954-3856 |
小麦HSP17终止子 | 3862-4071 |
玉米GIb 1终止子 | 4091-5084 |
LKR抑制元件 | 5091-6038 |
玉米L3的启动子和前导序列 | 6051-7089 |
土壤农杆菌T-DNA左边界 | 7146-7587 |
草甘膦抗性选择性标记 | 7594-10865 |
土壤农杆菌复制起点 | 10917-11313 |
大肠杆菌ColE1抑制蛋白 | 12822-13013 |
大肠杆菌复制起点,ColE1 | 13441-14029 |
细菌抗生素选择性标记 | 14560-15448 |
实施例3
本实施例阐述实施例1的质粒的用途,用于生产具有提高的游离赖氨酸水平的转基因玉米细胞、植物和可育的转基因玉米的种子。利用土壤农杆菌介导转化方法将质粒pMON99142插入玉米愈伤组织,以产生玉米细胞的多个转基因事件,这些事件被选择为草甘膦除草剂抗性(基于在T-DNA边界的一侧插入草甘膦抗性选择性标记的DNA)。转基因植物(RO)由多个转基因事件中的每一个的转化细胞生长而来。用与质粒的PCR扩增片段杂交的荧光标记探针分析RO转基因玉米植物,以确定草甘膦抗性标记DNA和基因抑制重组DNA构建体的存在。从代表转基因事件的多个植物中选择以下植物,其在单一位点上具有草甘膦抗性选择性标记DNA以及在另一单一位点上具有T-DNA的单一拷贝,所述T-DNA包含SDH和LKR基因抑制重组DNA构建体。单一拷贝-单一位点T-DNA植物与非转基因近交(inbred)玉米品系杂交,生产分离的后代种子,分析所述的种子以鉴定具有基因抑制重组体DNA并且没有草甘膦抗性选择性标记DNA的种子。用胚乳切片的DNA进行种子分析。被鉴定的种子长成植物,与几个近交玉米品系杂交几代,以将包含SDH和LKR基因抑制重组DNA构建体的T-DNA渗入到所述近交玉米品系中,以生产在胚乳和胚中均具有抑制赖氨酸分解代谢蛋白LKR/SDH的DNA的近交玉米品系。
分析该近交玉米品系的转基因玉米植物的种子中游离赖氨酸含量,所述的玉米自交品系在胚乳和胚中均具有抑制赖氨酸分解代谢蛋白LKR/SDH的DNA,并且确定赖氨酸含量大于1300ppm。具有选中的转基因事件细胞的近交玉米品系植物的种子被鉴定为含有大于1500ppm的游离赖氨酸、大于2000ppm的游离赖氨酸、大于3000ppm的游离赖氨酸和大于4000ppm的游离赖氨酸。
实施例4
本实施例阐述实施例2的质粒的用途,用于生产游离赖氨酸水平提高的转基因玉米细胞、植物和可育的转基因玉米的种子。使用质粒pMON99143基本上重复实施例3的转化、分析、选择和渗入(introgressing),以提供在胚乳和胚中均含有抑制赖氨酸分解代谢蛋白LKR/SDH的DNA并表达棒杆菌DHDPS酶的近交玉米品系。具有选中的转基因事件细胞的近交玉米品系植物的种子,被鉴定为含有大于1500ppm的游离赖氨酸、大于2000ppm的游离赖氨酸、大于3000ppm的游离赖氨酸和大于4000ppm的游离赖氨酸。
Claims (3)
1.一种生产具有至少1300ppm游离赖氨酸的提高的游离赖氨酸水平的玉米籽粒的方法,该方法是通过繁殖具有转基因细胞的玉米植物,该玉米植物是具有重组DNA的转基因玉米种子细胞的后代,该重组DNA包含胚特异性启动子、胚乳特异性启动子和抑制内源LKR-SDH蛋白产生的DNA,其中所述胚特异性启动子和所述胚乳特异性启动子与所述抑制内源LKR-SDH蛋白产生的DNA可操作性连接,并且其中所述重组DNA包含pMON99142或pMON99143的T-DNA边界间的DNA,所述pMON99142的序列示于SEQ ID NO:1,所述pMON99143的序列示于SEQ ID NO:2,并且其中所述pMON99142的T-DNA边界是SEQ ID NO:1所示序列的1-331位和5235-5676位的核苷酸,并且所述pMON99143的T-DNA边界是SEQ ID NO:2所示序列的1-357位和7146-7587位的核苷酸。
2.如权利要求1所述的方法,其中所述的重组DNA进一步包含编码在赖氨酸生物合成中有活性的蛋白的DNA,该DNA与所述的胚乳特异性启动子可操作性连接。
3.如权利要求2所述的方法,其中所述的重组DNA包含pMON99143的T-DNA边界间的DNA,其中所述pMON99143的T-DNA边界是SEQ ID NO:2所示序列的1-357位和7146-7587位的核苷酸。
Applications Claiming Priority (5)
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US11/311,892 US20060150286A1 (en) | 2004-12-23 | 2005-12-19 | Gene suppression in transgenic plants using multiple constructs |
US11/311,892 | 2005-12-19 | ||
US11/394,567 US20060242736A1 (en) | 2004-12-23 | 2006-03-31 | Dissimilar promoters for gene suppression |
US11/394,567 | 2006-03-31 | ||
PCT/US2006/041439 WO2007073445A1 (en) | 2005-12-19 | 2006-10-23 | Transgenic corn seed with enhanced free lysine |
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CN101389212A CN101389212A (zh) | 2009-03-18 |
CN101389212B true CN101389212B (zh) | 2013-05-01 |
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CNA200680052963XA Pending CN101374943A (zh) | 2005-12-19 | 2006-03-31 | 用于基因抑制的不同启动子 |
CN200680047649.2A Active CN101389212B (zh) | 2005-12-19 | 2006-10-23 | 具有增加的游离赖氨酸的转基因玉米种子 |
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US (1) | US20060150286A1 (zh) |
EP (1) | EP1963488A4 (zh) |
CN (2) | CN101374943A (zh) |
AR (2) | AR053076A1 (zh) |
AU (1) | AU2006327232A1 (zh) |
WO (1) | WO2007073394A1 (zh) |
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US20070074311A1 (en) * | 2005-08-30 | 2007-03-29 | Pioneer Hi-Bred International, Inc. | Compositions and methods for modulating expression of gene products |
US20070130642A1 (en) * | 2005-11-14 | 2007-06-07 | Pioneer Hi-Bred International, Inc. | Methods and compositions for reducing the expression of a polynucleotide of interest |
WO2018005491A1 (en) * | 2016-06-28 | 2018-01-04 | Monsanto Technology Llc | Methods and compositions for use in genome modification in plants |
CN110288076B (zh) * | 2019-07-09 | 2020-03-27 | 中央民族大学 | 指示信号在不同伴随信号下优先级的细菌细胞计算部件 |
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- 2006-03-31 CN CNA200680052963XA patent/CN101374943A/zh active Pending
- 2006-03-31 WO PCT/US2006/011946 patent/WO2007073394A1/en active Application Filing
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EP1963488A1 (en) | 2008-09-03 |
AU2006327232A1 (en) | 2007-06-28 |
AR086210A2 (es) | 2013-11-27 |
CN101389212A (zh) | 2009-03-18 |
AR053076A1 (es) | 2007-04-18 |
WO2007073394A1 (en) | 2007-06-28 |
CN101374943A (zh) | 2009-02-25 |
US20060150286A1 (en) | 2006-07-06 |
EP1963488A4 (en) | 2009-02-04 |
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