CN1871353A - 来自报春的脂肪酸去饱和酶 - Google Patents
来自报春的脂肪酸去饱和酶 Download PDFInfo
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- CN1871353A CN1871353A CNA2004800310731A CN200480031073A CN1871353A CN 1871353 A CN1871353 A CN 1871353A CN A2004800310731 A CNA2004800310731 A CN A2004800310731A CN 200480031073 A CN200480031073 A CN 200480031073A CN 1871353 A CN1871353 A CN 1871353A
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Abstract
本发明一般地涉及关于调节长链多不饱和脂肪酸(LC-PUFA′s)中双键数目和位置的去饱和酶的方法和组合物。特别地,本发明涉及用于改进植物产品中ω-3脂肪酸谱的方法和组合物,以及使用去饱和酶和编码这种酶的核酸的部分。在特别的实施方案中,去饱和酶是报春Δ6-去饱和酶。也提供了改进的大豆油组合物,其具有SDA和有益的ω-3脂肪酸相对于ω-6脂肪酸的总含量。
Description
发明背景
本申请要求2003年8月21日提交的美国临时专利申请Ser.No.60/496,751的优先权,通过引用将其全部公开内容特别引入此处。
1.发明领域
本发明一般地涉及调节长链多不饱和脂肪酸(LC-PUFA′s)中双键数目和位置的去饱和酶。特别是,本发明涉及使用去饱和酶和编码这种去饱和酶的核酸对脂肪酸谱的改进。
2.现有技术描述
大多数生物体中的脂肪酸生物合成的主要产物是16-和18-碳化合物。这些脂肪酸的链长和不饱和度的相对比在物种间变化很大。例如,哺乳动物主要产生饱和和单不饱和脂肪酸,而大多数高等植物产生具有一个、两个或三个双键的脂肪酸,后二者包括多不饱和脂肪酸(PUFA′s)。
PUFAs的两个主要家族是ω-3脂肪酸(也表示为“n-3”脂肪酸)、以二十碳五烯酸(EPA,20:4,n-3)为例,以及ω-6脂肪酸(也表示为“n-6”脂肪酸)、以花生四烯酸(ARA,20:4,n-6)为例。PUFAs是细胞和脂肪组织的质膜重要成分,它们可能分别以诸如磷脂或甘油三酯这样的形式在其中被存在。PUFAs对哺乳动物正常发育、特别是对婴儿大脑发育以及组织形成和修复是必需的。
几种紊乱对脂肪酸治疗有反应。补充PUFAs已经显示出降低血管成形术后的再狭窄速度。用于心血管疾病和类风湿性关节炎的某些饮食ω-3脂肪酸的健康益处也得到了很好的证明(Simopoulos,1997;James,et al.,2000)。此外,PUFAs被建议用于哮喘和银屑病的治疗。证据显示PUFAs可能参与钙代谢,暗示PUFAs可能对骨质疏松症和肾或尿道结石的治疗或预防是有用的。大多数对于健康益处的证据适用于鱼和鱼油中的长链ω-3脂肪、EPA和二十二碳六烯酸(DHA,22:6)。根据这个证据,加拿大(Scientific Review Committee,1990,Nutrition Recommendations,Minister of National Health and Welfare,Canada,Ottowa)、欧洲(de Deckerer,et al.,1998)、英国(The BritishNutrition Foundation,1992,Unsaturated fatty-acids-nutritional andphysiological significance:The report of the British NutritionFoundation′s Task Force,Chapman and Hall,London)以及美国(Simopoulos et al.,1999)的健康权威和营养学家推荐了增加这些PUFAs的饮食摄入。
PUFAs也可以用于治疗糖尿病(美国专利No.4,826,877;Horrobinet al.,1993)。改变了的脂肪酸代谢和组成已经在糖尿病动物中得到了证明。这些改变被认为参与了糖尿病引起的一些长期并发症,包括视网膜病、神经病、肾病和生殖系统损伤。含有γ-亚麻酸(GLA,18:3,Δ6,9,12)的月见草油(primrose oil)显示出预防和逆转糖尿病性神经损伤。
诸如亚油酸(LA,18:2,Δ9,12)和α-亚麻酸(ALA,18:3,Δ9,12,15)的PUFAs被认为是饮食中的必需脂肪酸,因为哺乳动物缺乏合成这些酸的能力。然而在被摄食后,哺乳动物具有代谢LA和ALA形成n-6和n-3家族的长链多不饱和脂肪酸(LC-PUFA)的能力。这些LC-PUFA′s是赋予膜流动性的重要细胞成分,并作为生物活性的类二十烷酸前体,诸如前列腺素、前列环素和白三烯,它们调节正常的生理功能。花生四烯酸是包括白三烯、前列腺素以及血栓素在内的类二十烷酸合成的主要前体,它也在炎症过程中起作用。施用诸如SDA的ω-3脂肪酸已经显示出抑制白三烯的生物合成(美国专利No.5,158,975)。SDA的消耗显示出导致促炎细胞因子TNF-α和IL-1β的血液水平的降低(PCTUS 0306870)。
在哺乳动物中,LC-PUFA的形成受到将LA转化为γ-亚麻酸(GLA,18:3,Δ6,9,12)以及将ALA转化为SDA(18:4,Δ6,9,12,15)的Δ6去饱和作用步骤的限速。许多生理和病理条件显示出抑制了这个代谢步骤甚至还因而抑制了LC-PUFA的产生。为克服限速步骤并增加EPA的组织水平,可以消耗大量ALA。然而刚好适量SDA的消耗提供了EPA的有效来源,因为SDA在人体内增加组织EPA水平的效率大约是ALA的4倍(共同未决的美国申请Ser.No.10/384,369)。在同样的研究中,SDA的施用也能够增加二十二碳六烯酸(DPA)的组织水平,它是EPA的延长产物。或者,通过EPA或DHA的饮食补充绕过Δ6-去饱和作用可有效缓解与低水平PUFA有关的许多病理疾病。然而,如下文更详细的阐述,由于许多原因,目前能得到的PUFA来源是不合意的。对可靠且经济的PUFA′s来源的需求激发了对PUFA′s替代来源的兴趣。
重要的主要长链PUFAs包括最初是在鱼油的不同类型中发现的DHA和EPA,,以及在诸如被孢霉的丝状真菌中发现的ARA。对于DHA,许多来源为商业生产存在,包括各种海洋生物体、从冷水海鱼获得的油、以及蛋黄组分。SDA的商业来源包括植物属毛束草属、Borago(琉璃苣)和蓝蓟属。然而,存在几个与天然来源PUFAs的商业生产有关的不利因素。PUFAs的天然来源,诸如动物和植物,往往具有高度异质的油组成。因此从这些来源获得的油可能需要大量纯化以分离出一种或多种期望的PUFAs或产生富含一种或多种PUFAs的油。
PUFAs的天然来源也受到供应方面不可控制的波动的影响。鱼类可能经历自然变异或由于过量捕鱼而灭绝。另外,即使有它们治疗上好处的大量证据,关于ω-3脂肪酸的饮食推荐还未被注意过。鱼油具有让人讨厌的味道和气味,也许不可能经济地与期望的产物分开,并可能使得这种产物无法作为食物补充剂被接受。动物油,特别是鱼油可能积累环境污染物。食物也许可富含鱼油,但是再一次地,由于成本以及衰减中的全世界鱼类储量,这种富集是有问题的。这个问题也是消费和摄取整个鱼类的障碍。尽管如此,如果要增加鱼类摄取的健康信息被社会接受,在满足对鱼类的需求方面可能是个问题。此外,还存在维持该产业的问题,其严重依赖野生鱼类储量来供给水产养殖(Naylor et al.,2000)。
其它自然限制促成了生产ω-3脂肪酸的新颖方法。天气和疾病可引起鱼和植物来源的产量波动。可获得的用于生产替代性产油农作物的农田受到人口稳定扩张以及与在剩余耕地上对粮食生产的增长性需求相关的竞争性影响。产生PUFAs的农作物诸如琉璃苣,还没有适应商业生长,可能不会在单一栽培中表现良好。在可以生长更有利并更好确定的农作物时,这些农作物的生长就没有经济竞争力。诸如被孢霉的生物体的大规模发酵也是很昂贵的。天然动物组织含有少量ARA并且难于加工。诸如紫球藻和被孢霉的微生物体很难商业规模培养。
许多酶参与PUFAs的生物合成。LA(18:2,Δ9,12)是通过Δ12-去饱和酶从油酸(OA,18:1,Δ9)产生的,而ALA(18:3,Δ9,12,15)是通过Δ15-去饱和酶从LA产生的。SDA(18:4,Δ6,9,12,15)和GLA(18:3,Δ6,9,12)是通过Δ6-去饱和酶从LA和ALA产生的。但是,如上所述,哺乳动物不能去饱和超过Δ9位置,因此不能将油酸转化为LA。同样地,哺乳动物不能合成ALA。其它真核生物,包括真菌和植物,具有在碳12和碳15位置上去饱和的酶。因此动物的主要多不饱和脂肪酸是通过膳食LA和ALA随后的去饱和并延伸而源自膳食的。
已经描述了各种编码去饱和酶的基因。例如,美国专利No.5,952,544描述了编码脂肪酸去饱和酶的从欧洲油菜分离和克隆的脂肪酸片段。′544专利的核酸片段的表达导致了ALA的积累。然而在表达欧洲油菜Δ15-去饱和酶的转基因植物中,大量的LA仍然没有被去饱和酶转化。转化更大量LA和ALA的更有活性的酶将是有利的。当与编码Δ15-去饱和酶的核酸共表达时,增加的ALA水平允许Δ6-去饱和酶作用于ALA从而产生更高水平的SDA。因为SDA的多种有利用途,需要产生SDA产量的大量增加。
已经试图将许多来源的核酸用于增加SDA产量。然而仍然需要创新以允许基于陆地的农作物商业生产的改进(参见例如Reed et al.,2000)。此外,从诸如秀丽新杆线虫(Meesapyodsuk et al.,2000)的生物体得到的去饱和酶多核苷酸的使用对富含植物种子油的商业生产并不理想。已经从报春属的有粉报春和高穗花报春两个物种中分离了编码Δ6-去饱和酶的基因,发现它们在酵母中是有活性的,但是没有显示在植物中的功能(Sayanova et al.,2003)。
因此,获得参与PUFA生物合成的遗传物质并在植物系统中表达分离的物质将是有利的,特别是在基于陆地的陆生农作物植物系统中,它可以被操作以提供一种或多种PUFA′s的商业数量的生产。也需要增加人和动物中ω-3脂肪的摄取。因而需要提供大范围的富含ω-3的食物和食物补充剂,以便受试者能够选择适合他们通常饮食习惯的饲料、饲料成分、食物和食物成分。特别有利的是具有增加的SDA的种子油。
目前在植物油中可获得的只有一种ω-3脂肪酸ALA。然而,摄取的ALA到诸如EPA和DHA的长链ω-3脂肪酸的转化很差。这已经在共同未决的美国申请Ser.No.10/384,369“炎症的治疗和预防”中证明,通过使用亚麻籽油将ALA摄取从群体平均值的1g/天增加到14g/天,仅仅适度增加了血浆磷脂EPA水平。ALA摄取增加14倍导致了血浆磷脂EPA增加2倍(Manzioris et al.,1994)。因此,需要使用脂肪酸去饱和酶、编码它们的基因以及生产它们的重组方法来有效地并在商业上可行地生产PUFAs。也存在对含有更高相对比的特定PUFAs以及含有它们的食物组合物和补充剂的需求。也存在对生产特定PUFAs的可靠的、经济的方法的需求。
尽管有上述的低效率和低产量,通过陆地食物链生产的ω-3脂肪酸对大众健康是有很大益处的,特别是SDA的生产。SDA是重要的,这是因为如上所述ALA到EPA的低转化。这是因为在转化中的起始酶Δ6-去饱和酶在人体中具有低活性且是限速的。通过研究提供了Δ6-去饱和酶是限速的证据,这证明了在小鼠和大鼠中,其底物ALA的转化比其产物SDA转化为EPA的效率低(Yamazaki,et al.,1992;Huang,1991)。
基于这些研究,可以发现在商业性油种子农作物,诸如canola、大豆、玉米、向日葵、红花或亚麻中,代表它们的种子油的单和多不饱和脂肪酸的一些组分转化为SDA需要多种去饱和酶的种子特异性表达,包括Δ6-、Δ12-和/或Δ15-去饱和酶。从表达高水平Δ6、Δ12和Δ15-去饱和酶的植物获得的油富含SDA和其它ω-3脂肪酸。这些油可以用来生产富含ω-3脂肪酸的食物或食物补充剂,摄食这些食物有效地增加了EPA和DHA的组织水平。食物和食品,诸如奶、人造黄油和香肠,都是用富含ω-3的油来制造或制备的,这将会产生治疗性好处。已经表明,通过利用含有富含ω-3的脂肪酸的油的食物,受试者可以具有比得上至少1.8g/天的EPA和DHA的ω-3摄取,而不会改变他们的饮食习惯。因此,存在对用于具有富含PUFAs的油的转基因农作物的Δ6-去饱和酶的新核酸以及由此生产的改进的油的强烈需求。
发明概述
一方面,本发明提供编码能够在碳6上使脂肪酸分子去饱和的多肽(Δ6-去饱和酶)的分离核酸。这些核酸可以用来转化细胞或修饰植物或由植物生产的油的脂肪酸组成。本发明的一个实施方案是从具有独特去饱和酶活性的报春物种分离的分离多核苷酸序列。在某些实施方案中,分离的多核苷酸是从例如Primula juliae、P.alpicola、P.waltonii、有粉报春或P.florindae分离的。在本发明某些进一步的实施方案中,多核苷酸编码与SEQ ID NO:4、SEQ ID NO:5、SEQ IDNO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:46或SEQ IDNO:48的多肽序列具有至少90%序列同一性的多肽,包括与这些序列至少大约92%、95%、98%和99%的同源性。本领域技术人员将认识到,由于这些序列是相关的,特定的多肽可同时与超过一个的这些多肽序列具有90%或更高的同源性。在某些实施方案中,由本发明提供的序列具有相对于亚油酸来说对α-亚麻酸的底物选择性,如这里所描述的一样。在进一步的实施方案中,具有至少2∶1的α-亚麻酸相对于亚油酸的底物选择性,包括从大约2∶1到大约2.9∶1。
另一方面,本发明提供编码具有在脂肪酸分子碳6上去饱和的去饱和酶活性的多肽的分离多核苷酸,其包括选自下组的序列:(a)编码SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:22、SEQ ID NO:24、SEQ IDNO:26、SEQ ID NO:46或SEQ ID NO:48的多肽的多核苷酸;(b)包含SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:21、SEQ ID NO:23、SEQ IDNO:25、SEQ ID NO:45或SEQ ID NO:47的核酸序列的多核苷酸;(c)在5X SSC、50%甲酰胺和42℃的条件下,与SEQ ID NO:2、SEQ IDNO:3、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:45或SEQ ID NO:47,或其互补序列杂交的多核苷酸;以及(d)编码与SEQID NO:4、SEQ ID NO:5、SEQ ID NO:22、SEQ ID NO:24、SEQ IDNO:26、SEQ ID NO:46或SEQ ID NO:48的多肽序列具有至少90%序列同一性的多肽的多核苷酸。
还有另一方面,本发明提供包含根据本发明的分离多核苷酸的重组载体。这里所使用的术语“重组载体”包括任何希望引入宿主细胞、组织和/或生物体中的重组DNA片段,特别包括从起始多核苷酸分离的表达盒。重组载体可以是线性或环状的。在各种方面,重组载体可包含至少一个选自下组的附加序列:可操作性连接到多核苷酸上的调节序列;可操作性连接到多核苷酸上的选择标记;可操作性连接到多核苷酸上的标记序列;可操作性连接到多核苷酸上的纯化部分;以及可操作性连接到多核苷酸上的导向序列。
还有另一方面,本发明提供细胞,诸如用本发明多核苷酸转化的哺乳动物、植物、昆虫、酵母和细菌细胞。在进一步的实施方案中,用除本发明多核苷酸以外,还含有组成型和组织特异性的启动子的重组载体转化细胞。在本发明某些实施方案中,这些细胞可进一步限定为用编码具有在碳12和/或15上使脂肪酸分子去饱和的去饱和酶活性的多肽的核酸序列转化。
本发明也提供包含SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:46或SEQ ID NO:48的氨基酸序列的多肽;或其具有在碳6上使脂肪酸分子去饱和的去饱和酶活性的片段。
本发明还有另一方面提供生产含有来自植物种子ω-3脂肪酸的种子油的方法,包括步骤:(a)获得根据本发明的植物种子;和(b)从所述种子提取油。这些植物的例子包括canola、黄豆、大豆、油菜籽、向日葵、棉花、可可、花生、红花、椰子、亚麻、油棕榈、油籽欧洲油菜和玉米。转化这些植物细胞的优选方法包括使用土壤杆菌的Ti和Ri质粒、电穿孔以及高速轰击。
还有另一方面,本发明提供生产包含种子油的植物的方法,所述种子油包含改变的ω-3脂肪酸水平,包括将本发明的重组载体引入产油植物中。在这个方法中,重组载体的引入可包括遗传转化。在实施方案中,转化包括步骤:(a)用本发明的重组载体转化植物细胞;和(b)从植物细胞再生植物,其中,相对于同一基因型的未用载体转化的相应植物,该植物具有改变水平的ω-3脂肪酸。在这个方法中,植物可以是例如选自拟南芥、油籽芸苔、油菜籽、向日葵、红花、canola、玉米、大豆、棉花、亚麻、西蒙德木、乌桕树、烟草、可可树、花生、水果植物、柑橘植物以及产生坚果和浆果的植物。植物可被进一步限定为用编码具有在碳12和/或15上使脂肪酸分子去饱和的去饱和活性的多肽的核酸序列转化。植物可包含增加的SDA。该方法可进一步包括将重组载体引入许多产油植物并筛选具有遗传的重组载体的植物或其后代以得到具有期望的ω-3脂肪酸谱的植物。
还有另一方面,本发明提供SDA含量大约5%到大约50%且γ-亚油酸含量小于大约10%的内源性大豆种子油。在某些实施方案中,SDA的含量可被进一步限定为从大约5%到大约32%,从大约5%到大约35%,从大约15%到大约30%,从大约22%到大约30%,以及从大约22%到大约40%。在进一步的实施方案中,γ-亚油酸含量可被进一步限定为小于大约10、8、5和/或大约3%。在特定实施方案中,十八碳四烯酸含量大约从15%到大约35%,γ-亚油酸含量小于5%。在更进一步的实施方案中,种子可包含ω-3与ω-6脂肪酸的比是从大约0.35∶1到大约3.5∶1,包括从大约1∶1到大约3.5∶1以及从大约2∶1到大约3.5∶1。
还有另一方面,本发明提供增加人或动物消耗的可食用产品的营养价值的方法,包括加入由本发明提供的大豆种子油到可食用产品中。在某些实施方案中,产品是人和/或动物食物。可食用产品也可以是动物饲料和/或食物补充剂。在这个方法中,大豆种子油可增加可食用产品的SDA含量和/或可增加可食用产品的ω-3与ω-6脂肪酸的比。该可食用产品在添加大豆种子油之前可能缺乏SDA。
还有另一方面,本发明提供生产食物或饲料的方法,包括加入由本发明提供的大豆种子油到起始食物或饲料成分中以生产食物或饲料。在某些实施方案中,此方法被进一步限定为生产食物和/或饲料的方法。本发明也提供由此方法制造的食物或饲料。
还有另一方面,本发明还包含向人或动物提供SDA的方法,包括将权利要求1的大豆种子油给予所述人或动物。在此方法中,大豆种子油可提供在可食用组合物中,包括食物或饲料。食物的例子包括饮料、浸制食品、酱油、调味品、色拉调料、果汁、糖浆、甜点、糖霜和馅、软冻产品、糖果或半成品食物。可食用组合物可以基本上是液体或固体。可食用组合物也可以是食物补充剂和/或保健食品(nutraleutical)。在这个方法中,大豆种子油可给予人和/或动物。可给予大豆种子油的动物的例子包括家畜或家禽。
附图简述
下面的附图构成本说明书一部分并包括在内以进一步证明本发明的某些方面。通过参考这些附图的一个或多个并结合这里所提供的特定实施方案的详述,可更好的理解本发明。
图1显示Primula juliae Δ6去饱和酶PjD6D-1和PjD6D-2(SEQID NOs:4和5)、Primula alpicola Pa6D-1和Pa6D-2(SEQ ID NOs:22和24)、Primula waltonii PwD6D(SEQ ID NO:26)、有粉报春D6D-2(SEQ ID NO:46)、Primula florindae D6D(SEQ ID NO:48)、Boragooficinalis D6D(SEQ ID NO:59)以及Echium gentianoides D6D(SEQ IDNO:60)的比对。
图2显示载体pMON67011的图谱。
图3显示载体pMON83950的图谱。
图4显示载体pMON77245的图谱。
图5显示载体pMON77247的图谱。
图6显示载体pMON82821的图谱。
图7显示载体pMON82822的图谱。
图8显示载体pMON83961的图谱。
图9显示载体pMON83962的图谱。
图10显示载体pMON83963的图谱。
图11显示载体pMON83964的图谱。
图12显示载体pMON83965的图谱。
图13显示载体pMON83966的图谱。
发明详述
本发明通过提供产生具有改进的PUFA含量的植物的方法和组合物而克服了现有技术的局限性。诸如植物的生物体的脂肪酸含量的修饰存在很多有利条件,包括改善的营养和健康益处。脂肪酸含量的修饰可用于实现在植物、植物部分和包括植物种子油在内的植物产品中期望的PUFA′s有益水平或谱。例如,当期望的PUFA′s在植物种子组织中产生时,油可从种子分离,这将典型地产生期望的PUFAs很高的油或者具有期望的脂肪酸含量或谱的油,其又可用于在食品和其它产品中提供有益特性。本发明在特定实施方案中提供具有SDA同时也含有有益的油酸成分的内源性大豆油。
本发明的各方面包括修饰细胞的PUFA含量的方法和组合物,例如,植物细胞PUFA含量的修饰。与本发明相关的组合物包括新的分离的多核苷酸序列、多核苷酸构建体以及由本发明的多核苷酸转化的植物和/或植物的部分。分离的多核苷酸可编码报春脂肪酸去饱和酶,特别是可编码报春Δ6-去饱和酶。可使用宿主细胞表达编码将脂肪酸去饱和的去饱和酶多肽的多核苷酸。
本发明的一些方面包括去饱和酶多肽和编码它的多核苷酸。本发明的各种实施方案可使用去饱和酶多核苷酸和所编码的多肽的组合,其典型地依赖宿主细胞、底物的可获得性以及期望的终产物。“去饱和酶”是指可去饱和或催化一个或多个脂肪酸的连续碳之间的双键的形成以产生单-或多-不饱和脂肪酸或其前体的多肽。特别感兴趣的是能够催化油酸转化为LA、LA转化为ALA、或者ALA转化为SDA的多肽,包括在12、15或6位去饱和的酶。术语“多肽”是指氨基酸的任何链,不考虑长度或翻译后修饰(如糖基化或磷酸化)。选择具有去饱和酶活性的特定多肽的考虑事项包括但不限于:多肽的最佳pH、这个多肽是否是限速酶或其组分、所使用的去饱和酶对期望PUFA的合成是否是必需的、和/或多肽是否需要辅因子。表达的多肽优选具有与它在宿主细胞中所在位置的生化环境相容的特征。例如,多肽可能不得不争夺底物。
所述多肽的Km和比活性的分析可在确定特定多肽在特定宿主细胞中修饰PUFA(s)生产、水平或谱的适合性中被考虑。用于特定情况下的多肽是一种典型的可在预期的宿主细胞中存在的条件下起作用的多肽,但是另外可以是任何具有期望特征或能够修饰期望PUFA(s)的相关生产、水平或谱或者这里所讨论的任何其它期望特征的去饱和酶多肽。表达的酶的底物可由宿主细胞产生或者可外源提供。为实现表达,本发明的多肽由如下文所描述的多核苷酸编码。
发明人已经分离并生产了来自报春的显示Δ6-去饱和酶活性的酶。编码Δ6-去饱和酶的序列可在转基因植物、微生物或动物中表达以实现更多的SDA合成。也可使用与这里所提供的Δ6-去饱和酶多核苷酸基本上相同的、或者编码与Δ6-去饱和酶多肽基本上相同的多肽的其它多核苷酸。“基本上相同”是指氨基酸序列或核酸序列按增加的优选顺序显示出与SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:22、SEQ IDNO:24、SEQ ID NO:26、SEQ ID NO:46或SEQ ID NO:48的Δ6-去饱和酶多肽序列或者编码这些多肽的序列至少90%、95%、98或99%的同一性。可以使用序列分析软件来进行多肽或多核苷酸比较,例如GCGWisconsin Package的序列分析软件包(Accelrys,San Diego,CA)、MEGAlign(DNAStar,Inc.,1228 S.Park St.,Madison,Wis.53715)以及MacVector(Oxford Molecular Group,2105 S.Bascom Avenue,Suite200,Campbell,Calif.95008)。这些软件通过确定相似性或同一性程度来匹配相似序列。
本发明包括相关的去饱和酶,包括在报春的相同或不同种中天然存在的所公开的Δ6-去饱和酶的变体。相关的去饱和酶可通过它们与所公开的去饱和酶有基本上相同功能的能力来识别;就是说具有Δ6-去饱和酶活性。相关的去饱和酶的识别也可通过筛选序列数据库中与所公开的去饱和酶同源的序列,通过基于所公开的去饱和酶的探针与从来源生物体构建的文库的杂交,或者通过使用来自来源生物体的mRNA和基于所公开的去饱和酶的引物的RT-PCR。因此本发明提供了在严格条件下与这里所描述的编码去饱和酶的序列杂交的核酸。本领域技术人员明白,通过增加盐浓度和降低温度能提供更不严格的条件。因此杂交条件很容易操控,从而通常将是依赖于期望结果的方法选择。高严格条件的例子是5X SSC、50%甲酰胺以及42℃。通过在这种条件下漂洗例如10分钟,在这些条件下不能与特定靶序列杂交的序列可被除去。
在本发明的另一方面,含有核酸或其片段、含有启动子、Δ6-去饱和酶编码序列以及终止区域的载体可被转移到生物体中,启动子和终止区域在其中是有功能的。这样,本发明提供了产生重组Δ6-去饱和酶的生物体。本发明的另一方面提供分离的Δ6-去饱和酶,其可通过蛋白质纯化的标准方法从重组生物体纯化。(例如,参见Ausubel et al.,1994)。
本发明的各方面包括这里所描述的编码去饱和酶的核酸序列。核酸可以是从报春分离的,包括SEQ ID NO:2、SEQ ID NO:3、SEQ IDNO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:45或SEQ IDNO:47以及类似物。设计来扩增被确定为潜在的脂肪酸去饱和酶的序列的基于寡核苷酸引物的克隆策略、基于基因组DNA数据库的BLAST检索的克隆策略可用于对各个克隆测序。然后这些克隆可被功能性表征。
可提供整合到宿主细胞基因组或自主的在宿主细胞复制(例如游离复制)的核酸构建体。对于ALA和/或SDA的生产,通常使用的表达盒(即可操作性连接到指导多核苷酸表达的核酸序列上的编码蛋白质的多核苷酸)包括提供来表达编码Δ6-去饱和酶的多核苷酸的表达盒。在某些实施方案中,宿主细胞可含有野生型油酸成分。
当考虑到这里所提供的教导时,用于表达报春去饱和酶的表达载体构建的方法和组合物对本领域的普通技术人员来说将是显而易见的。如这里所描述的,表达载体是工程化以用于受控表达期望的多核苷酸的DNA或RNA分子,例如编码Δ6-去饱和酶的多核苷酸。载体的例子包括质粒、噬菌体、粘粒或病毒。穿梭载体,例如(Wolk et al.1984;Bustos et al.,1991)也是根据本发明所预期的。载体以及制备和使用它们的方法的综述可以在Sambrook et al.(2001);Goeddel(1990);和Perbal(1988)中找到。能够影响多核苷酸表达的序列元件包括启动子、增强子元件、上游活化序列、转录终止信号和聚腺苷酸化位点。
编码去饱和酶的多核苷酸可置于强启动子的转录控制下。在一些情况下,这将引起表达的去饱和酶的量上增加,并伴随着作为酶所催化的反应而产生的脂肪酸的增加。有大量的各种植物启动子序列可以用来驱动编码去饱和酶的多核苷酸在转基因植物中的组织特异性表达。事实上,在本发明的特定实施方案中,所使用的启动子是种子特异性启动子。这种启动子的例子包括来自诸如napin(Kridl et al.,SeedSci.Res.1:209:219,1991)、菜豆蛋白(Bustos,et al.,Plant Cell,1(9):839-853,1989)、大豆胰蛋白酶抑制剂(Riggs,et al.,Plant Cell1(6):609-621,1989)、ACP(Baerson et al.,Plant Mol.Biol.,22(2):255-267,1993)、硬脂酰-ACP去饱和酶(Slocombe et al.,Plant Physiol.104(4):167-176,1994)、大豆的β-伴大豆球蛋白a’亚单位(P-Gm7S,参见例如Chen et al.,Proc.Natl.Acad.Sci.83:8560-8564,1986)、蚕豆USP(P-Vf.Usp,参见例如SEQ ID NO:1,2,和3,美国专利申请10/429,516)、球蛋白启动子(参见例如Belanger and Kriz,Genet.129:863-872(1991)、大豆的β-伴大豆球蛋白α亚单位(7Sα)(美国专利申请10/235,618,引入作为参考)以及玉米L3油质蛋白启动子(P-Zm.L3,参见例如Hong et al.,Plant Mol.Biol.,34(3):549-555,1997)基因的5′调节区域。也包括玉米蛋白,它是在玉米胚乳中发现的一组贮存蛋白质。玉米蛋白基因的基因组克隆已经被分离(Pedersen et al.,Cell 29:1015-1026(1982),以及Russell et al.,Transgenic Res.6(2):157-168),也可使用来自这些克隆的启动子,包括15kD、16kD、19kD、22kD、27kD以及基因。
普通技术人员能够确定适合在特定宿主细胞中表达的载体和调节元件(包括可操作性连接的启动子和编码区域)。本文中的“可操作性连接”的意思是启动子和终止子序列有效的起到调节转录的作用。作为进一步的例子,适于Δ6-去饱和酶在转基因植物中表达的载体包括来自半日花素、napin或大豆球蛋白的种子特异性启动子序列,其可操作性连接到Δ6-去饱和酶编码区域上,并进一步可操作性连接到种子贮存蛋白质终止信号或胭脂碱合酶终止信号上。作为更进一步的例子,用于植物中Δ6-去饱和酶表达的载体可包含组成型启动子或组织特异性启动子,其可操作性连接到Δ6-去饱和酶编码区域上,并进一步可操作性连接到组成型或组织特异性终止子或胭脂碱合酶终止信号上。
保持这里所预期功能的这里所公开的核苷酸序列或调节元件的修饰在本发明的范围之内。这种修饰包括插入、取代和缺失,特别是反映遗传密码简并性的取代。
构建这种重组载体的标准技术对本领域普通技术人员来说是公知的,可在诸如Sambrook et al.(2001)的参考文献,或者广泛可获得的许多关于重组DNA技术的任何实验室手册中找到。可得到各种连接DNA片段的策略,它的选择取决于DNA片段的末端性质。根据本发明进一步预期在核酸载体中包括有助于克隆、表达或加工的其它核苷酸序列元件,例如编码信号肽的序列、将蛋白质滞留在内质网中所需要的编码KDEL的序列或者编码引导Δ-6去饱和酶到叶绿体的转运肽的序列。这种序列是本领域普通技术人员所已知的。优化的转运肽由例如Van den Broeck et al.(1985)所描述。原核和真核信号序列由例如Michaelis et al.(1982)所公开。
编码期望的去饱和酶的多核苷酸可以用多种方式识别。作为例子,期望的去饱和酶的来源,例如来自报春的基因组或cDNA库,用可检测的酶促或化学合成的探针进行筛选,所述探针可从DNA、RNA、或非天然生成的核苷酸或其混合物制造。探针可从已知的去饱和酶的多核苷酸酶促合成,用于正常的或降低严格性的杂交方法。寡核苷酸探针也可以用于筛选来源,可基于已知去饱和酶的序列,包括已知去饱和酶中保守的序列,或基于从期望的纯化蛋白质获得的肽序列。基于氨基酸序列的寡核苷酸探针可以是简并的以包含遗传密码的简并,或者是偏倚的,以有利于来源生物体的优选密码子。寡核苷酸也可以用作来自已知或可疑来源的逆转录mRNA的PCR引物;PCR产物可以是全长cDNA或者可用来产生探针以获得期望的全长cDNA。或者,期望的蛋白质可被完全测序并进行编码多肽的DNA的全合成。
一旦期望的基因组或cDNA被分离,可通过已知方法测序。现有技术中承认这种方法易受误差影响,所以同一区域的多次测序是常规的,不过还是期望在得到的推导序列中引起的错误率是可测的,特别是在具有重复结构域、大范围二级结构或者不常见的碱基组成的区域,例如高GC碱基含量的区域。当产生矛盾时,可进行重新测序并采用特殊方法。特殊方法可包括改变测序条件,这是通过使用:不同温度;不同酶;改变寡核苷酸形成高度有序结构能力的蛋白质;改变的核苷酸例如ITP或甲基化dGTP;不同凝胶组成,例如加入甲酰胺;不同引物或者位于问题区域不同距离的引物;或者不同模板,例如单链DNAs。也可以采用mRNA测序。
具有去饱和酶活性的多肽的一些或所有编码序列可来自天然来源。然而,在一些情况下,希望通过采用宿主优选的密码子来修饰所有的或部分的密码子,例如以增强表达。宿主优选的密码子可从特定宿主物种和/或感兴趣的组织中最大量表达的蛋白质中最高频率的密码子来确定。这样,具有去饱和酶活性的多肽的编码序列可以全部或部分合成。DNA的全部或部分也可以合成以去除在转录的mRNA中存在的二级结构的任何去稳定序列或区域。也可以合成DNA的全部或部分以将碱基组成改变为希望的宿主细胞中更优选的。合成序列以及组合序列的方法是文献中非常确定的。可采用体外诱变和选择、定点诱变或者其它方法来获得天然存在的去饱和酶基因的突变以产生在体内具有去饱和酶活性的多肽,其具有更希望的在宿主细胞中起作用的物理和动力学参数,例如更长的半衰期或者生产期望的多不饱和脂肪酸的更高速度。
一旦获得编码去饱和酶多肽的多核苷酸,就将它置于能够在宿主细胞中复制的载体中,或者通过诸如PCR或长PCR技术体外增殖。复制载体可包括质粒、噬菌体、病毒、粘粒和类似物。理想的载体包括那些适用于感兴趣基因的诱变或者用于在宿主细胞中表达感兴趣基因的那些。长PCR技术使体外增殖大构建体成为可能,以便修饰感兴趣的基因,例如诱变或加入表达信号,得到的构建体的增殖可在不使用复制载体或宿主细胞时而在体外完整发生。
对于去饱和酶多肽的表达来说,功能性转录和翻译起始和终止区域可操作性连接到编码去饱和酶多肽的多核苷酸上。多肽编码区的表达可在体外或者宿主细胞中发生。转录和翻译起始和终止区源自各种非唯一来源,包括要表达的多肽、已知或者怀疑能够在期望系统中表达的基因、表达载体、化学合成、或者来自宿主细胞中的内源性基因座。
在宿主细胞中的表达可以以瞬时或稳定方式实现。瞬时表达可通过将含有在宿主细胞中有功能的表达信号引入构建体而发生,但是该构建体在宿主细胞中不复制并很少整合,或者其中的宿主细胞不增殖。瞬时表达也可通过诱导可操作性连接到感兴趣基因的可调节启动子的活性而实现,尽管这种可诱导系统常常显示出很低的基础水平表达。稳定表达可通过引入能够整合入宿主基因组的构建体或者在宿主细胞中自主复制的构建体而实现。感兴趣基因的稳定表达可通过使用位于表达构建体上的或者用表达构建体转染的选择标记,接着选择表达标记的细胞而进行选择。当稳定表达是由于整合时,构建体的整合可随机发生在宿主基因组中或者可通过使用含有足以靶定与宿主基因座的重组的与宿主基因组同源的区域的构建体而靶定。在构建体被靶定到内源性基因座时,转录和翻译调节区的所有或一些可由内源性基因座提供。
当希望在来源生物体中增加去饱和酶多肽的表达时,可采用几种方法。可将编码去饱和酶多肽的附加基因引入宿主生物体中。来自天然去饱和酶基因座的表达也可通过同源重组而增加,例如通过将更强的启动子插入宿主基因组中以引起增加的表达,通过将去稳定序列从mRNA或编码的蛋白质中去除——其通过将那些信息从宿主基因组中删除,或者通过加入稳定序列到mRNA中(美国专利No.4,910,141)。
预期可通过使用游离的或整合的表达载体将编码去饱和酶的多于一种的多核苷酸或者编码多于一种去饱和酶的多核苷酸引入到宿主细胞中并在其中增殖。在由独立的复制载体表达两种或更多基因时,可期望每种载体具有不同的复制方式。无论是否整合,每种引入的构建体应该具有不同的选择方式,应该缺少与其它构建体的同源性以保持稳定表达并防止构建体之间的元件重配。调节区域的明智选择、引入构建体的选择方式以及增殖方法可经实验确定,以便所有引入的多核苷酸以必需水平表达以提供期望产物的合成。
当对转化来说必要时,可将本发明的编码Δ6-去饱和酶的序列插入植物转化载体,例如由Bevan(1984)描述的二元载体。植物转化载体可通过修饰根癌土壤杆菌的天然基因转移体系而得到。该天然体系包括含有大片段的大Ti(肿瘤诱导)-质粒,即T-DNA,它是被转移到转化植物中的。Ti质粒的另一片段vir区负责T-DNA转移。T-DNA区毗接末端重复。在修饰的二元载体中,肿瘤诱导基因已经被缺失,利用vir区的功能来转移由T-DNA边界序列毗接的外源DNA.T区域也含有抗生素抗性选择标记,以及插入转移序列的多克隆位点。这样设计的菌株被称为“解除武装”的根癌土壤杆菌菌株,允许T区域毗接的序列有效转化到植物核基因组中。
本发明有许多应用。基于本发明多核苷酸的探针可在用于分离相关分子的方法或者检测表达去饱和酶的生物体的方法中找到用处。当用作探针时,多核苷酸或寡核苷酸必须是可检测的。这通常是通过附着标记而实现的,要么在内部位点例如通过掺入修饰的残基,要么在5’或3’末端。这种标记能够直接检测到,能够结合到被可检测标记的第二分子上,或者能够结合到未标记的第二分子以及被可检测标记的第三分子上;该过程可延长有效长的时间以获得满意地可检测的信号而没有不可接受水平的背景信号。第二、第三或者桥接体系可包括针对任何其它分子的抗体的使用,包括标记或其它抗体,或者可涉及互相结合的任何分子,例如生物素-链霉抗生物素/抗生物素体系。可检测的标记典型地包括放射性同位素、化学或酶促发光的或改变光的分子、产生可检测反应产物的酶、磁性分子、荧光分子或者在结合时荧光或发光特性改变的分子。标记方法的例子可在美国专利No.5,011,770中找到。或者,靶分子的结合可通过经等温滴定量热法测定探针结合到靶上时溶液中热量的改变而直接检测,或者通过将探针或靶包被在表面上并分别检测由靶或探针结合而产生的表面光散射的改变,这可以用BIAcore系统进行。
可通过标准技术将包含感兴趣基因的构建体引入宿主细胞中。为方便起见,通过任何方法被操纵来摄入DNA序列或构建体的宿主细胞在这里将被称为“转化的”或“重组的”。该宿主将至少具有一个拷贝的表达构建体,可能具有两个或更多,这取决于例如基因是被整合到基因组中、扩增,还是存在于具有多拷贝数的染色体外元件上。
转化的宿主细胞可通过对包含在引入的构建体上的标记进行选择而被鉴别。或者,可将期望的构建体引入单独的标记构建体,因为许多转化技术可引入许多DNA分子到宿主细胞中。典型地,可对转化的宿主在选择培养基上生长的能力进行选择。选择培养基可混入抗生素或者缺少对未转化宿主生长所必需的因子,例如营养或者生长因子。因而引入的标记基因可赋予抗生素抗性,或者编码必须的生长因子或酶,当在转化宿主中表达时允许在选择培养基上生长。转化宿主的选择也可在能够直接或间接检测表达的标记蛋白质的时候进行。标记蛋白质可单独或者作为与另一蛋白质的融合物表达。标记蛋白质可通过其酶活性检测;例如β-半乳糖苷酶可将底物X-gal转化为有色产物,荧光素酶可将荧光素转化为发光产物。标记蛋白质可通过其发光的或改变的特征检测;例如维多利亚水母的绿色荧光蛋白在用蓝光照射时发荧光。抗体可用于检测标记蛋白质或例如感兴趣蛋白质上的分子标签。表达标记蛋白质或标志的细胞可例如直观选择或者通过诸如FACS或使用抗体的淘选这样的技术选择。期望地,对卡那霉素和氨基糖苷G418的抗性是感兴趣的,在缺少尿嘧啶、亮氨酸、赖氨酸或色氨酸的培养基上生长的能力也是感兴趣的。
特别感兴趣的是Δ6-去饱和酶介导的真核宿主细胞中PUFA’s的生产。真核细胞包括植物细胞诸如来自产油作物植物的那些,以及其它受遗传操纵的细胞,包括真菌细胞。可将细胞培养或者形成为包括植物在内的宿主生物的部分或者整体。在优选的实施方案中,宿主是产生和/或能够同化外源提供的Δ6-去饱和酶底物的植物细胞,优选产生大量的一种或多种底物。
将转化的宿主细胞生长在适宜于期望的最终结果的适当条件下。对于培养物中生长的宿主细胞来说,将条件典型地优化为产生最大的或者最经济产量的PUFA’s,这与所选去饱和酶活性有关。可被优化的培养基条件包括:碳源、氮源、底物的加入、加入底物的最终浓度、加入底物的形式、需氧或厌氧生长、生长温度、诱导剂、诱导温度、诱导时的生长阶段、收获时的生长阶段、pH、密度以及选择的维持。
本发明的另一方面提供了含有本发明的分离DNA的转基因植物或植物后代。预期包括单子叶植物和双子叶植物两者。用编码Δ6-去饱和酶的分离DNA经任何植物转化方法转化植物细胞。通过本领域普通技术人员公知的方法(例如Horsch et al.,1985)将转化的植物细胞——经常在愈伤组织培养物或叶盘中——再生为完整的转基因植物。在一个实施方案中,转基因植物选自拟南芥、canola、黄豆、大豆、油菜籽、向日葵、棉花、可可、花生、红花、椰子、亚麻、油棕榈、油籽欧洲油菜、玉米、西蒙德木、乌桕、烟草、水果类植物、柑桔类植物或者产生坚果和浆果的植物。由于转化的植物的后代遗传了编码Δ6-去饱和酶的多核苷酸,所以转基因植物的种子或插条也可用于维持转基因植物品系。
本发明进一步提供了用于提供具有增加含量的ALA和/或SDA的转基因植物的方法。该方法包括例如将编码Δ6-去饱和酶的DNA引入缺少或者具有低水平的SDA但是含有ALA的植物细胞中,由转基因细胞再生具有增加的SDA含量的植物。在本发明某些实施方案中,也可将编码Δ15-和/或Δ12-去饱和酶的DNA引入植物细胞中。这种植物可能包含或者也可能不包含内源性Δ12和/或Δ15-去饱和酶活性。在某些实施方案中,预期修饰的商业上种植的作物植物作为转基因生物,包括但不限于拟南芥、canola、黄豆、大豆、油菜籽、向日葵、棉花、可可、花生、红花、椰子、亚麻、油棕榈、油籽欧洲油菜、玉米、西蒙德木、乌桕、烟草、水果类植物、柑桔类植物或者产生坚果和浆果的植物。
本发明进一步提供了用于提供可包含增加水平的ALA和/或SDA的转基因植物的方法,其中所述增加的水平比非转化植物中发现的水平更高。包含编码Δ6-去饱和酶和/或Δ12-去饱和酶和/或Δ-15去饱和酶的DNA的表达载体可通过本领域普通技术人员已知的重组技术方法(Sambrook et al.,2001)进行构建。特别地,商业上种植的作物植物预期作为转基因生物,包括但不限于拟南芥、canola、黄豆、大豆、油菜籽、向日葵、棉花、可可、花生、红花、椰子、亚麻、油棕榈、油籽欧洲油菜以及玉米。
对于饮食补充来说,可将纯化的PUFAs、转化的植物或植物部分、或者它们的衍生物混入配制的食用油、脂肪或人造黄油中,以便在正常使用时接受者能接受期望的量。也可将PUFAs混入婴儿配方、营养补充剂或其它食品中,可发现作为抗炎症或降低胆固醇的试剂的用途。
如这里使用的,“可食用组合物”被定义为可被哺乳动物摄取的组合物,诸如食品、营养物质和药学组合物。如这里使用的,“食品”是指可被使用来或制备来用作哺乳动物食物的物质,包括可被用于食物制备(诸如油炸油)或食物添加剂的物质。例如,食品包括用于人类食用的动物或者任何来自它们的产物例如蛋。典型的食品包括但不限于饮料(例如软饮、碳酸饮料、现混饮料)、浸制食物(例如水果和蔬菜)、酱油、调味品、色拉调料、果汁、糖浆、甜点(例如布丁、明胶、糖霜和馅、烘烤食品和冷冻甜点诸如冰激凌和冰糕)、软冻产品(例如软冻奶油、软冻冰激凌和酸奶、软冻浇头诸如牛奶或非牛奶的搅拌浇头)、油和乳化产品(例如起酥油、人造黄油、蛋黄酱、黄油、食用油和色拉调料)以及半成品含水食物(例如米饭和狗食)。
此外,这里描述的可食用组合物也可作为包含在食物和饮料中的添加剂或补充剂而被摄取。这些可以与诸如各种维生素和矿物质的营养物质配制在一起并混入诸如营养饮料、豆奶和汤的基本上液体的组合物;基本上固体的组合物;以及明胶或者用作粉末形式混入各种食物中。在这种功能食品或健康食品中的有效成分的含量可与典型药学试剂中含有的剂量类似。
纯化的PUFAs、转化的植物或植物部分也可被混入动物特别是家畜饲料中。这样,动物自身可受益于富含PUFA的饮食,而由这种家畜生产的食品的人类食用者也可受益。预期在某些实施方案中SDA将在动物中转化为EPA,因而这种动物可通过食用SDA而受益于EPA增加。
对于药学用途(人或兽医的)来说,组合物一般可口服给药,但是可以通过可成功吸收它们的任何途径给药,例如经肠胃外(即经皮下、经肌肉或者经静脉)、经直肠、经阴道或者经局部给药,例如作为皮肽软膏或者洗剂。本发明的转化PUFAs的植物或者植物部分可被单独给药或者与药学可接受的载体或赋形剂组合给药。在可得到时,明胶胶囊是口服给药的优选形式。如上所述的饮食补充剂也可提供口服给药途径。本发明的不饱和酸可以以缀合形式给药,或者作为脂肪酸的盐、酯、酰胺或前药。本发明包括任何药学可接受的盐,特别优选的是钠盐、钾盐或锂盐。也包括N-烷基多羟胺盐,诸如在PCT公布WO 96/33155中发现的N-甲基葡萄糖胺。优选的酯是乙酯。作为固体盐,PUFAs也可以片剂形式给药。对于静脉给药,PUFAs或其衍生物体可混入商业制剂例如Intralipids中。
如果希望,对去饱和酶活性重要的去饱和酶多肽区域的确定可通过常规诱变然后表达得到的突变蛋白并确定它们的活性。突变可包括取代、缺失、插入和点突变,或者它们的组合。取代可在保守的疏水性或亲水性(Kyte and Doolittle,1982)的基础上进行,或者在呈现类似的多肽二级结构(Chou and Fasman,1978)的能力的基础上进行。典型的功能分析用缺失诱变开始以确定对功能来说必需的蛋白质N和C末端的界限,然后进行内部缺失、插入或点突变以进一步确定对功能来说必需的区域。也可以使用诸如盒诱变或全合成的其它技术。缺失诱变通过例如使用外切核酸酶顺序去除5’或3’编码区而实现。可得到用于这种技术的试剂盒。缺失之后,编码区通过将含有起始或终止密码子的寡核苷酸分别连接到5’或3’缺失后的缺失编码区而补全。或者,将编码起始或终止密码子的寡核苷酸插入编码区,其通过各种方法,包括定点诱变、诱变性PCR或者通过连接到在现有限制性位点被消化的DNA上。
内部缺失同样可通过各种方法进行,包括使用DNA中存在的限制性位点,通过使用诱变引物经定点诱变或者诱变性PCR。插入可通过诸如接头扫描诱变、定点诱变或者诱变性PCR方法进行。点突变可通过诸如定点诱变或者诱变性PCR技术进行。也可使用化学诱变来鉴别对活性重要的去饱和酶多肽区域。这种结构-功能分析可确定哪些区域可被缺失、哪些区域允许插入、哪些点突变允许突变蛋白以与天然去饱和酶基本上同样的方式起作用。所有这种突变蛋白和编码它们的核苷酸序列在本发明的范围之内。
如上所述,本发明的某些实施方案涉及植物转化构建体。例如,本发明的一个方面是包含一种或多种去饱和酶基因或cDNA的植物转化载体。本发明使用的典型的编码序列包括Primula juliae Δ6-去饱和酶(SEQ ID NOs:2-3)。在某些实施方案中,本发明也可采用反义去饱和酶序列。典型的编码去饱和酶的核酸包括SEQ ID NO:2、SEQ IDNO:3、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:45或SEQ ID NO:47的至少20、40、80、120、300以及直到全长核酸序列。在某些方面,核酸可编码1、2、3、4或更多种去饱和酶。在特定实施方案中,核酸可编码Δ6-和Δ15-去饱和酶。
用于植物转化的载体可以包括例如质粒、粘粒、YACs(酵母人工染色体)、BACs(细菌人工染色体)或者任何其它合适的克隆体系,以及它们的DNA片段。这样当使用术语“载体”或“表达载体”的时候,包括所有前述类型的载体以及从中分离的核酸序列。预期高插入能力的克隆体系的利用将允许引入包含一个以上所选基因的大DNA序列。根据本发明,这可以用于引入各种编码去饱和酶的核酸。通过使用细菌或酵母人工染色体(分别为BACs或YACs)或者甚至植物人工染色体可能有助于这种序列的引入。例如,用于土壤杆菌介导的转化的BACs的使用由Hamilton et al.(1996)披露。
特别适用于转化的是从这种载体分离的表达盒。当然,用于转化植物细胞的DNA片段一般将包括人们希望引入并在宿主细胞中表达的cDNA或基因。根据需要,这些DNA片段可进一步包括诸如启动子、增强子、多接头或者甚至调节基因的结构。选择引入细胞的DNA片段或基因经常会编码在得到的重组细胞中表达的蛋白质,产生可筛选的或可选择的性状和/或给予得到的转基因植物改善的表型。然而,可能并不总是这种情况,本发明也包括引入不表达的转基因的转基因植物。可能包括在本发明中使用的载体中的优选的组分如下。
在本发明的一个实施方案中利用了某些启动子。本发明可使用的这种启动子的例子包括但不限于35S CaMV(花椰菜花叶病毒)、34SFMV(玄参花叶病毒)(参见例如美国专利No.5,378,619,其内容整体引入这里)、Napin(来自芸苔)、7S(来自大豆)、球蛋白和Lec(来自玉米)。在植物种子成熟期间进行调节的napin启动子是对使用本发明来说特别感兴趣的。所有这种启动子和转录调节元件,单独或者组合起来,预期用于该可复制的表达载体中,并且是本领域普通技术人员所已知的。
转录起始位点和编码序列起始点之间的DNA序列,即非翻译前导序列也可影响基因表达。那么人们可能希望采用带有本发明的转化构建体的特定前导序列。优选的前导序列预期包括那些包含了预知将引导连接基因优化表达的序列,即包括优选的可增加或保持mRNA稳定性并防止不当翻译起始的共有前导序列。根据本文的公开内容,这种序列的选择对本领域技术人员来说将是已知的。源自植物中高表达基因的序列将是典型优选的。
根据本发明制备的转化构建体将典型地包括3’末端DNA序列作为终止转录的信号并允许由可操作性连接到去饱和酶基因的编码序列(例如cDNA)产生的mRNA的多腺苷酸化。在本发明的一个实施方案中,使用去饱和酶基因的天然终止子。或者,异源的3’末端可以增强去饱和酶编码区的表达。认为有用的终止子的例子包括来自根癌土壤杆菌的胭脂碱合酶基因的那些终止子(nos 3’末端)(Bevan et al.,1983),来自根癌土壤杆菌的胭脂碱合酶基因的T7转录终止子,来自马铃薯或西红柿的蛋白酶抑制剂I或II基因的3’末端以及CaMV 35S终止子(tml3’)。如果希望,可进一步包括调节元件,诸如Adh内含子(Callis et al.,1987)、蔗糖合酶内含子(Vasil et al.,1989)或者TMV ω元件(Gallie et al.,1989)。
通过采用可选择的或者可筛选的标记蛋白质,人们可提供或者增强鉴别转化体的能力。“标记基因”是给予表达标记蛋白的细胞以独特表型,从而允许这种转化细胞与没有标记的细胞区分开的基因。这种基因可以编码可选择的或可筛选的标记,取决于标记是否赋予了人们能够通过化学方法“选择”的特性,即通过选择剂(例如除草剂、抗生素或类似物)的使用,或者它是否仅仅是人们通过观察或测试,即通过“筛选”(例如绿色荧光蛋白)就能够鉴别的特性。当然,合适的标记蛋白的许多例子是现有技术中已知的,可用于实施本发明。
适于转化本发明所使用的植物或其它细胞的方法据信包括实际上任何的可由此将DNA引入细胞的方法,诸如通过直接送递DNA,诸如通过PEG介导的原生质体转化(Omirulleh et al.,1993),通过脱水/抑制介导的DNA摄取(Potrykus et al.,1985),通过电穿孔(美国专利No.5,384,253,特别引入这里整体作为参考),通过用碳化硅纤维搅拌(Kaeppler et al.,1990;美国专利No.5,302,523,特别引入这里整体作为参考;以及美国专利No.5,464,765,特别引入这里整体作为参考),通过土壤杆菌介导的转化(美国专利No.5,591,616和美国专利No.5,563,055;两者均特别引入这里作为参考)以及通过DNA包被粒子的加速(美国专利No.5,550,318;美国专利No.5,538,877;以及美国专利No.5,538,880;每一篇均特别引入这里整体作为参考),等等。通过诸如这些技术的应用,实际上任何植物物种的细胞都可被稳定转化,并且这些细胞发育成为了转基因植物。
在实现外源DNA送递到受体细胞后,下一步通常涉及鉴别转化的细胞用于进一步培养和植物再生。为改善鉴别转化体的能力,人们可能希望采用可选择的或可筛选的带有根据本发明制备的转化载体的标记基因。在这种情况下,那么人们一般就可以通过将细胞暴露于选择剂来分析潜在的转化细胞群,或者人们会筛选细胞的期望标记基因性状。
幸免于选择剂暴露的细胞、或者在筛选分析中被评分为阳性的细胞可在支持植物再生的培养基中培养。在典型的实施方案中,MS和N6培养基可通过包括更多的诸如生长调节剂的物质而被修改。一种这样的生长调节剂是麦草畏或者2,4-D。然而,可采用其它生长调节剂,包括NAA、NAA+2,4-D或者毒莠定。已经发现以这些或者类似方式的培养基改进有助于细胞在特殊发育阶段的生长。组织可维持在有生长调节剂的基础培养基中直到可得到足够组织来开始植物再生的努力,或者在手工选择的重复循环之后直到组织形态适于再生,典型地至少2周,然后转移到对胚状体成熟有益的培养基中。每2周将培养物转移到这种培养基上。茎干发育将标志着转移到不含生长调节剂的培养基的时间。
为证实再生植物中外源DNA或“转基因”的存在,可进行各种分析。这种分析包括例如“分子生物学”分析,诸如Southern和Northern印迹以及PCRTM;“生物化学”分析,诸如检测蛋白质产物的存在,例如通过免疫学方法(ELISAs和Western印迹)或者通过酶功能;植物部分分析,诸如叶或根的分析;也包括通过分析整个再生植物的表型。
除了用根据本发明制备的构建体直接转化特定的植物基因型,转基因植物也可通过将具有本发明的所选DNA的植物与缺少该DNA的第二植物杂交而产生。植物繁殖技术也可用来引入多种去饱和酶,例如Δ6、Δ12和/或Δ15-去饱和酶到单株植物中。以这种方式,可有效上调Δ6-去饱和酶。通过产生Δ6-去饱和酶活性和/或其它去饱和酶活性(例如Δ12-和/或Δ15-去饱和酶活性)的纯合植物,可增加植物中的有益代谢物。
如上所述,所选去饱和酶基因可通过杂交被引入特定植物品种中,不需要总是直接转化那种特定品种的植物。因此,本发明不但包括直接转化的植物或者从根据本发明转化的细胞再生的植物,也包括这些植物的后代。这里使用的术语“后代”表示根据本发明制备的亲本植物的任意代的后代,其中后代包含根据本发明制备的所选DNA构建体。“杂交”植物以提供相对于起始植物品系具有一种或多种添加的转基因或等位基因的植物品系,如这里所公开的,定义为产生了引入植物品系中的特定序列的技术,其通过将起始品系与包含本发明的转基因或等位基因的供体植物品系杂交。为实现这点,可以例如进行下面的步骤:(a)种植第一(起始品系)和第二(包含期望的转基因或等位基因的供体植物品系)亲本植物的种子;(b)将第一和第二亲本植物的种子生长成带花的植物;(c)用来自第二亲本植物的花粉给来自第一亲本植物的花授粉;以及(d)收获具有受精过的花的亲本植物上产生的种子。
回交在这里被定义为包括下面步骤的过程:(a)将含有期望的基因、DNA序列或元件的第一基因型植物与缺少所述期望的基因、DNA序列或元件的第二基因型植物杂交;(b)选择含有期望的基因、DNA序列或元件的一个或多个后代植物;(c)将该后代植物与第二基因型植物杂交;以及(d)为了将来自第一基因型植物的期望的DNA序列转移到第二基因型植物,重复步骤(b)和(c)。
DNA元件对植物基因型的渐渗被定义为回交转化过程的结果。被DNA序列渐渗的植物基因型可被称为回交转化的基因型、品系、近交系或者杂种。类似的,缺乏期望DNA序列的植物基因型可被称为未转化的基因型、品系、近交系或者杂种。
实施例
引入下面的实施例来举例说明本发明的实施方式。本领域技术人员应当意识到,下面实施例中公开的技术代表由发明人发现的在本发明的实施中很好起作用的技术。然而,本领域技术人员根据该公开内容应当意识到,在公开的具体实施方案中能够进行许多改变并仍然获得相似或类似的结果,这没有脱离本发明的概念、精神和范围。更特别地,显而易见的是,某些化学上和生理上相关的试剂可替换这里描述的试剂,但是会获得同样或类似的结果。所有这样的对本领域技术人员显而易见的类似替换和修改被认为是在如所附的权利要求所限定的本发明的精神、范围和概念之内的。
实施例1
Primula juliae Δ6去饱和酶序列的克隆
Primula juliae Δ6去饱和酶(PjD6D)的克隆是通过使用简并寡核苷酸对部分内部基因组DNA区域进行PCR扩增、接着进行双向基因组步移而完成的。使用DNeasy Plant Mini试剂盒(Qiagen,Valencia,CA)根据厂商程序从P.juliae(Collector’s Nursery,Battleground WA)分离总基因组DNA。最初,使用如Garcia-Maroto et al.(2002)描述的简并寡核苷酸BO-1 For和BO-2 Rev分离到了相应于SEQ ID NO:1的687到1238位的552bp的片段。将片段克隆进pCR4-TOPO(Invitrogen,Carlsbad,CA)以产生载体pMON83955,对插入片段测序。引物序列BO-1 For和BO-2 Rev如下:
BO-1 For:5’-ATMAGYATYGGTTGGTGGAARTGG-3’
(SEQ ID NO:6)
BO-2 Rev:5’-AATCCACCRTGRAACCARTCCAT-3’
(SEQ ID NO:7)
为确定pMON83955插入片段的基因组侧翼序列,采用了UniversalGenome Walker KitTM(BD Biosciences,Palo Alto,CA),根据厂商程序进行。用四种限制酶EcoRV、PvuII、StuI和DraI消化DNA产生了四种P.juliae基因组文库。在纯化步骤后,将消化产物与试剂盒中提供的接头连接。然后程序涉及两次PCR反应,每次都使用基因特异性引物和接头引物。第二次PCR反应使用首次PCR反应产物的稀释物作为模板。对于5’方向,首次和第二次PCR反应分别使用引物PD6D R8和PD6D R2。对于3’方向,首次和第二次PCR反应分别使用引物PD6DF8和PD6D F3。引物序列如下所示:
PD6D R8:5’-CACACATGACCGGATAAAACGACCAGT-3’
(SEQ ID NO:8)
PD6D R2:5’-GGGAATGTACTGGAGGTCAGGGTCGTA-3’
(SEQ ID NO:9)
PD6D F8:5’-CGTGCAGTTCAGCTTGAACCATTTCTC-3’
(SEQ ID NO:10)
PD6D F3:5’-TGCAGGGACACTCAACATATCGTGCCC-3’
(SEQ ID NO:11)
沿5’方向的基因组步移从EcoRV文库产生了574bp的片段。将该产物克隆进pCR4-TOPO(Invitrogen)得到pMON83956,对插入片段测序。得到的序列不包含推定的Δ6去饱和酶基因的起始密码子,这样另一组PCR反应是使用基因特异性引物进行的,所述引物设计来从pMON83956插入片段沿5’方向步移。用于第二组沿5’方向基因组步移的引物分别是用于第一和第二PCR反应的PD6D R15和PD6D R14。序列如下:
PD6D R15:5’-GTAGGTTGGTGGAGAAGGGAGGGAGGA-3’
(SEQ ID NO:12)
PD6D R14:5’-GGAAGGGGGATGGTAAGCGAGGAAAGC-3’
(SEQ ID NO:13)
将来自StuI文库的328bp长度的产物克隆进pCR4-TOPO(Invitrogen)得到pMON83958,对插入片段测序。该插入片段包含2个潜在的起始密码子,相隔44个碱基。第一个起始密码子相应于SEQ IDNO:1的87位,第二个相应于135位。沿3’方向的基因组步移由DraI文库产生了773bp的片段。将该产物克隆进pCR4-TOPO,得到pCR4-TOPO。对插入片段测序,发现其含有推定的Δ6去饱和酶基因编码区的292bp,接着是相应于SEQ ID NO:1第1473位的终止密码子。
将插入片段pMON83955、pMON83956、pMON83957和pMON83958比对形成组合序列SEQ ID NO:1。设计三种引物来PCR扩增2种不同长度的来自P.juliae基因组DNA的编码序列,反映了pMON83958中发现的两种起始密码子。两种序列中较长的PjD6D-1使用正向引物Pj D6D F2和反向引物Pj D6D R1进行扩增。两种序列中较短的PjD6D-2使用正向引物PjD6D F1和反向引物PjD6D R1进行扩增。然后将两种推定的Δ6去饱和酶编码序列的每一种连接到酵母表达载体pYES2.1-TOPO中。当测序时,含有PjD6D-1的质粒命名为pMON83950(SEQ ID NO:3),含有质粒PjD6D-2的质粒命名为pMON67011(SEQ ID NO:2)。引物序列如下:
Pj D6D F2:5’-GTCGACATGGAAAACACATTTTCACCACCACCT-3’
(SEQ ID NO:14)
Pj D6D F1:5’-GTCGACATGACTAAGACCATTTACATAACCAGC-3’
(SEQ ID NO:15)
Pj D6D R1:5’-CCTGCAGGTCACCCGACATTTTTAACAGCCTCCC-3’
(SEQ ID NO:16)
两种PjΔ6去饱和酶克隆PjD6D-2和PjD6D-1编码潜在的446个氨基酸和462个氨基酸的多肽,分别示于SEQ ID NO:4和SEQ IDNO:5。较短的肽序列(PjD6D-2)的起始MET位点位于较长序列(PjD6D-1)第一个MET位点的下游16个氨基酸处。第二个MET的3’序列是同样的。这些序列具有与其它植物Δ6去饱和酶(图1)的高相似性,包括在所有前端去饱和酶(Napier et al.,2003)中发现的N-末端细胞色素b5结构域。在细胞色素b5结构域中发现了表征细胞色素b5超家族的八个不变残基以及H-P-G-G的血红素结合基序,这显示出对于酶活性是必需的(Napier et al.,1997,Sayanova et al,1999,Sperlingand Heinz 2001)。在推定的PjD6D去饱和酶的去饱和酶结构域中,三个保守的组氨酸盒是所有膜结合去饱和酶的特征(Shanklin et al.,1994)。在所有前端去饱和酶中发现的区别特征在于,三个组氨酸盒在第一个位置含有谷氨酰胺(Q-x-x-H-H)而不是组氨酸(Napier et al.,1997,Napier et al.,2003,Sperling and Heinz 2001)。推导的PjD6D氨基酸序列具有与Primula vialii和有粉报春去饱和酶大约88%的同一性以及与Echium pitardii和E.gentianoides去饱和酶大约64%的同一性。对图1所示的多序列比对的目测表明P.juliae Δ6去饱和酶序列不含任何内含子。这也在来自报春和蓝蓟物种的Δ6去饱和酶中观察到了(Sayanova etal.,2003,Garcia-Maroto et al.,2002)。
实施例2
酵母转化和表达
使用用于pYES2.1/V5-His-TOPO的Invitrogen手册中描述的PEG/Li Ac方案,将构建体pMON83950(图3)和pMON67011(图2)引入尿嘧啶营养缺陷型宿主菌株酿酒酵母INVSc1(Invitrogen)中。转化体的选择在由不含尿嘧啶的有2%葡萄糖的SC极限培养基制成的平板上进行。将转化体菌落用于接种5ml的不含尿嘧啶的有2%葡萄糖的SC极限培养基,在30℃生长过夜。对于诱导,将静止期酵母细胞沉淀,重悬浮于不含尿嘧啶的有2%半乳糖的SC极限培养基中,在15℃生长3天。当提供外源脂肪酸给培养物时,加入带有乳化剂0.1%Tergitol的0.01%LA(Δ9,12-18:2)。将培养物在15℃生长3天,随后通过离心收获。将细胞沉淀用无菌TE缓冲液pH7.5漂洗一次,去除培养基,冻干24h。用含有LacZ基因的载体转化的宿主株用作所有研究中的阴性对照。
通过加入0.1mL甲苯并在室温温育过夜而将脂质从冻干的酵母沉淀中提取出来。通过加入0.5mL的0.6N的溶于甲醇中的甲醇钠并温育45min而将提取的脂在原处转化为脂肪酸甲酯(FAMEs)。通过加入0.8mL的10%(w/v)NaCl和0.15mL庚烷而提取FAMEs。将含有FAMEs的庚烷层取出,直接用于气相色谱(GC)。在配备火焰-离子化探测器和毛细管柱(ωwax 250;30m×0.25mm i.d.×0.25μm;Supelco,Bellefonte,PA)的Hewlett-Packard 5890 II Plus GC(Hewlett-Packard,Palo Alto,CA)上鉴别FAMEs。使用100∶1的分流比进行注射。注射器维持在250℃,火焰离子化探测器维持在270℃。在注射后将柱温度维持在180℃ 1.5min,随后以40℃/min的速度增加到240℃,在245℃保持3.38min。
表1显示了表达P.juliae克隆pMON67011(PjD6D-2)、pMON83950(PjD6D-1)或者高山被孢霉Δ6去饱和酶pMON77205的酵母脂肪酸组成。在两种P.juliae克隆中都观察到了LA和ALA的Δ6去饱和的期望产物(表1,分别为GLA和SDA),证明pMON67011和pMON83950中含有的克隆是Δ6去饱和酶。底物选择性通过供给等量的LA和ALA来确定。高山被孢霉是累积高水平的n-6脂肪酸花生四烯酸的丝状真菌,预期具有带有n-6选择性的Δ6去饱和酶。表2显示了P.juliae和高山被孢霉Δ6去饱和酶的n-3∶n-6底物选择性。观察到了高山被孢霉Δ6去饱和酶的n-3∶n-6选择性为约0.8。观察到了两种P.juliae Δ6去饱和酶的n-3∶n-6选择性为约1.5-1.9。
表1:表达不同Δ6去饱和酶的酵母的脂肪酸组成的比较
载体 | 基因 | 培养基中的FA | LA* | GLA* | ALA* | SDA* |
pMON67011pMON67011pMON67011pMON67011pMON67011pMON67011pMON67011pMON67011 | P.juliae D6D-2P.juliae D6D-2P.juliae D6D-2P.juliae D6D-2P.juliae D6D-2P.juliae D6D-2P.juliae D6D-2P.juliae D6D-2 | --LALAALAALALA+ALALA+ALA | 2.02.525.728.40.30.322.724.3 | 0.00.014.016.80.10.16.05.8 | 0.00.10.00.124.430.618.020.4 | 0.00.00.00.016.819.08.58.9 |
pMON83950pMON83950pMON83950pMON83950pMON83950pMON83950pMON83950pMON83950 | P.juliae D6D-1P.juliae D6D-1P.juliae D6D-1P.juliae D6D-1P.juliae D6D-1P.juliae D6D-1P.juliae D6D-1P.juliae D6D-1 | --LALAALAALALA+ALALA+ALA | 2.32.326.323.50.60.718.816.9 | 0.00.015.016.60.20.14.34.8 | 0.30.20.00.037.333.917.115.7 | 0.00.00.00.017.517.49.49.8 |
pMON77205pMON77205pMON77205 | 高山被孢霉D6D高山被孢霉D6D高山被孢霉D6D | --LA | 1.71.056.8 | 0.00.06.0 | 0.20.00.0 | 0.00.00.0 |
pMON77205 | 高山被孢霉D6D | LA | 25.4 | 4.6 | 0.2 | 0.0 |
pMON77205pMON77205pMON77205pMON77205 | 高山被孢霉D6D高山被孢霉D6D高山被孢霉D6D高山被孢霉D6D | ALAALALA+ALALA+ALA | 0.50.934.818.7 | 0.00.01.32.8 | 69.223.039.718.4 | 2.65.01.12.2 |
**报导为包括在GC-FID色谱图中的所有分析物的总量的%,包括但是未显示(16:0,16:1,18:0,20:0,20:1,20:2,22:0,22:1,22:2
表2.P.juliae和高山被孢霉Δ6去饱和酶的n-3∶n-6底物选择性比较
载体 | 基因 | 培养基中的FA | %转化GLA* | %转化SDA* | n-3∶n-6比** |
pMON67011pMON67011 | P.juliae D6D-2P.juliae D6D-2 | LA+ALALA+ALA | 21.019.2 | 32.130.3 | 1.531.58 |
pMON83950pMON83950 | P.juliae D6D-1P.juliae D6D-1 | LA+ALALA+ALA | 18.722.2 | 35.438.4 | 1.891.73 |
pMON77205pMON77205 | 高山被孢霉D6D高山被孢霉D6D | LA+ALALA+ALA | 3.612.8 | 2.810.5 | 0.780.82 |
*转化为GLA的百分比是通过用GLA的值(表1)除以LA和GLA值(表1)的和而计算的。对SDA用ALA和SDA(表1)的和进行同样的计算。
**n-3∶n-6比是通过%转化SDA除以%转化GLA而计算的。
实施例3
Primula juliae Δ6-去饱和酶的植物转化和表达
Primula juliae Δ6-去饱和酶活性是通过将它与来自粗糙链孢霉(NcD15D)pMON77245(图4)或者构巢曲霉(AnD15D)pMON77247(图5)的Δ15-去饱和酶组合在大豆中而估计。载体pMON77245按三步构建。首先通过用Sse8387 I消化pMON67011、接着去除3’突出端和SalI而将P.juliae Δ6-desaturase(PjD6D-2)置于种子特异性7S α’启动子后面,然后将得到的片段连接到表达载体pMON68527的EcoRI和填平的XhoI位点中,产生质粒pMON77243。第二步,通过用NotI消化、接着经填平反应将PjD6D-2表达盒从pMON77243上移除,然后将得到的片段连接到2T二元载体pMON77244的EcoRV位点中。最后,通过用NotI消化pMON77227,将在7Sα种子特异性启动子控制下的密码子优化的NcD15D(SEQ ID NO:17)与PjD6D-2组合,其然后将得到的NcD15D表达盒片段连接到NotI消化的pMON77344中,得到pMON77245(图4)。载体pMON77247(图5)的构建通过用NotI消化载体pMON77242,将得到的包含连接到7Sα启动子的密码子优化的AnD15D(SEQ ID NO:18)的表达盒片段连接到pMON77244的NotI位点中。将载体pMON77245和pMON77247使用Martinell et al.的方法转化到大豆中(美国专利No.6,384,301,其公开内容整体引入这里作为参考)。
PjD6D-2编码序列表达的测定通过确定未成熟(大约开花后30天)的R1转基因大豆种子中的脂肪酸组成进行,所述种子包括纯合子和杂合子,所述组成确定通过脂质甲酯衍生物的气相色谱进行(PCTUS03/16144,2003年5月21日提交,其完整公开内容特别引入这里作为参考)。PA(棕榈酸,16:0)、SA(硬脂酸,18:0)、OA、LA、GLA、ALA和SDA的水平被表达为测定脂肪酸总重量的百分比,示于表3和表4中。非转基因对照品系是A3525。只要有可能,在每个事件中分析五种个体种子。
发现了来自大部分pMON77245转基因事件的个体种子累积可测定量的SDA。在所有情况下,SDA的水平比GLA水平更高,每个事件的平均SDA∶GLA比在2∶1到高达8∶1的范围。在事件GM_A38083中观察到了最高的单个种子值,其含有32.0%的SDA和2.6%的GLA,SDA∶GLA比为12∶1。下列12种事件中,有9种在至少五分之一的种子中SDA值>10%。当SDA值增加时,PA、SA和OA水平与对照水平没有明显不同;然而,对LA来说具有很强的负相关。在累积SDA的种子中,GLA水平仍然很低,在2.3到5.5%之间。ALA水平随SDA水平而增加。
表3:来自单个的pMON77245转化的R1种子的相对区百分比结果(大约的重量百分比)
pMON77245 | 脂肪酸(重量百分比) | ||||||
谱系 | PA | SA | OA | LA | GLA | ALA | SDA |
A3525 | 11.47 | 5.21 | 16.5 | 56.75 | 0 | 9.15 | 0 |
A3525 | 11.66 | 4.53 | 18.54 | 54.9 | 0 | 9.51 | 0 |
A3525 | 11.8 | 5.42 | 16.66 | 56.04 | 0 | 9.14 | 0 |
A3525 | 11.41 | 4.91 | 17.64 | 56 | 0 | 9.08 | 0 |
A3525 | 11.56 | 4.36 | 17.86 | 56.55 | 0 | 8.77 | 0 |
GM_A38005 | 12.57 | 4.19 | 18.45 | 53.99 | 0 | 10.8 | 0 |
GM_A38005 | 13.73 | 4.77 | 19.32 | 52.42 | 0 | 9.76 | 0 |
GM_A38005 | 14.81 | 4.74 | 19.09 | 36.84 | 5.23 | 10.3 | 8.98 |
pMON77245 | 脂肪酸(重量百分比) | ||||||
GM_A38005 | 13.4 | 4.71 | 18.34 | 53.26 | 0 | 10.29 | 0 |
GM_A38005 | 13.21 | 4.38 | 19.97 | 52.19 | 0 | 10.25 | 0 |
GM_A38005 | 13.08 | 4.78 | 17.99 | 53.56 | 0 | 10.59 | 0 |
GM_A38013 | 12.91 | 4.45 | 19.72 | 40.8 | 4.57 | 9.56 | 7.99 |
GM_A38013 | 12.45 | 4.38 | 18.9 | 55.04 | 0 | 9.23 | 0 |
GM_A38013 | 13.04 | 4.68 | 17.38 | 40.36 | 4.66 | 10.27 | 9.61 |
GM_A38013 | 13.26 | 4.34 | 17.14 | 40.03 | 4.6 | 10.17 | 10.46 |
GM_A38013 | 11.67 | 4.26 | 22.5 | 44.26 | 3.3 | 8.95 | 5.05 |
GM_A38021 | 12.95 | 4.33 | 19.39 | 53.48 | 0 | 9.85 | 0 |
GM_A38021 | 13.07 | 4.87 | 18.12 | 54.1 | 0 | 9.84 | 0 |
GM_A38021 | 13.14 | 4.27 | 22.76 | 34.62 | 2.3 | 13.7 | 9.2 |
GM_A38021 | 12.98 | 4.08 | 21.58 | 39.6 | 1.6 | 13.7 | 6.45 |
GM_A38021 | 13.21 | 4.34 | 17.24 | 29.03 | 1.78 | 19.07 | 15.31 |
GM_A38043 | 13.1 | 4.26 | 19.58 | 52.44 | 0 | 10.62 | 0 |
GM_A38043 | 13.09 | 4.3 | 20.01 | 52.83 | 0 | 9.77 | 0 |
GM_A38043 | 14.01 | 4.35 | 22.05 | 29.98 | 4.39 | 12.18 | 13.05 |
GM_A38043 | 13.32 | 4.26 | 19.41 | 51.85 | 0 | 11.16 | 0 |
GM_A38043 | 12.8 | 4.34 | 19.81 | 53 | 0 | 10.05 | 0 |
GM_A38048 | 13.44 | 5.5 | 18.01 | 44.46 | 2.28 | 10.7 | 5.61 |
GM_A38048 | 13.43 | 4.8 | 18.57 | 44.25 | 2.34 | 10.93 | 5.68 |
GM_A38048 | 13.14 | 4.47 | 18.88 | 44.97 | 2.33 | 10.78 | 5.44 |
GM_A38048 | 12.98 | 4.89 | 17.79 | 44.92 | 2.43 | 11.23 | 5.76 |
GM_A38048 | 13.3 | 4.56 | 17.95 | 35.88 | 3.41 | 13.15 | 11.75 |
GM_A38060 | 12.73 | 4.94 | 17.37 | 43.16 | 4.01 | 10.4 | 7.39 |
GM_A38060 | 12.85 | 5.19 | 15.27 | 35.1 | 5.32 | 11.88 | 14.39 |
GM_A38060 | 12.73 | 4.99 | 16.41 | 43.44 | 3.95 | 10.25 | 8.23 |
GM_A38060 | 13.06 | 5.34 | 16.06 | 42.75 | 4.04 | 10.32 | 8.43 |
GM_A38060 | 12.85 | 5.25 | 16.45 | 42.68 | 4.01 | 10.39 | 8.36 |
GM_A38064 | 13.32 | 5 | 18.8 | 42 | 3.86 | 10.16 | 6.87 |
GM_A38064 | 13.07 | 4.72 | 18.97 | 42.1 | 3.59 | 9.95 | 7.6 |
pMON77245 | 脂肪酸(重量百分比) | ||||||
GM_A38064 | 13.45 | 4.84 | 19.7 | 41.67 | 3.8 | 9.92 | 6.62 |
GM_A38064 | 12.66 | 4.61 | 19.09 | 43.21 | 3.52 | 9.85 | 7.05 |
GM_A38064 | 13.03 | 4.73 | 19.58 | 36.38 | 4.94 | 11.28 | 10.06 |
GM_A38069 | 12.9 | 4.71 | 21.24 | 41.12 | 2.64 | 11.43 | 5.97 |
GM_A38069 | 12.74 | 4.76 | 20.35 | 51.21 | 0 | 10.94 | 0 |
GM_A38069 | 12.93 | 4.77 | 20.5 | 51.27 | 0 | 10.53 | 0 |
GM_A38069 | 13.18 | 4.69 | 18.85 | 38.76 | 3.3 | 12.34 | 8.87 |
GM_A38069 | 13.08 | 4.79 | 19.16 | 52.08 | 0 | 10.89 | 0 |
GM_A38083 | 13.33 | 5.28 | 21.73 | 27.31 | 2.48 | 15.28 | 13.35 |
GM_A38083 | 12.8 | 4.96 | 16.85 | 11.52 | 2.64 | 18.11 | 32.02 |
GM_A38083 | 12.32 | 5.07 | 22.23 | 13.59 | 2.52 | 17.46 | 25.56 |
GM_A38083 | 13.22 | 4.26 | 20.83 | 15.89 | 3.81 | 14.69 | 26.12 |
GM_A38083 | 13.74 | 4.61 | 17.03 | 20.93 | 4.84 | 13.82 | 23.91 |
GM_A38084 | 12.9 | 4.04 | 22.66 | 41.63 | 3.37 | 9.07 | 5.28 |
GM_A38084 | 13.38 | 3.94 | 28.07 | 25.81 | 4.9 | 11.37 | 11.42 |
GM_A38084 | 13.92 | 3.75 | 31.36 | 32.26 | 2.89 | 9.23 | 5.51 |
GM_A38084 | 14.42 | 4.12 | 27.17 | 33.26 | 3.28 | 11.57 | 5.77 |
GM_A38084 | 12.74 | 3.95 | 22.59 | 40.82 | 3.3 | 9.68 | 5.91 |
GM_A38089 | 13.05 | 4.48 | 22.37 | 42.63 | 2.55 | 9.3 | 4.59 |
GM_A38089 | 13.15 | 4.63 | 18.82 | 53.48 | 0 | 9.03 | 0 |
GM_A38089 | 12.67 | 4.41 | 20.59 | 51.87 | 0 | 9.42 | 0.07 |
GM_A38089 | 12.64 | 4.29 | 20.56 | 52.58 | 0 | 8.96 | 0 |
GM_A38089 | 12.72 | 4.57 | 21.81 | 50.79 | 0 | 9.16 | 0 |
GM_A38094 | 12.62 | 4.57 | 18.97 | 52.96 | 0 | 9.9 | 0.11 |
GM_A38094 | 13.3 | 5.08 | 17.08 | 34.49 | 5.35 | 11.39 | 12.35 |
GM_A38094 | 13.08 | 4.52 | 18.38 | 38.95 | 5.41 | 9.88 | 8.82 |
GM_A38094 | 13.41 | 5 | 17.27 | 38.5 | 5.49 | 10.26 | 9.1 |
GM-A38094 | 12.58 | 4.46 | 20.06 | 40.28 | 4.88 | 9.5 | 7.25 |
来自pMON77247转基因事件的个体种子累积了与pMON77245相比的相似量的SDA,只是事件GM_A38083累积了明显更高水平的SDA。PA、SA、OA和LA水平与表3所示的对照水平相似。通常SDA水平比GLA水平更高,每个事件的平均SDA∶GLA比在1∶1到1.6∶1的范围,这比在pMON77245中现察到的更小。
表4:来自单个的pMON77247转化的R1种子的相对区百分比结果(大约的重量百分比)
pMON77247 | 脂肪酸(重量百分比) | ||||||
谱系 | PA | SA | OA | LA | GLA | ALA | SDA |
GM_A38909 | 12.18 | 4.19 | 20.66 | 44.94 | 3.52 | 8.65 | 4.87 |
GM_A38909 | 12.25 | 3.84 | 22.37 | 44.89 | 2.95 | 8.22 | 4.46 |
GM_A38909 | 12.06 | 4.67 | 22.95 | 43.37 | 3.31 | 8.32 | 4.86 |
GM_A38909 | 12.64 | 4.63 | 17.61 | 45.99 | 3.66 | 9.01 | 5.44 |
GM_A38909 | 12.28 | 4.2 | 19.42 | 46.1 | 3.1 | 9.01 | 4.82 |
GM_A38941 | 13.95 | 4.87 | 18.03 | 40.2 | 7.08 | 7.87 | 6.92 |
GM_A38941 | 13.76 | 4.38 | 19.72 | 33.62 | 8.94 | 8.57 | 9.95 |
GM_A38941 | 13.15 | 4.91 | 17.89 | 52.06 | 0.75 | 9.52 | 0.8 |
GM_A38941 | 12.73 | 4.27 | 22.23 | 42.14 | 4.98 | 7.44 | 5.15 |
GM_A38941 | 12.73 | 4.34 | 19.37 | 52.34 | 0.36 | 9.53 | 0.37 |
GM_A38946 | 13.02 | 4.68 | 17.4 | 44.66 | 4.54 | 8.83 | 5.89 |
GM_A38946 | 13.17 | 4.42 | 17.35 | 43.71 | 5.01 | 8.91 | 6.49 |
GM_A38946 | 13.63 | 4.24 | 18.96 | 38.16 | 6.36 | 8.89 | 8.75 |
GM_A38946 | 13.32 | 4.6 | 17.76 | 43.37 | 4.8 | 8.94 | 6.2 |
GM_A38946 | 13.32 | 4.5 | 18.07 | 43.24 | 4.71 | 8.95 | 6.23 |
GM_A38977 | 13.43 | 5.18 | 21.3 | 40.54 | 4.43 | 8.51 | 5.62 |
GM_A38977 | 13.6 | 4.92 | 21.44 | 40.95 | 4.26 | 8.41 | 5.42 |
GM_A38977 | 13.17 | 4.23 | 21.61 | 38.02 | 5.45 | 8.38 | 8.07 |
GM_A38977 | 13.06 | 4.97 | 21.93 | 37.82 | 5.75 | 8.63 | 6.86 |
GM_A38977 | 13.33 | 4.5 | 22.96 | 37.43 | 5.54 | 8.42 | 6.76 |
GM_A39047 | 13.22 | 4.21 | 20.95 | 31.88 | 7.8 | 9.01 | 11.72 |
pMON77247 | 脂肪酸(重量百分比) | ||||||
GM_A39047 | 13.34 | 4.47 | 19.14 | 31.1 | 7.54 | 9.9 | 13.35 |
GM_A39047 | 13.79 | 4.32 | 18.82 | 32.97 | 8.26 | 9.07 | 11.68 |
GM_A39047 | 13.16 | 4.38 | 19.34 | 29.61 | 7.94 | 9.97 | 14.44 |
GM_A39047 | 12.65 | 4.25 | 17.48 | 50.71 | 1.49 | 9.92 | 2.45 |
实施例4
Primula juliae Δ6-去饱和酶与粗糙链孢霉Δ15-去饱和酶
在canola中组合的活性
Primula juliae Δ6-去饱和酶与粗糙链孢霉Δ15-去饱和酶组合的活性通过用MON82822(FIG.7)转化canola来评估。将含有天然NcD15D(SEQ ID NO:19)以及PjD6D-2的pMON82822两者都插入在napin启动子控制下的种子特异性表达盒中(PCT US03/16144,其公开内容特别引入这里作为参考)。
pMON82822载体的构建通过首先用PmeI和BamHI(填平的)消化pMON77214(PCT US03/16144),并将得到的天然NcD15D napin盒连接到2T二元载体pMON71801的EcoRV位点中以产生pMON82820。接下来用NotI消化pMON82819,末端填平,将得到的PjD6D-2 napin表达盒连接到pMON82820的填平的AscI中以产生pMON82822。
第二个载体pMON82821也构建为含有密码子优化的NcD15D(SEQ ID NO:17)和PjD6D-2 pMON82821,其通过首先用SalI和Sse8387I消化pMON67011,将得到的PjD6D-2片段连接到pMON82800中的napin表达盒的SalI和XhoI(填平的)位点中,产生pMON82819。含有密码子优化NcD15D的napin盒的构建通过用PmeI和BamHI(填平的)消化pMON67024,将得到的片段连接到EcoRV消化的2T二元载体pMON71801中,得到pMON82801。最后,用NotI消化pMON82819,填平,将得到的PjD6D-2 napin表达盒连接到pMON82801的填平的NotI位点中,得到pMON82821。
使用由Radke et al.,(Plant Cell Reports 11:499-505,1992)描述的修正方案将pMON82822转化到canola(欧洲油菜)中。简要的说,将栽培品种′Ebony′(Monsanto Canada,Inc.,Winnipeg,Canada)的canola种子消毒,按Radke et al.,1992所述进行体外发芽。用烟草加料板进行预先共培养,用含有期望载体的根癌土壤杆菌株ABI(Koncz and Schell,Mol Gen Genet 204:383-396(1986))进行外植体制备和外植体接种,按所述用含有75mg/l壮观霉素、25mg/l氯霉素和50mg/l卡那霉素的LB培养基(固体或液体)中维持的土壤杆菌进行。对于植物转化来说,包括愈伤组织诱导、茎干再生、成熟和生根、使用草甘膦选择而不是如Radke et al.,1992所述使用卡那霉素。特别地,B5-1愈伤组织诱导培养基补加500mg/l羧苄青霉素和50mg/l的Timentin(DuchefaBiochemie BV)以抑制土壤杆菌生长,并将卡那霉素从培养基中省去。B5BZ茎干再生培养基另外含有500mg/l羧苄青霉素、50mg/l的Timentin以及45mg/l的草甘膦,每两周将外植体转移到新鲜培养基中。
将草甘膦选择的茎干转移到无激素的B5-0茎干成熟培养基中两周,培养基中含有300mg/l羧苄青霉素和45mg/l草甘膦,最后将茎干转移到含有45mg/l草甘膦的B5生根诱导培养基中。将生根的绿色小植株移植到盆栽土中,使其适应温室条件。将植物保持在标准条件下的温室中。按上述对大豆转化体所进行的那样,通过甲酯衍生的脂质的GC分析来确定成熟种子的脂肪酸组成。用pMON82822转化的植物的canola种子的GC分析产生了199个事件,SDA水平在0.12到4.49%的范围(重量%,100种子集合)。
实施例5
PjD6D表达载体的构建以及对大豆、玉米和canola的转化
PjD6D序列的单独表达在种子特异性启动子表达下对canola、玉米和大豆进行了在植物中的评估。大豆表达载体的构建通过用NotI消化pMON77243,将得到的含有PjD6D-2的片段连接到二元载体pMON17227的NotI位点中。canola表达载体的构建通过用SalI和Sse8387I消化pMON83950(成为平端),将得到的含有PjD6D-1编码区的片段连接到种子特异性napin表达盒载体pMON82800的SalI和XhoI(填平的)位点中。然后将得到的质粒用NotI消化,接着将得到的napin-PjD6D-1表达盒连接到二元载体pMON17227的NotI位点中。玉米表达载体的构建通过用SalI(填平的)和Sse83872I(成为平端)消化pMON83950,将得到的PjD6D-1片段连接到pMON71084中球蛋白表达盒的SfiI(成为平端)位点中。然后将得到的载体用PmeI和HindIII消化,然后将表达盒连接到二元载体pMON30167的HpaI和HindIII位点中。
玉米中P.juliaeΔ6-去饱和酶的活性与进行玉米密码子优化的粗糙链孢霉Δ15-去饱和酶(NcD15Dnno)(SEQ ID NO:20)进行组合评估。用SalI和Sse8387I消化载体pMON67011(成为平端),将得到的PjD6D-2片段连接到pMON71084中球蛋白表达盒的SfiI(填平的)位点中以得到pMON82823。接下来用PmeI和HindIII消化pMON82806(PCTUS03/16144),将得到的球蛋白NcD15Dnno盒连接到1T二元载体pMON30167的NotI(填平的)和HindIII位点中以得到pMON82824。最后通过用PmeI和HindIII消化pMON82823而将球蛋白PjD6D-2盒与球蛋白NcD15Dnno组合,将得到的片段连接到pMON82824的SmaI和HindIII位点中以得到pMON82825。通过本领域技术人员已知的根癌土壤杆菌介导的转化,如美国专利No.6,603,061的描述,将得到的载体引入玉米中。
实施例6
Primula waltonii和Primula alpicola Δ6去饱和酶序列的克隆
Primula waltonii Δ6去饱和酶(PwD6D)和P.alpicola Δ6去饱和酶(PaD6D)基因的克隆通过使用简并寡核苷酸对部分内部基因组DNA区域进行PCR扩增、然后通过双向基因组步移而实现。按照厂商程序,使用DNeasy Plant Mini试剂盒(Qiagen)从P.waltonii和P.alpicola(Collector’s Nursery)分离总基因组DNA。按照Garcia-Maroto et al.2002所述,使用简并寡核苷酸BO-1 For和BO-2 Rev从P.alpicola基因组DNA分离两个片段:
BO-1 For:5’-ATMAGYATYGGTTGGTGGAARTGG-3’
(SEQ ID NO:6)
BO-2 Rev:5’-AATCCACCRTGRAACCARTCCAT-3’
(SEQ ID NO:7)
第一个P.alpicola片段长550bp,相应于SEQ ID NO:21的553到1103位。将该片段克隆到pCR4-TOPO(Invitrogen)中以产生载体pMON83977(无内含子)。第二个P.alpicola片段长550bp,相应于SEQ ID NO:23的763到1313位。将该片段克隆到pCR4-TOPO(Invitrogen)中以产生载体pMON83975(含有内含子)。从P.waltonii获得一个长550bp的片段,相应于SEQ ID NO:25的763到1313位。将该片段克隆到pCR4-TOPO(Invitrogen)中以产生载体pMON83976。由SEQ ID NOs:21、23和25编码的多肽序列分别示于SEQ ID NOs:22、24和26。
为确定pMON83975、pMON83976和pMON83977插入片段的基因组侧翼序列,利用Universal Genome Walker KitTM(BD Biosciences)根据厂商程序进行。通过用四种限制酶:EcoRV、PvuII、StuI和DraI消化DNA,从每种报春物种产生四种基因组文库。在纯化步骤后,将消化产物连接到试剂盒中提供的接头上。然后程序涉及两次PCR反应,每次都用基因特异性引物和接头引物。第二次PCR反应使用第一次PCR反应产物的稀释物作为模板。
A.pMON83975(PaD6D-2)
对于5’方向,分别使用引物PD6D R7和PD6D R1用于第一次和第二次PCR反应。对于3’方向,分别使用引物PD6D F7和PD6D F1用于第一次和第二次PCR反应。引物序列如下:
PD6D R7:5’-CACACATGACCGGATAAAACGTCCAGT-3’
(SEQ ID NO:27)
PD6D R1:5’-AGGGATATACTGGAGGTCGGGGTCGTA-3’
(SEQ ID NO:28)
PD6D F7:5’-GAGCTATTCCGTTACGGGGATACAACA-3’
(SEQ ID NO:29)
PD6D F1:5’-TGCAGGGACACTTAACATATCGTGCCC-3’
(SEQ ID NO:30)
沿5’方向的基因组步移从EcoRV文库产生了751bp的片段。将该产物克隆到pCR4-TOPO(Invitrogen)中,得到pMON83978,对插入片段测序。得到的序列不含推定的Δ6去饱和酶基因的起始密码子,因此使用设计用于沿5’方向步移的基因特异性引物从pMON83978插入片段进行另一组PCR反应。用于沿5’方向的第二组基因组步移的引物分别是用于第一次和第二次PCR反应的PD6D R17和PD6D R16。序列如下:
PD6D R17:5’-GTGAAAGTTGTTGAGGAGGGATCGGTA-3’
(SEQ ID NO:31)
PD6D R16:5’-GTGGAAGGAGGATGGTAAGCGAGGAAA-3’
(SEQ ID NO:32)
将来自PvuII文库的长473bp的产物克隆到pCR4-TOPO中,得到pMON83980,并且对插入片段测序。该插入片段含有相应于SEQID NO:23位置1的起始密码子。沿3’方向的基因组步移从DraI文库产生了942bp的片段。将该产物克隆到pCR4-TOPO中,得到pMON83979。对插入片段测序,发现含有推定的Δ6去饱和酶基因的编码区的294bp,接下来是相应于SEQ ID NO:23位置1549的终止密码子。
将插入片段pMON83975、pMON83978、pMON83980和pMON83979进行序列比对以形成推定的P.alpicola Δ6去饱和酶基因的组合序列,得到PaD6D-2,SEQ ID NO:23。设计两个引物来PCR扩增P.alpicola基因组DNA的全部开放读码框。引物序列如下:
Pa D6D F1:5’-GTCGACATGGCTAACAAATCTCAAACAGGTTAC-3’
(SEQ ID NO:33)
Pa D6D R1:5’-CCTGCAGGTCACCCGAGAGTTTTAACAGCCTCC-3”
(SEQ ID NO:34)
然后将PCR扩增片段(SEQ ID NO:23)连接到酵母表达载体pYES2.1-TOPO中,得到载体pMON83968。
B.pMON83976(PwD6D)
使用上面显示的引物Pa D6D F1(SEQ ID NO:33)和Pa D6D R1(SEQ ID NO:34)将推定的Δ6去饱和酶从P.waltonii基因组DNA进行PCR扩增。然后将PCR扩增的片段(SEQ ID NO:25)连接到酵母表达载体pYES2.1-TOPO中,得到载体pMON83967。
C.pMON83977(PaD6D-1)
对于5’方向,分别使用引物PD6D R9和PD6D R4用于第一次和第二次PCR反应。对于3’方向,分别使用引物PD6D F9和PD6D F4用于第一次和第二次PCR反应。引物序列如下:
PD6D R9:5’-CACACATTACCGGATAAAACGTCCAGT-3’
(SEQ ID NO:35)
PD6D R4:5’-AGGAATATACTGGAGGTCTGGGTCGTA-3’
(SEQ ID NO:36)
PD6D F9:5,-ATTTTTCTTCGGACGTATACATGGGCC-3’
(SEQ ID NO:37)
PD6D F4:5’-TTCGGGGACACTGAACATATCGTGCCC-3’
(SEQ ID NO:38)
沿5’方向的基因组步移从StuI文库产生了979bp的片段。将该产物克隆到pCR4-TOPO(Invitrogen)中,得到pMON83981,对插入片段测序。得到的序列含有推定的Δ6去饱和酶相应于SEQ ID NO:21位置1的起始密码子。沿3’方向的基因组步移从DraI文库产生了1028bp的片段。将该产物克隆到pCR4-TOPO中,得到pMON83982。对插入片段测序,发现含有推定的Δ6去饱和酶基因的编码区的295bp,接下来是相应于SEQ ID NO:21位置1339的终止密码子。
将插入片段pMON83977、pMON83981和pMON83982进行序列比对以形成第二种推定的P.alpicola Δ6去饱和酶基因的组合序列,得到PaD6D-1,SEQ ID NO:21。设计两个引物来PCR扩增P.alpicola基因组DNA的全部开放读码框。引物序列如下:
PfD6D-F2:5’-GTCGACATGGCCAACACTAGTTACATTTCCAGCT-3’
(SEQ ID NO:39)
PfD6D-R2:5’-GATATCACCCCAGAGTGTTAACAGCTTCCCAG-3’
(SEQ ID NO:40)
然后将PCR扩增片段连接到酵母表达载体pYES2.1-TOPO中,得到载体pMON67026(SEQ ID NO:21)。
PaD6D-2与PwD6D(也缩写为PRIwaD6D)与其它特征性植物Δ6去饱和酶的比对显示,这两个基因含有相应于SEQ ID NO:23中476到676位以及SEQ ID NO:25中476到651位的单一内含子。这在来自报春和蓝蓟物种的Δ6去饱和酶中也观察到了(Sayanova et al.,2003,Garcia-Maroto et al.,2002)。
三个Δ6去饱和酶克隆编码PaD6D-1(SEQ ID NO:22)的446个氨基酸、PaD6D-2(SEQ ID NO:24)的449个氨基酸以及PwD6D(SEQ ID NO:26)的449个氨基酸的可能多肽。这些序列与其它植物Δ6去饱和酶具有高相似性(图1),包括N末端的细胞色素b5结构域,这在所有前端去饱和酶中都被发现(Napier et al.,2003)。在细胞色素b5结构域中发现了表征细胞色素b5超家族的八个不变残基以及H-P-G-G血红素结合基序,其显示出对酶活性是必需的(Napier et al.,1997,Sayanova et al,1999,Sperling and Heinz 2001)。在PaD6D-1、PaD6D-2和PwD6D去饱和酶的去饱和酶结构域中是三个保守的组氨酸盒,这是所有膜结合去饱和酶的特征(Shanklin et al.,1994)。在所有前端去饱和酶中发现的区别特征在于,第三个组氨酸盒在第一位置含有谷氨酰胺(Q-x-x-H-H)而不是组氨酸(Napier et al.,1997,Napier et al.,2003,Sperling andHeinz 2001)。推定的PaD6D-1氨基酸序列具有与PaD6D-2大约80%的同一性以及与PwD6D大约80%的同一性。然而,两个含有内含子的基因PaD6D-2和PwD6D彼此之间,比两个P.alpicola基因PaD6D-1和PaD6D彼此之间更类似,具有大约97%的同一性。
实施例7
另外的报春Δ6去饱和酶序列的克隆
使用Sarkosyl/CTAB裂解系统从有粉报春和P.florindae分离基因组DNA。将来自每个物种的五克组织在研钵和乳钵中用液氮研磨直到磨成细粉。然后将粉状组织重悬浮在裂解缓冲液(140mM山梨糖醇,220mM Tris-HCl、pH8.0,22mM乙二胺四乙酸(EDTA),800mM氯化钠(NaCl),1% N-十二烷基肌氨酸以及0.8%溴化十六烷基三甲铵(CTAB))中,在65℃温育1小时,每10分钟缓慢颠倒一下。温育之后,将10ml氯仿加入裂解悬浮液,室温缓慢摇动温育20分钟。将裂解悬浮液在12,000Xg离心10分钟。将水层转移到干净的试管中,用0.6%异丙醇沉淀核酸。将核酸沉淀重悬浮在4ml含有10mM Tris-HCl、pH8.0、1mM EDTA、1M NaCl以及20mg蛋白酶K的溶液中。然后将重悬浮的核酸在63℃温育2小时。然后经75℃温育20分钟使蛋白酶K热灭活。向溶液中加入RNase(2.5μg),在37℃温育1小时。用等体积的苯酚∶氯仿∶异戊醇(25∶24∶1)萃取溶液2次。然后将纯化的基因组DNA用乙醇沉淀。
大约地,将3μg基因组DNA在独立的反应中用限制性内切酶EcoRI、HindIII、KpnI、SalI和XhoI进行消化。消化之后,按照厂商方案,使用QIAquick PCR纯化试剂盒(Qiagen,Valencia,CA)纯化每个反应产物。使用100μl试剂盒中提供的洗脱缓冲液从纯化柱上洗脱消化的基因组DNA。有助于分子内相互作用的连接在200μl体积中进行,其中在有800单位连接酶(M0202L)(New England Biolabs,Beverly MA)、不含PEG的连接反应中使用20μl洗脱的消化的基因组DNA,在16℃过夜,然后在75℃热灭活10分钟。连接之后,使用QIAquick PCR纯化试剂盒再次纯化反应产物。使用6-20ng纯化的连接DNA和10-20pg引物以及扩展长模板PCR系统(Roche AppliedScience,Indianapolis,IN)进行反向PCR。引物示于表5,是结合使用可得到的序列数据和包括去饱和酶结构域的数据来设计的。热循环条件由94℃初始温育2分钟;10个循环的94℃ 20秒、52℃ 30秒和68℃ 8分钟;接着是25个循环的94℃ 30秒、52℃ 30秒和68℃ 8分钟,每个循环加10秒。循环完成后,68℃进行再温育7分钟组成。按照厂商方案,将琼脂糖电泳后可见的反向PCR文库产物克隆到pCR2.1-TOPO或者pCR4Blunt-TOPO(Invitrogen)中。克隆了下面的反向文库片段(近似大小):有粉报春-EcoRI(6kb)和P florindae-HindIII(3kb)。DNA测序在Applied Biosystems 3730xl DNA分析仪上使用BigDyeTerminator v3.0进行。
表5.在确定Δ6去饱和酶基因5’和3’区域的反向PCR中使用的引物和片段。
物种 | 引物 | 序列 | SEQ ID NO: |
有粉报春P.florindae | Pf1107F1Pf1107R1Pf1113-1F2Pf1113-1R2 | TGGAGGTCTGGGTCGTAATCCTTCGGACGTATACATGGGCTCGTAATCCAGGCTATTGCATTTTCTTCGGACGTCCATGT | 41424344 |
用公开数据对推定序列进行比对以确定每个基因包括的开放读码框(ORF)的大致区域。扩增每个基因ORF的引物是基于比对的反向PCR数据而设计的。使用校对聚合酶扩增推定的Δ6基因以确保最终产物的保真。用于最后克隆推定的Δ6去饱和酶基因的引物示于表6。将产物克隆到pUC19或者pCR4Blunt-TOPO(Invitrogen)中。DNA测序在Applied Biosystems 3730xl DNA分析仪上使用Big DyeTerminator v3.0进行。克隆了两个推定的Δ6去饱和酶基因:有粉报春(PfaD6D)(pMON84809)(SEQ ID NO:45)和P.florindae(PflD6D)(pMON84810)(SEQ ID NO:47)。
表6.用于扩增Δ6去饱和酶基因的引物
引物 | 序列 | SEQ ID NO: |
Pfarinosa754FPfarinosa2447RPflorindaestartFPflorindaestopR | GACGATTTTTGAGTGAGAGTTAATTTGAGTCAATAATACGACATCATAGACAATCATCAAGACACCGTATACCCCCTCAAAACACCCCCAAATCTCAATATCACCCGAGAGTTTTAACAGCCT | 49505152 |
设计了两个引物从pMON84809扩增完整的PfaD6D开放读码框。将得到的片段连接到酵母表达载体pYES2.1-TOPO,得到pMON67065。这两个引物如下:
Pfar F1:5’-GTCGACAACA
ATGTCCAACACATATCCACCAAATC-3’
(SEQ ID NO:53)
Pfar R1:5’-CCTGCAGGTCACCCCAGAGTGTTAACAGCTTC-3’
(SEQ ID NO:54)
设计了两个引物从pMON84810扩增含有两个外显子和一个内含子的完整的PflD6D基因。将得到的片段连接到酵母表达载体pYES2.1-TOPO中,得到pMON67067。这两个引物如下:
Pw F1:5’-GTCGAC
ATGGCTAACAAATCTCAAAC-3’
(SEQ ID NO:55)
Pw R2:5’-CCTGCAGGTCACCCGAGAGT-3’
(SEQ ID NO:56)
两个Δ6去饱和酶克隆编码PfaD6D(SEQ ID NO:46)的454个氨基酸和PflD6D(SEQ ID NO:48)的449个氨基酸的可能多肽。这些序列与其它植物Δ6去饱和酶具有高相似性(图1),包括N末端的细胞色素b5结构域,这在所有前端去饱和酶中都被发现(Napier et al.,2003)。在细胞色素b5结构域中发现了表征细胞色素b5超家族的八个不变残基以及H-P-G-G血红素结合基序,其显示出对酶活性是必需的(Napier et al.,1997,Sayanova et al,1999,Sperling and Heinz 2001)。在推定的PflD6D和PfaD6D去饱和酶的去饱和酶结构域中是三个保守的组氨酸盒,这是所有膜结合去饱和酶的特征(Shanklin et al.,1994)。在所有前端去饱和酶中发现的区别特征是,第三个组氨酸盒在第一位置含有谷氨酰胺残基(Q-x-x-H-H)而不是组氨酸(Napier et al.,1997,Napier et al.,2003,Sperling and Heinz 2001)。
实施例8
内含子去除
三个报春克隆PaD6D-2(SEQ ID NO:22)、PwD6D(SEQ ID NO:25)和PflD6D(SEQ ID NO:47)的比对显示出DNA序列之间的广泛相似性。PaD6D-2具有与PwD6D大约97%的同一性,以及与PflD6D大约98%的同一性。PwD6D具有与PflD6D大约98%的同一性。利用2步PCR程序去除每个基因的内含子区域。简要地说,程序需要在独立的PCR反应中扩增两个外显子,接着通过第二轮PCR扩增将两个外显子组合在一起。因为三种Δ6去饱和酶基因之间的广泛相似性,对每个基因扩增使用同一组引物。
设计两组引物来从pMON83967中的PwD6D插入片段扩增外显子1。扩增产物的大小是475bp,相应于PwD6D的外显子1。两个引物如下。
Pw F1:5’-GTCGAC
ATGGCTAACAAATCTCAAAC-3’
(SEQ ID NO:55)
Pw R1:5’-GTAATGCCCAGAGTCGTGACCTATCCATCCGCACTGGATCC-3’
(SEQ ID NO:57)
使用下面显示的引物序列从pMON83967PCR扩增外显子2。扩增产物的大小是875bp。
Pw F2:5’-GATCCAGTGCGGATGGATAGGTCACGACTCTGGGCATTACCG-3’
(SEQ ID NO:58)
Pw R2:5’-CCTGCAGGTCACCCGAGAGT-3’(SEQ ID NO:56)
然后将扩增的外显子1和外显子2产物与引物Pw F1和Pw R2组合起来以PCR扩增没有起始内含子的ORF。将得到的1350bp的片段连接到酵母表达载体pYES2.1-TOPO中,得到pMON67062。
内含子区域从pMON83968中的PaD6D-2以及pMON84810中的PflD6D去除是利用如上所述对PwD6D同样的程序进行的。外显子的大小与PwD6D的相同。将得到的1350bp的组合外显子片段连接到酵母表达载体pYES2.1-TOPO中,得到对于PaD6D-2的pMON67063以及对于PflD6D的pMON67064。
实施例9
酵母转化和表达
使用酿酒酵母EasyComp转化试剂盒(Invitrogen),将构建体pMON83950(图3)、pMON67011(图2)、pMON67026、pMON67062、pMON67064和pMON67065引入到尿嘧啶营养缺陷型酿酒酵母株INVSc1(Invitrogen)。转化体在由不含尿嘧啶的、有2%葡萄糖的SC基本培养基组成的平板上进行选择。将转化体菌落用于接种5ml的不含尿嘧啶的、有2%葡萄糖的SC基本培养基,在30℃生长过夜。对于诱导来说,将稳定期酵母细胞沉淀并重悬浮在补充了2%半乳糖、不含尿嘧啶的SC基本培养基中,在25℃生长1天,接着在15℃生长3天。当提供外源脂肪酸给培养物时,将0.01%(v/v)LA(Δ9,12-18:2)和0.01% ALA(Δ9,12,15-18:3)与乳化剂0.1%(w/v)Tergitol一起加入。将培养物在25℃生长1天,接着在15℃生长3天,随后经离心收获。用无菌TE缓冲液pH7.5漂洗细胞沉淀一次以去除培养基,冻干24h用含有LacZ基因的载体转化的宿主菌株在所有研究中用作阴性对照。
通过用0.5mL含有0.075mg/mL 2,6-二-叔丁基-4-甲氧基苯酚的甲醇中的5%(v/v)H2SO4在90℃进行转甲基作用90min,从冻干的酵母沉淀制备FAMEs。通过加入0.9mL 10%(w/v)NaCl和0.3mL庚烷提取FAMEs。将含有FAMEs的庚烷层取出,如实施例2所述直接用于GC。
示于表7的结果证明,P.juliae克隆pMON67011和pMON83950、P.alpicola克隆pMON67026和pMON67063、P.waltonii克隆pMON67062、P.florindae克隆pMON67064以及有粉报春克隆pMON67065在酵母表达系统中显示出Δ6去饱和酶活性。表8中的数据证明,每个报春克隆都编码具有n-3或者n-6底物脂肪酸选择性的蛋白质。
表7:报春克隆在酵母表达系统中的Δ6去饱和酶活性
载体 | 基因 | 培养基中的FA | LA* | GLA* | ALA* | SDA* |
LacZ-1LacZ-2LacZ-3LacZ-1LacZ-2LacZ-3 | LacZLacZLacZLacZLacZLacZ | ---LA+ALALA+ALALA+ALA | 0.00.00.023.520.329.1 | 0.00.00.00.00.00.0 | 0.00.00.020.616.628.0 | 0.00.00.00.00.00.0 |
pMON67011pMON67011pMON67011pMON67011pMON67011pMON67011 | P.juliae D6D-2P.juliae D6D-2P.juliae D6D-2P.juliae D6D-2P.juliae D6D-2P.juliae D6D-2 | ---LA+ALALA+ALALA+ALA | 0.00.00.218.714.718.6 | 0.00.00.06.55.45.1 | 0.00.00.012.29.614.6 | 0.00.00.08.47.68.8 |
pMON67026pMON67026pMON67026pMON67026pMON67026pMON67026 | P.alpicola D6D1P.alpicola D6D-1P.alpicola D6D-1P.alpicola D6D-1P.alpicola D6D-1P.alpicola D6D-1 | ---LA+ALALA+ALALA+ALA | 0.00.00.023.019.122.6 | 0.00.00.03.63.73.1 | 0.00.00.021.817.924.1 | 0.00.00.01.51.51.5 |
pMON83950pMON83950pMON83950pMON83950pMON83950pMON83950 | P.juliae D6D-1P.juliae D6D-1P.juliae D6D-1P.juliae D6D-1P.juliae D6D-1P.juliae D6D-1 | ---LA+ALALA+ALALA+ALA | 0.00.00.021.213.921.7 | 0.00.00.04.04.24.3 | 0.00.00.014.98.816.8 | 0.00.00.06.76.08.3 |
pMON67062pMON67062pMON67062pMON67062pMON67062 | P.waltonii D6DP.waltonii D6DP.waltonii D6DP.waltonii D6DP.waltonii D6D | ---LA+ALALA+ALA | 0.00.00.017.512.8 | 0.00.00.05.74.8 | 0.00.00.012.18.6 | 0.00.00.07.16.0 |
载体 | 基因 | 培养基中的FA | LA* | GLA* | ALA* | SDA* |
pMON67062 | P.waltonii D6D | LA+ALA | 20.9 | 5.2 | 16.8 | 8.4 |
pMON67063pMON67063pMON67063pMON67063pMON67063pMON67063 | P.alpicola D6D-2P.alpicola D6D-2P.alpicola D6D-2P.alpicola D6D-2P.alpicola D6D-2P.alpicola D6D2- | ---LA+ALALA+ALALA+ALA | 0.00.00.019.916.019.8 | 0.00.00.03.73.63.6 | 0.00.00.013.49.514.9 | 0.00.00.06.75.67.8 |
pMON67064pMON67064pMON67064pMON67064pMON67064pMON67064 | P.florindae D6DP.florindae D6DP.florindae D6DP.florindae D6DP.florindae D6DP.florindae D6D | ---LA+ALALA+ALALA+ALA | 0.00.00.017.412.817.1 | 0.00.00.05.64.84.5 | 0.00.00.012.08.314.6 | 0.00.00.06.95.98.3 |
pMON67065pMON67065pMON67065pMON67065pMON67065pMON67065 | 有粉报春D6D有粉报春D6D有粉报春D6D有粉报春D6D有粉报春D6D有粉报春D6D | ---LA+ALALA+ALALA+ALA | 0.00.00.022.128.821.1 | 0.00.00.00.90.80.8 | 0.00.00.019.727.522.7 | 0.00.00.00.30.20.3 |
*报导为GC-FID色谱图中包括的所有被分析物总量的%,包括(16:0,16:1,18:0,20:0,20:1,20:2,22:0,22:1,22:2)
表8:报春Δ6去饱和酶的n-3∶n-6底物选择性比较
样品 | 载体 | 基因 | %转化GLA* | %转化SDA* | n-3∶n-6比 |
123 | LacZ-1LacZ-2LacZ-3 | LacZLacZLacZ | 0.000.000.00 | 0.000.000.00 | 0.000.000.00 |
456 | pMON67011pMON67011pMON67011 | P.juliae D6D-2P.juliae D6D-2P.juliae D6D-2 | 25.7526.9821.64 | 40.6444.0737.78 | 1.581.631.75 |
789 | pMON67026PMON67026pMON67026 | P.alpicola D6D-1P.alpicola D6D-1P.alpicola D6D-1 | 13.6016.0612.12 | 6.397.835.83 | 0.470.490.48 |
101112 | pMON83950pMON83950pMON83950 | P.juliae D6D-1P.juliae D6D-1P.juliae D6D-1 | 15.8223.2316.58 | 31.1440.7232.92 | 1.971.751.99 |
131415 | pMON67062pMON67062pMON67062 | P.waltonii D6DP.waltonii D6DP.waltonii D6D | 24.4627.0519.77 | 36.8041.1533.41 | 1.501.521.69 |
161718 | pMON67063pMON67063pMON67063 | P.alpicola D6D-2P.alpicola D6D-2P.alpicola D6D-2 | 15.7418.5315.37 | 33.4836.8234.39 | 2.131.992.24 |
192021 | pMON67064pMON67064pMON67064 | P.florindae D6DP.florindae D6DP.florindae D6D | 24.3427.2920.96 | 36.7241.5636.13 | 1.511.521.72 |
222324 | pMON67065pMON67065pMON67065 | 有粉报春D6D有粉报春D6D有粉报春D6D | 4.072.773.70 | 1.250.791.09 | 0.310.290.29 |
*转化为GLA的百分比是通过用GLA的值(表1)除以LA和GLA值(表1)的和而计算的。对SDA用ALA和SDA(表1)的和进行同样的计算。
**n-3∶n-6比是通过%转化SDA除以%转化GLA而计算的。
实施例10
拟南芥克隆、转化和表达
确认报春Δ6去饱和酶在酵母中的活性之后,然后将基因克隆到pMON73273(含有组成型35S CaMV启动子的二元载体)中,在拟南芥中表达以确定在植物中的活性。PwD6D和PaD6D-2都完整克隆了内含子。将下面的载体转化到拟南芥中:pMON83961(MaD6D)(图8),pMON83962(PjD6D-1)(图9),pMON83963(PaD6D-2)(图10),pMON84964(PjD6D-2)(图11),pMON84965(PaD6D-1)(图12)以及pMON83966(PwD6D)(图13)。
通过将种子播种在含有反渗透水(ROW)饱和的MetroMix 200(The Scotts Company,Columbus,OH)的4英寸罐中而生长拟南芥植物。植物春化通过将罐放入有盖的平台中,在4-7℃的生长室中、每天光照8小时,进行4-7天。将平台转移到生长室中,22℃、55%相对湿度、以160-200μEinstein/s/m2的平均强度每天光照16小时。在发芽后将盖子抬起,滑回1英寸,然后当真叶形成时将盖子移去。当需要时,用ROW在底部给植物浇水,直到发芽后2-3周。如果需要,然后用Plantex 15-15-18溶液(Plantex Corporation Ottawa,Canada)在50ppmN2下在底部给植物浇水。对罐进行间苗,使得在发芽后2-3周时,每个罐中剩余1株植物。一旦植物开始开花,就对最初的花序进行修剪以助长腋生部位的生长。
转基因拟南芥植物的获得按照由Bent et al.,Science,265:1856-1860,1994或者Bechtold et al.,C.R.Acad.Sci,Life Sciences,316:1194-1199,1993所述进行。将含有转化载体pMON69804、pMON69812或者pMON69815之一的根癌土壤杆菌株ABI的培养物在LB(10%细菌用胰蛋白胨、5%酵母提取物以及10% NaCl,有卡那霉素(75mg/L)、氯霉素(25mg/L)以及壮现霉素(100mg/L))中生长过夜。离心细菌培养物,重悬浮在5%葡萄糖+.05% Silwet-77溶液中。将整个拟南芥Columbia植物(大约5-7周龄)的地上部分浸入到得到的溶液中2-3秒。通过将植物在纸巾上吸干而除去过量溶液。将浸泡过的植物以它们的侧面放在有盖的平台上,转移到19℃的生长室。16到24小时后将盖子移去,将植物向上放置。当植物达到成熟时,在种子收获前停止给水2-7天。将收获的种子通过不锈钢网筛(40孔/英寸)以除去碎片。
将上述收获的种子播种在含有ROW饱和的MetroMix 200(TheScotts Company)平台上。植物如上所述进行春化和发芽。在真叶出现后,用草甘膦喷洒幼苗以选择转化的植物。
成熟种子(R2)的脂肪酸组成通过如上面对大豆种子进行的甲基酯衍生脂质GC分析确定。来自每个转基因事件的混合种子的值示于表9。在酵母分析中观察到的n-3或n-6底物选择性在植物中确认。
表9.拟南芥种子的脂肪酸分析
基因 | 谱系 | 构建体 | PA | SA | OA | LA | GLA | ALA | SDA |
MaD6D | At_S54435:@ | pMON83961 | 7.47 | 3.73 | 14.18 | 26.31 | 2.3 | 17.3 | 0.72 |
MaD6D | At_S54436:@ | pMON83961 | 7.44 | 3.91 | 14.72 | 25.51 | 1.72 | 18.57 | 0.44 |
MaD6D | At_S54437:@ | pMON83961 | 7.65 | 3.72 | 14.51 | 28.49 | 0.37 | 17.97 | 0 |
MaD6D | At_S54438:@ | pMON83961 | 7.65 | 3.53 | 13.55 | 25.48 | 2.09 | 19.18 | 0.87 |
MaD6D | At_S54439:@ | pMON83961 | 7.7 | 3.51 | 13.69 | 27.81 | 1.63 | 17.07 | 0.45 |
MaD6D | At_S54440:@ | pMON83961 | 7.38 | 3.55 | 14.42 | 25.95 | 1.6 | 18.26 | 0.53 |
MaD6D | At_S54441:@ | pMON83961 | 7.24 | 3.54 | 13.53 | 24.24 | 4.4 | 17.68 | 1.52 |
MaD6D | At_S54442:@ | pMON83961 | 7.29 | 3.6 | 14.7 | 25.31 | 3.58 | 16.45 | 0.98 |
MaD6D | At_S54443:@ | pMON83961 | 7.01 | 3.61 | 14.46 | 27.25 | 0.44 | 18.49 | 0 |
MaD6D | At_S54444:@ | pMON83961 | 7.68 | 3.75 | 14.34 | 27.89 | 1.19 | 17.95 | 0.05 |
PjD6D-1 | At_S54446:@ | pMON83962 | 7.5 | 3.34 | 13.52 | 25.05 | 2.06 | 13.81 | 5.93 |
PjD6D-1 | At_S54447:@ | pMON83962 | 7.29 | 3.15 | 14.03 | 26.18 | 1.64 | 14 | 5.25 |
PjD6D-1 | At_S54448:@ | pMON83962 | 7.2 | 3.08 | 13.37 | 27.24 | 0.49 | 17 | 2.72 |
PjD6D-1 | At_S54449:@ | pMON83962 | 7.24 | 3.17 | 14.28 | 27.52 | 0.46 | 16.65 | 2.44 |
PjD6D-1 | At_S54450:@ | pMON83962 | 7.24 | 3.18 | 13.38 | 26.3 | 1.32 | 15.16 | 4.92 |
PjD6D-1 | At_S54451:@ | pMON83962 | 7.53 | 3.04 | 14.49 | 28.01 | 1.8 | 13.03 | 4.79 |
PjD6D-1 | At_S54452:@ | pMON83962 | 7.59 | 3.44 | 13.16 | 25.54 | 1.72 | 13.3 | 6.69 |
PiD6D-1 | At_S54453:@ | pMON83962 | 7.22 | 3.21 | 14.05 | 26.72 | 1.14 | 14.35 | 4.48 |
PjD6D-1 | At_S54454:@ | pMON83962 | 6.98 | 3.23 | 13.48 | 25.12 | 2.27 | 12.62 | 6.55 |
PjD6D-1 | At_S54455:@ | pMON83962 | 7.34 | 3.18 | 14.63 | 27.07 | 0.16 | 18.57 | 1.1 |
PjD6D-1 | At_S54456:@ | pMON83962 | 7.26 | 3.44 | 15.8 | 27.83 | 0.5 | 15.81 | 2.45 |
基因 | 谱系 | 构建体 | PA | SA | OA | LA | GLA | ALA | SDA |
PjD6D-1 | At_S54457:@ | pMON83962 | 7.41 | 3.11 | 14.03 | 27.39 | 1.92 | 12.97 | 4.95 |
PjD6D-1 | At_S54458:@ | pMON83962 | 7.2 | 3.26 | 13.38 | 26.18 | 1.31 | 14.54 | 5.1 |
PjD6D-1 | At_S54459:@ | pMON83962 | 7.23 | 3.16 | 13.25 | 26.38 | 1.32 | 15.07 | 4.46 |
PjD6D-1 | At_S54460:@ | pMON83962 | 7.21 | 3.19 | 13.48 | 26.35 | 1.32 | 14.36 | 5.16 |
PjD6D-1 | At_S54461:@ | pMON83962 | 7.18 | 3.34 | 13.5 | 26.64 | 0.79 | 15.65 | 3.96 |
PJD6D-1 | At_S54462:@ | pMON83962 | 7.11 | 3.15 | 13.88 | 27.28 | 1.12 | 15.02 | 3.84 |
PjD6D-1 | At_S54463:@ | pMON83962 | 7.4 | 3.19 | 13.37 | 26.35 | 0.61 | 17.58 | 2.93 |
PjD6D-1 | At_S54464:@ | pMON83962 | 7.57 | 3.34 | 13.72 | 26.12 | 1.24 | 15.26 | 4.69 |
PaD6D-2 | At_S54466:@ | pMON83963 | 7.25 | 3.18 | 14.44 | 26.54 | 1.46 | 14.44 | 4.45 |
PaD6D-2 | At_S54467:@ | pMON83963 | 7.28 | 3.07 | 14.66 | 27.82 | 0.31 | 17.25 | 1.59 |
PaD6D-2 | At_S54468:@ | pMON83963 | 7.34 | 3.22 | 15.05 | 26.37 | 2.01 | 13.14 | 4.86 |
PaD6D-2 | At_S54469:@ | pMON83963 | 6.91 | 2.94 | 14.35 | 26.77 | 1.32 | 14.33 | 4.38 |
PaD6D-2 | At_S54470:@ | pMON83963 | 7.36 | 3.26 | 13.31 | 27.8 | 1.36 | 13.39 | 4.52 |
PaD6D-2 | At_S54471:@ | pMON83963 | 7.14 | 3.07 | 14.38 | 25.73 | 3.26 | 11.32 | 6.18 |
PaD6D-2 | At_S54472:@ | pMON83963 | 7.67 | 3.28 | 14.01 | 27.82 | 0 | 19.54 | 0.3 |
PaD6D-2 | At_S54473:@ | pMON83963 | 7.48 | 3.27 | 13.95 | 26.26 | 2.12 | 13.24 | 5.57 |
PaD6D-2 | At_S54474:@ | pMON83963 | 7.22 | 3.01 | 14.95 | 27.87 | 1.02 | 14.5 | 3.48 |
PaD6D-2 | At_S54475:@ | pMON83963 | 7.44 | 3.07 | 13.33 | 26.46 | 1.58 | 14.27 | 5.24 |
PaD6D-2 | At_S54476:@ | pMON83963 | 7.35 | 3.17 | 14.22 | 27.48 | 0.8 | 15.51 | 3.25 |
PaD6D-2 | At_S54477:@ | pMON83963 | 8.01 | 2.7 | 15.85 | 30.18 | 0 | 16.8 | 0 |
PaD6D-2 | At_S54478:@ | pMON83963 | 7.45 | 3.05 | 13.47 | 27.48 | 0.13 | 19.53 | 0.84 |
PaD6D-2 | At_S54479:@ | pMON83963 | 7.14 | 2.99 | 15.32 | 27.71 | 0.24 | 17.74 | 0.9 |
PaD6D-2 | At_S54480:@ | pMON83963 | 7.37 | 3.1 | 14.8 | 27.87 | 0.07 | 18.64 | 0.45 |
PaD6D-2 | At_S54481:@ | pMON83963 | 7.39 | 3.2 | 13.49 | 27.32 | 0.1 | 19.9 | 0.6 |
PaD6D-2 | At_S54482:@ | pMON83963 | 7.29 | 3.1 | 13.72 | 27.63 | 0.25 | 17.96 | 1.63 |
PaD6D-2 | At_S54483:@ | pMON83963 | 7.04 | 2.97 | 15.2 | 28.08 | 0 | 18.71 | 0.1 |
PaD6D-2 | At_S54484:@ | pMON83963 | 7.09 | 2.89 | 14.89 | 28.18 | 0.05 | 19.73 | 0 |
PaD6D-2 | At_S54485:@ | pMON83963 | 7.17 | 2.93 | 15.33 | 27.21 | 1.52 | 13.48 | 4.57 |
PjD6D-2 | At_S54487:@ | pMON83964 | 7.18 | 3.06 | 14.91 | 27.66 | 0.79 | 15.58 | 3 |
PjD6D-2 | At_S54488:@ | pMON83964 | 7.36 | 3.09 | 14.13 | 27.75 | 1.34 | 14.21 | 4.15 |
基因 | 谱系 | 构建体 | PA | SA | OA | LA | GLA | ALA | SDA |
PjD6D-2 | At_S54489:@ | pMON83964 | 7.48 | 2.9 | 13.86 | 27.52 | 0.6 | 16.94 | 2.95 |
PjD6D-2 | At_S54490:@ | pMON83964 | 7.39 | 3.08 | 14.12 | 27.93 | 0.63 | 16.23 | 2.88 |
PjD6D-2 | At_S54491:@ | pMON83964 | 7.35 | 3.05 | 15.03 | 28.07 | 0 | 19.04 | 0.16 |
PjD6D-2 | At_S54492:@ | pMON83964 | 7.59 | 3.07 | 14.84 | 27.99 | 0 | 19.18 | 0.33 |
PjD6D-2 | At_S54493:@ | pMON83964 | 7.36 | 2.97 | 13.57 | 28.18 | 0.68 | 16.38 | 2.96 |
PjD6D-2 | At_S54494:@ | pMON83964 | 7.39 | 3.03 | 13.37 | 27.5 | 0.98 | 15.71 | 3.96 |
PjD6D-2 | At_S54495:@ | pMON83964 | 7.46 | 2.98 | 13.59 | 26.97 | 1.02 | 16.11 | 3.83 |
PjD6D-2 | At_S54496:@ | pMON83964 | 7.65 | 3.02 | 14.54 | 27.83 | 0.35 | 17.43 | 1.87 |
PjD6D-2 | At_S54497:@ | pMON83964 | 7.62 | 2.94 | 13.64 | 28.44 | 0.89 | 15.27 | 3.61 |
PjD6D-2 | At_S54498:@ | pMON83964 | 7.55 | 3.06 | 14.06 | 27.53 | 1.01 | 14.89 | 4.37 |
PjD6D-2 | At_S54499:@ | pMON83964 | 7.19 | 3.12 | 14.62 | 26.77 | 1.55 | 13.28 | 5.14 |
PjD6D-2 | At_S54500:@ | pMON83964 | 7.42 | 2.9 | 13.83 | 27.84 | 0.39 | 17.55 | 2.3 |
PjD6D-2 | At_S54501:@ | pMON83964 | 7.51 | 3.09 | 14.23 | 28.21 | 0 | 19.5 | 0.1 |
PjD6D-2 | At_S54502:@ | pMON83964 | 7.41 | 3 | 13.56 | 27.41 | 0.81 | 16.36 | 3.33 |
PjD6D-2 | At_S54503:@ | pMON83964 | 7.33 | 2.95 | 13.46 | 26.74 | 1.09 | 15.92 | 4.28 |
PaD6D-1 | At_S54505:@ | pMON83965 | 7.24 | 2.97 | 14.25 | 27.24 | 0.96 | 19.3 | 0.21 |
PaD6D-1 | At_S54506:@ | pMON83965 | 7.37 | 3.12 | 14.25 | 26.81 | 1.26 | 19.08 | 0.24 |
PaD6D-1 | At_S54507:@ | pMON83965 | 7.48 | 3.03 | 15.61 | 26.86 | 0.52 | 18.75 | 0.09 |
PaD6D-1 | At_S54508:@ | pMON83965 | 7.61 | 3.07 | 13.41 | 25.28 | 2.2 | 19.67 | 0.51 |
PaD6D-1 | At_S54509:@ | pMON83965 | 7.33 | 3.24 | 14.21 | 25.71 | 2.32 | 18.64 | 0.48 |
PaD6D-1 | At_S54510:@ | pMON83965 | 7.66 | 3.09 | 15.86 | 24.84 | 1.1 | 18.88 | 0.23 |
PaD6D-1 | At_S54511:@ | pMON83965 | 7.55 | 3.08 | 15.2 | 25.25 | 0.94 | 19.36 | 0.21 |
PaD6D-1 | At_S54512:@ | pMON83965 | 7.43 | 3.16 | 13.51 | 26 | 1.37 | 19.63 | 0.29 |
PaD6D-1 | At_S54513:@ | pMON83965 | 8.11 | 3.3 | 14.94 | 24.33 | 0.45 | 20.26 | 0.12 |
PaD6D-1 | At_S54514:@ | pMON83965 | 7.35 | 3.14 | 14.18 | 26.35 | 1.36 | 19.27 | 0.36 |
PaD6D-1 | At_S54515:@ | pMON83965 | 7.52 | 2.95 | 12.14 | 26.65 | 0.63 | 22.45 | 0.21 |
PaD6D-1 | At_S54516:@ | pMON83965 | 7.86 | 3.29 | 15.13 | 23.72 | 0.74 | 20.03 | 0.21 |
PaD6D-1 | At_S54517:@ | pMON83965 | 7.2 | 3.49 | 15.25 | 27.77 | 0.26 | 18.14 | 0 |
PaD6D-1 | At_S54518:@ | pMON83965 | 7.17 | 2.81 | 15.7 | 23.13 | 0.06 | 20.4 | 0 |
PaD6D-1 | At_S54519:@ | pMON83965 | 6.9 | 3.07 | 15.34 | 26.65 | 0.14 | 19.19 | 0 |
基因 | 谱系 | 构建体 | PA | SA | OA | LA | GLA | ALA | SDA |
PaD6D-1 | At_S54520:@ | pMON83965 | 8.64 | 3.7 | 15.97 | 20.96 | 0.97 | 18.39 | 0.28 |
PaD6D-1 | At_S54521:@ | pMON83965 | 7.2 | 3.19 | 13.39 | 26.03 | 1.63 | 19.36 | 0.32 |
PaD6D-1 | At_S54522:@ | pMON83965 | 8.77 | 3.69 | 15.83 | 20.94 | 0 | 18.92 | 0 |
PaD6D-1 | At_S54523:@ | pMON83965 | 7.43 | 3.33 | 14.1 | 26.94 | 0.23 | 19.93 | 0 |
PwD6D | At_S54524:@ | pMON83966 | 7.37 | 3.17 | 15.27 | 25.68 | 2.72 | 13.22 | 4.36 |
PwD6D | At_S54525:@ | pMON83966 | 7.15 | 3.38 | 14.38 | 25.61 | 2.86 | 12.9 | 4.82 |
PwD6D | At_S54526:@ | pMON83966 | 6.87 | 3.6 | 14.68 | 27.25 | 0.23 | 17.54 | 1.19 |
PwD6D | At_S54527:@ | pMON83966 | 7.01 | 3.45 | 15.06 | 26.18 | 1.43 | 14.72 | 3.88 |
PwD6D | At_S54528:@ | pMON83966 | 7.21 | 3.04 | 14.6 | 27.87 | 0.11 | 18.52 | 0.65 |
PwD6D | At_S54530:@ | pMON83966 | 7.59 | 3.17 | 15.34 | 21.81 | 0.77 | 17.64 | 2.92 |
PwD6D | At_S54531:@ | pMON83966 | 7.4 | 3.58 | 14.39 | 26.71 | 0.4 | 17.68 | 1.74 |
PwD6D | At_S54532:@ | pMON83966 | 6.28 | 3.44 | 14.76 | 24.09 | 2.51 | 12.87 | 6.1 |
PwD6D | At_S54533:@ | pMON83966 | 7.01 | 3.48 | 14.15 | 25.54 | 2.01 | 12.98 | 5.54 |
PwD6D | At_S54534:@ | pMON83966 | 7.35 | 3.35 | 14.6 | 26.37 | 2.25 | 13.61 | 4.32 |
PwD6D | At_S54535:@ | pMON83966 | 7.24 | 3.56 | 14.59 | 27.04 | 0.45 | 17.02 | 2.17 |
PwD6D | At_S54536:@ | pMON83966 | 7.22 | 3.54 | 13.14 | 25.92 | 1.49 | 15.35 | 4.53 |
PwD6D | At_S54537:@ | pMON83966 | 7.18 | 3.6 | 13.51 | 26.27 | 1.61 | 15.03 | 4.02 |
PwD6D | At_S54538:@ | pMON83966 | 7.75 | 3.29 | 13.57 | 25.43 | 2.33 | 13.96 | 5.35 |
PwD6D | At_S54539:@ | pMON83966 | 7.15 | 3.13 | 14.86 | 26.63 | 0.16 | 18.99 | 0.35 |
PwD6D | At_S54540:@ | pMON83966 | 7.66 | 3.28 | 14.22 | 26.2 | 0.97 | 16.45 | 3.24 |
PwD6D | At_S54541:@ | pMON83966 | 7.39 | 2.98 | 13.83 | 27.29 | 0 | 20.28 | 0 |
PwD6D | At_S54542:@ | pMON83966 | 7.39 | 3.32 | 14.71 | 26.08 | 1.56 | 14 | 4.18 |
control | At_S54543:@ | pMON26140 | 6.82 | 3.04 | 14.82 | 25.91 | 0 | 20.07 | 0 |
control | At_S54544:@ | pMON26140 | 7.49 | 3.23 | 13.69 | 27.33 | 0 | 19.77 | 0 |
control | At_S54545:@ | pMON26140 | 7.32 | 3.23 | 15.05 | 27.47 | 0 | 18.6 | 0 |
control | At_S54546:@ | pMON26140 | 7.52 | 3.3 | 13.73 | 27.15 | 0 | 19.86 | 0 |
control | At_S54547:@ | pMON26140 | 7.44 | 3.21 | 14.21 | 27.43 | 0 | 19.36 | 0 |
control | At_S54548:@ | pMON26140 | 7.39 | 3.25 | 14.1 | 27.05 | 0 | 19.59 | 0 |
control | At_S54549:@ | pMON26140 | 7.71 | 3.28 | 13.61 | 27.98 | 0 | 20 | 0 |
control | At_S54550:@ | pMON26140 | 7.62 | 3.24 | 13.58 | 28.28 | 0 | 18.83 | 0 |
基因 | 谱系 | 构建体 | PA | SA | OA | LA | GLA | ALA | SDA |
control | At_S54551:@ | pMON26140 | 7.52 | 3.18 | 14.73 | 27.27 | 0 | 19.78 | 0 |
control | At_S54552:@ | pMON26140 | 7.44 | 3.21 | 14.95 | 27.69 | 0 | 18.43 | 0 |
control | At_S54553:@ | pMON26140 | 7.72 | 3.26 | 13.74 | 27.2 | 0 | 19.94 | 0 |
control | At_S54554:@ | pMON26140 | 7.3 | 3.11 | 15.09 | 27.73 | 0 | 18.75 | 0 |
control | At_S54555:@ | pMON26140 | 7.44 | 2.99 | 14.51 | 29.21 | 0 | 18.34 | 0 |
control | At_S54556:@ | pMON26140 | 7.52 | 3.19 | 15.22 | 27.24 | 0 | 18.92 | 0 |
control | At_S54557:@ | pMON26140 | 7.49 | 3.07 | 14.6 | 28.87 | 0 | 18.17 | 0 |
control | At_S54558:@ | pMON26140 | 7.45 | 3.11 | 14.72 | 27.88 | 0 | 18.88 | 0 |
control | At_S54559:@ | pMON26140 | 7.63 | 3.26 | 14.39 | 27.12 | 0 | 19.57 | 0 |
control | At_S54560:@ | pMON26140 | 7.74 | 3.15 | 13.17 | 28.5 | 0 | 19.61 | 0 |
control | At_S54561:@ | pMON26140 | 7.39 | 3.15 | 14.34 | 27.06 | 0 | 19.42 | 0 |
control | At_S54562:@ | pMON26140 | 7.25 | 3.12 | 15.78 | 27.96 | 0 | 17.93 | 0 |
control | At_S54563:@ | pMON26140 | 7.59 | 3.24 | 14.32 | 27.2 | 0 | 19.54 | 0 |
control | At_S54564:@ | pMON26140 | 6.73 | 2.82 | 16.17 | 26.66 | 0 | 18.63 | 0 |
control | At_S54565:@ | pMON26140 | 7.2 | 3 | 15.14 | 27.78 | 0 | 18.66 | 0 |
control | At_S54566:@ | pMON26140 | 7.33 | 3.16 | 14.6 | 27.28 | 0 | 19.26 | 0 |
实施例10
canola转化和表达
根据实施例4的方法,将实施例9中描述的载体pMON83961、pMON83962、pMON83963和pMON83964也转化到canola中。引入pMON70500作为阴性对照。叶的脂肪酸组成通过甲基酯衍生脂质的GC分析确定。数据示于表10。再一次确认了在酵母和拟南芥中观察到的底物选择性。
表10canola叶组织的脂肪酸分析
采样 | 构建体 | PA | SA | OA | LA | GLA | ALA | SDA |
BN_G8912 | pMON70500 | 11.64 | 0.63 | 0.39 | 10.68 | 0 | 53.2 | 0 |
BN_G8913 | pMON70500 | 12.31 | 0.79 | 0.57 | 11.93 | 0 | 53.87 | 0 |
BN_G8914 | pMON70500 | 16.59 | 1.72 | 2.09 | 20.81 | 0 | 47.72 | 0 |
BN_G8915 | pMON70500 | 11.74 | 0.82 | 0.27 | 7.86 | 0 | 58.66 | 0 |
BN_G8918 | pMON70500 | 10.14 | 0.59 | 0.35 | 11.18 | 0 | 52.94 | 0 |
BN_G8919 | pMON70500 | 10.47 | 0.75 | 0.43 | 13.63 | 0 | 50.63 | 0 |
BN_G8925 | pMON70500 | 11.3 | 0.72 | 0.51 | 13.69 | 0 | 50.95 | 0 |
BN_G8926 | pMON70500 | 11.61 | 0.84 | 0.77 | 15.8 | 0 | 49.08 | 0 |
BN_G8928 | pMON70500 | 10.93 | 0.69 | 0.63 | 16.22 | 0 | 49.41 | 0 |
BN_G8929 | pMON70500 | 15.53 | 2.06 | 2.18 | 13.04 | 0 | 47.53 | 0 |
BN_G9007 | pMON83961 | 14.54 | 1.83 | 2.23 | 11.27 | 3.3 | 46.08 | 1.5 |
BN_G9008 | pMON83961 | 16.91 | 2.38 | 1.41 | 10.26 | 3.81 | 46.21 | 2.01 |
BN_G9009 | pMON83961 | 17.11 | 1.86 | 3.04 | 16.21 | 0.48 | 47.15 | 0.23 |
BN_G9011 | pMON83961 | 18.45 | 2.27 | 3.2 | 19.45 | 7.25 | 37.69 | 1.95 |
BN_G9013 | pMON83961 | 17.95 | 2.39 | 2.66 | 20.5 | 1.29 | 44.84 | 0.37 |
BN_G9014 | pMON83961 | 16.65 | 1.94 | 1.83 | 12 | 4.73 | 42.26 | 2.79 |
BN_G9033 | pMON83962 | 16.89 | 2.23 | 1.16 | 16.35 | 0 | 50.45 | 2.52 |
BN_G9034 | pMON83962 | 15.83 | 2.16 | 1.64 | 15.89 | 0 | 50.66 | 1.11 |
BN_G9035 | pMON83962 | 16.36 | 3.18 | 2.74 | 23 | 0 | 40.73 | 3.14 |
BN_G9036 | pMON83962 | 17.01 | 2.65 | 2.4 | 21.09 | 0.37 | 41.23 | 5.12 |
BN_G9037 | pMON83962 | 16.08 | 2.64 | 1.82 | 17.68 | 0.17 | 44.39 | 3.29 |
BN_G8828 | pMON83963 | 13.18 | 1.32 | 2.58 | 14 | 0.15 | 47.07 | 4.1 |
BN_G8829 | pMON83963 | 11.56 | 1.34 | 1.42 | 12.07 | 0.66 | 37.31 | 7.55 |
BN_G8830 | pMON83963 | 12.49 | 1.37 | 1.31 | 12.24 | 0.31 | 41.45 | 5.87 |
BN_G9020 | pMON83963 | 16.66 | 2.54 | 4.3 | 23.54 | 1.42 | 41.6 | 0 |
BN_G9021 | pMON83963 | 16.72 | 1.91 | 2.01 | 14.58 | 0 | 47.55 | 1.36 |
BN_G9024 | pMON83964 | 18.32 | 2.63 | 2.14 | 25.17 | 0.63 | 37.29 | 5.34 |
BN_G9025 | pMON83964 | 18.41 | 2.42 | 3.16 | 26.57 | 0 | 39.23 | 0.51 |
BN_G9026 | pMON83964 | 12.23 | 1.53 | 1.8 | 15.08 | 0.14 | 42.48 | 2.99 |
***
这里公开和要求保护的所有组合物和方法都可以根据本公开内容不经过过度实验而被制造和实施。尽管已经根据优选实施方案描述了本发明的组合物和方法,但是对本领域技术人员来说显而易见的是,在不背离本发明的概念、精神和范围时,对这里描述的组合物和方法以及在步骤中或者在方法步骤的顺序中可以进行变化。更明确地,显而易见的是,化学上和生理上相关的某些试剂可用于替换这里描述的试剂,只要能获得同样或相似的结果。对本领域技术人员来说显而易见的所有这些相似替换和修改都被认为在本发明的精神、范围和概念之内,如所附的权利要求所限定的。
参考文献
下列参考文献加入这里作为参考,在此范围内,它们补充、解释、提供了背景,或者教导了这里采用的方法、技术和/或组合物。
美国专利4,826,877
美国专利4,910,141
美国专利5,011,770
美国专利5,158,975
美国专利5,302,523
美国专利5,378,619
美国专利5,384,253
美国专利5,464,765
美国专利5,538,877
美国专利5,538,880
美国专利5,550,318
美国专利5,563,055
美国专利5,591,616
美国专利5,952,544
美国专利6,603,061
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序列表
<110>Ursin,Virginia
Froman,Byron
Gonzales,Jennifer
LaRosa,Thomas J.
Screen,Steven E.
Dong,Fenggao
<120>来自报春的脂肪酸去饱和酶
<130>MONS:044WO
<160>60
<140>未知
<141>2004-08-20
<140>60/496,751
<141>2003-08-21
<170>PatentIn version 3.1
<210>1
<211>1953
<212>DNA
<213>Primula juliae
<400>1
tatatatata tatatatata atcccaaaca aacactgtca cttgcaaaac aaactcaacc 60
cacgttactt atcccttttc cccaaaatgg aaaacacatt ttcaccacca cctactaaca 120
ccaattccaa ccccatgact aagaccattt acataaccag ctcagaactt gaasaacata 180
acaagccagg tgacctatgg atatcaattc acggtcaagt ttacgacgtt tcttcctggg 240
ctgcgcttca cccggggggc atcgctcccc tcctcgccct tgcaggacat gatgtgaccg 300
acgctttcct cgcttaccat ccccctycca cctcccgcct cctccctccc ttctccacca 360
acctacttct agaaaaacat tccgtgtccg agacctcttc cgactatcgc aaacttctag 420
acagctttca taagatgggc atgtttcgtg ccaggggcca cactgcctac gcgacctttg 480
tcattatgat acttatgttg gtttcctctg tgactggggt gctttgcagt gagaatccgt 540
gggtgcattt ggtttgtgga gcggcaatgg ggtttgcctg gatccagtgc ggatggatag 600
gtcatgattc cggacattac cggataatga ctgacaggaa atggaaccgg ttcgctcaga 660
tcctgagctc aaactgcctc caagggatta gtatcgggtg gtggaagtgg aaccacaacg 720
cgcaccacat tgcctgcaat agtctggagt acgaccctga cctccagtac attcccttgt 780
tggttgtgtc cccgaagttc tttaactccc tcacttctcg tttctacgac aagaagctga 840
acttcgacgg tgtgtcgagg tttttggttc aataccagca ctggtcgttt tatccggtca 900
tgtgtgttgc taggctgaac atgcttgcgc agtcgtttat actgcttttt tcgaggaggg 960
aggtggcgaa cagggtgcag gagattcttg gactagcggt tttttggctt tggtttccgc 1020
tcctgctttc ttgccttcct aattggggtg agagaataat gtttttgctc gcgagctact 1080
ccgttacggg gatacaacac gtgcagttca gcttgaacca tttctcatct gacgtttacg 1140
tgggcccacc cgtaggtaac gattggttta agaaacagac tgcagggaca ctcaacatat 1200
cgtgcccggc gtggatggat tggttccacg gtggattgca gtttcaggtc gagcaccact 1260
tgttcccgcg gatgcctagg ggtcagtttc ggaagatttc tccttttgtg agggatttgt 1320
gtaagaaaca caatttgact tacaatattg cgtcttttac taaagcaaat gtgttgacgc 1380
ttgagaccct gagaaacaca gccattgagg ctcgggacct ctctaatccg atcccaaaga 1440
atatggtgtg ggaggctgtt aaaaatgtcg ggtgaaattg actatgtgtt ttgctattgg 1500
agcttcaatt tcgtgattgt cgtttaaggg ggtatacaca atcaccagat aatcaaacgt 1560
tttctgttgt atttcgttct tgttatttac atttgtagag tggctcatgt aactgacttg 1620
tgtcgaatcg ttaagcctaa atacaagtgt aacaatttag tttctgtcca atttgagaaa 1680
tagaaaagtt tggttgagcc ttttttttct tctaatttct tcaacaggct tattgagtgc 1740
cttatttgcc acatacttaa gcgaaatgct ccaagtgcgc tagccgcaga tgtataaatt 1800
gtctttttcg gcttcaagtt ttaactgtat aacgtcattt cggcttatcg taatggttca 1860
aattagctgc ttttgttttg acaattgtcc taagcaggca ctgatcaaca ctatcagttg 1920
ttctttccct ggtaaaaaag aactgttgaa ttt 1953
<210>2
<211>1341
<212>DNA
<213>Primula juliae
<400>2
atgactaaga ccatttacat aaccagctca gaacttgaaa aacataacaa gccaggtgac 60
ctatggatat caattcacgg tcaagtttac gacgtttctt cctgggctgc gcttcacccg 120
gggggcatcg ctcccctcct cgcccttgca ggacatgatg tgaccgacgc tttcctcgct 180
taccatcccc cttccacctc ccgcctcctc cctcccttct ccaccaacct acttctagaa 240
aaacattccg tgtccgagac ctcttccggc tatcgcaaac ttctagacag ctttcataag 300
atgggcatgt ttcgtgccag gggccacact gcctacgcga cctttgtcat tatgatactt 360
atgttggttt cctctgtgac tggggtgctt tgcagtgaga atccgtgggt gcatttggtt 420
tgtggagcgg caatggggtt tgcctggatc cagtgcggat ggataggtca tgattccgga 480
cattaccgga taatgactga caggaaatgg aaccggttcg ctcagatcct gagctcaaac 540
tgcctccaag ggattagcat cgggtggtgg aagtggaacc acaacgcgca ccacattgcc 600
tgcaatagtc tggagtacga ccctgacctc cagtacattc ccttgttggt tgtgtccccg 660
aagttcttta actccctcac ttctcgtttc tacgacaaga agctgaactt cgacggtgtg 720
tcgaggtttt tggttcaata ccagcactgg tcgttttatc cggtcatgtg tgttgctagg 780
ctgaacatgc ttgcgcagtc gtttatactg cttttttcga ggagggaggt ggcgaacagg 840
gtgcaggaga ttcttggact agcggttttt tggctttggt ttccgctcct gctttcttgc 900
cttcctaatt ggggtgagag aataatgttt ttgctcgcga gctactccgt tacggggata 960
caacacgtgc agttcagctt gaaccatttc tcatctgacg tttacgtggg cccacccgta 1020
ggtaacgatt ggtttaagaa acagactgca gggacactca acatatcgtg cccggcgtgg 1080
atggattggt tccatggcgg gttgcagttt caggtcgagc accacttgtt cccgcggatg 1140
cctaggggtc agtttcggaa gatttctcct tttgtgaggg atttgtgtaa gaaacacaat 1200
ttgacttaca atattgcgtc ttttactaaa gcaaatgtgt tgacgcttga gaccctgaga 1260
aacacagcca ttgaggctcg ggacctctct aatccgatcc caaagaatat ggtgtgggag 1320
gctgttaaaa atgtcgggtg a 1341
<210>3
<211>1389
<212>DNA
<213>Primula juliae
<400>3
atggaaaaca cattttcacc accacctact aacaccaatt ccaaccccat gactaagacc 60
atttacataa ccagctcaga acttgaaaaa cataacaagc caggtgacct atggatatca 120
attcacggtc aagtttacga cgtttcttcc tgggctgcgc ttcacccggg gggcatcgct 180
cccctcctcg cccttgcagg acatgatgtg accgacgctt tcctcgctta ccatccccct 240
tccacctccc gcctcctccc tcccttctcc accaacctac ttctagaaaa acattccgtg 300
tccgagacct cttccgacta tcgcaaactt ctagacagct ttcataagat gggcatgttt 360
cgtgccagag gccacactgc ctacgcgacc tttgtcatta tgatacttat gttggtttcc 420
tctgtgactg gggtgctttg cagtgagaat ccgtgggtgc atttggtttg tggagcggca 480
atggggtttg cctggatcca gtgcggatgg ataggtcatg attccggaca ttaccggata 540
atgactgaca ggaaatggaa ccggttcgct cagatcctga gctcaaactg cctccaaggg 600
attagcatcg ggtggtggaa gtggaaccac aacgcgcacc acattgcctg caatagtctg 660
gagtacgacc ctgacctcca gtacattccc ttgttggttg tgtccccgaa gttctttaac 720
tccctcactt ctcgtttcta cgacaagaag ctgaacttcg acggtgtgtc gaggtttttg 780
gttcaatacc agcactggtc gttttatccg gtcatgtgtg ttgctaggct gaacatgctt 840
gcgcagtcgt ttatactgct tttttcgagg agggaggtgg cgaacagggt gcaggagatt 900
cttggactag cggttttttg gctttggttt ccgctcctgc tttcttgcct tcctaattgg 960
ggtgagagaa taatgttttt gctcgcgagc tactccgtta cggggataca acacgtgcag 1020
ttcagcttga accatttctc atctgacgtt tacgtgggcc cacccgtagg taacgattgg 1080
tttaagaaac agactgcagg gacactcaac atatcgtgcc cggcgtggat ggattggttc 1140
catggcgggt tgcagtttca ggtcgagcac cacttgttcc cgcggatgcc taggggtcag 1200
tttcggaaga tttctccttt tgtgagggat ttgtgtaaga aacacaattt gacttacaat 1260
attgcgtctt ttactaaagc aaatgtgttg acgcttgaga ccctgagaaa cacagccatt 1320
gaggctcggg acctctctaa tccgatccca aagaatatgg tgtgggaggc tgttaaaaat 1380
gtcgggtga 1389
<210>4
<211>446
<212>PRT
<213>Primula juliae
<400>4
Met Thr Lys Thr Ile Tyr Ile Thr Ser Ser Glu Leu Glu Lys His Asn
1 5 10 15
Lys Pro Gly Asp Leu Trp Ile Ser Ile His Gly Gln Val Tyr Asp Val
20 25 30
Ser Ser Trp Ala Ala Leu His Pro Gly Gly Ile Ala Pro Leu Leu Ala
35 40 45
Leu Ala Gly His Asp Val Thr Asp Ala Phe Leu Ala Tyr His Pro Pro
50 55 60
Ser Thr Ser Arg Leu Leu Pro Pro Phe Ser Thr Asn Leu Leu Leu Glu
65 70 75 80
Lys His Ser Val Ser Glu Thr Ser Ser Asp Tyr Arg Lys Leu Leu Asp
85 90 95
Ser Phe His Lys Met Gly Met Phe Arg Ala Arg Gly His Thr Ala Tyr
100 105 110
Ala Thr Phe Val Ile Met Ile Leu Met Leu Val Ser Ser Val Thr Gly
115 120 125
Val Leu Cys Ser Glu Asn Pro Trp Val His Leu Val Cys Gly Ala Ala
130 135 140
Met Gly Phe Ala Trp Ile Gln Cys Gly Trp Ile Gly His Asp Ser Gly
145 150 155 160
His Tyr Arg Ile Met Thr Asp Arg Lys Trp Ash Arg Phe Ala Gln Ile
165 170 175
Leu Ser Ser Asn Cys Leu Gln Gly Ile Ser Ile Gly Trp Trp Lys Trp
180 185 190
Asn His Asn Ala His His Ile Ala Cys Asn Ser Leu Glu Tyr Asp Pro
195 200 205
Asp Leu Gln Tyr Ile Pro Leu Leu Val Val Ser Pro Lys Phe Phe Asn
210 215 220
Ser Leu Thr Ser Arg Phe Tyr Asp Lys Lys Leu Asn Phe Asp Gly Val
225 230 235 240
Ser Arg Phe Leu Val Gln Tyr Gln His Trp Ser Phe Tyr Pro Val Met
245 250 255
Cys Val Ala Arg Leu Asn Met Leu Ala Gln Ser Phe Ile Leu Leu Phe
260 265 270
Ser Arg Arg Glu Val Ala Asn Arg Val Gln Glu Ile Leu Gly Leu Ala
275 280 285
Val Phe Trp Leu Trp Phe Pro Leu Leu Leu Ser Cys Leu Pro Asn Trp
290 295 300
Gly Glu Arg Ile Met Phe Leu Leu Ala Ser Tyr Ser Val Thr Gly Ile
305 310 315 320
Gln His Val Gln Phe Ser Leu Asn His Phe Ser Ser Asp Val Tyr Val
325 330 335
Gly Pro Pro Val Gly Asn Asp Trp Phe Lys Lys Gln Thr Ala Gly Thr
340 345 350
Leu Asn Ile Ser Cys Pro Ala Trp Met Asp Trp Phe His Gly Gly Leu
355 360 365
Gln Phe Gln Val Glu His His Leu Phe Pro Arg Met Pro Arg Gly Gln
370 375 380
Phe Arg Lys Ile Ser Pro Phe Val Arg Asp Leu Cys Lys Lys His Asn
385 390 395 400
Leu Thr Tyr Asn Ile Ala Ser Phe Thr Lys Ala Asn Val Leu Thr Leu
405 410 415
Glu Thr Leu Arg Asn Thr Ala Ile Glu Ala Arg Asp Leu Ser Asn Pro
420 425 430
Ile Pro Lys Asn Met Val Trp Glu Ala Val Lys Asn Val Gly
435 440 445
<210>5
<211>462
<212>PRT
<213>Primula juliae
<400>5
Met Glu Asn Thr Phe Ser Pro Pro Pro Thr Asn Thr Asn Ser Asn Pro
1 5 10 15
Met Thr Lys Thr Ile Tyr Ile Thr Ser Ser Glu Leu Glu Lys His Asn
20 25 30
Lys Pro Gly Asp Leu Trp Ile Ser Ile His Gly Gln Val Tyr Asp Val
35 40 45
Ser Ser Trp Ala Ala Leu His Pro Gly Gly Ile Ala Pro Leu Leu Ala
50 55 60
Leu Ala Gly His Asp Val Thr Asp Ala Phe Leu Ala Tyr His Pro Pro
65 70 75 80
Ser Thr Ser Arg Leu Leu Pro Pro Phe Ser Thr Asn Leu Leu Leu Glu
85 90 95
Lys His Ser Val Ser Glu Thr Ser Ser Asp Tyr Arg Lys Leu Leu Asp
100 105 110
Ser Phe His Lys Met Gly Met Phe Arg Ala Arg Gly His Thr Ala Tyr
115 120 125
Ala Thr Phe Val Ile Met Ile Leu Met Leu Val Ser Ser Val Thr Gly
130 135 140
Val Leu Cys Ser Glu Asn Pro Trp Val His Leu Val Cys Gly Ala Ala
145 150 155 160
Met Gly Phe Ala Trp Ile Gln Cys Gly Trp Ile Gly His Asp Ser Gly
165 170 175
His Tyr Arg Ile Met Thr Asp Arg Lys Trp Asn Arg Phe Ala Gln Ile
180 185 190
Leu Ser Ser Asn Cys Leu Gln Gly Ile Ser Ile Gly Trp Trp Lys Trp
195 200 205
Asn His Asn Ala His His Ile Ala Cys Asn Ser Leu Glu Tyr Asp Pro
210 215 220
Asp Leu Gln Tyr Ile Pro Leu Leu Val Val Ser Pro Lys Phe Phe Asn
225 230 235 240
Ser Leu Thr Ser Arg Phe Tyr Asp Lys Lys Leu Asn Phe Asp Gly Val
245 250 255
Ser Arg Phe Leu Val Gln Tyr Gln His Trp Ser Phe Tyr Pro Val Met
260 265 270
Cys Val Ala Arg Leu Asn Met Leu Ala Gln Ser Phe Ile Leu Leu Phe
275 280 285
Ser Arg Arg Glu Val Ala Asn Arg Val Gln Glu Ile Leu Gly Leu Ala
290 295 300
Val Phe Trp Leu Trp Phe Pro Leu Leu Leu Ser Cys Leu Pro Asn Trp
305 310 315 320
Gly Glu Arg Ile Met Phe Leu Leu Ala Ser Tyr Ser Val Thr Gly Ile
325 330 335
Gln His Val Gln Phe Ser Leu Asn His Phe Ser Ser Asp Val Tyr Val
340 345 350
Gly Pro Pro Val Gly Asn Asp Trp Phe Lys Lys Gln Thr Ala Gly Thr
355 360 365
Leu Asn Ile Ser Cys Pro Ala Trp Met Asp Trp Phe His Gly Gly Leu
370 375 380
Gln Phe Gln Val Glu His His Leu Phe Pro Arg Met Pro Arg Gly Gln
385 390 395 400
Phe Arg Lys Ile Ser Pro Phe Val Arg Asp Leu Cys Lys Lys His Asn
405 410 415
Leu Thr Tyr Asn Ile Ala Ser Phe Thr Lys Ala Asn Val Leu Thr Leu
420 425 430
Glu Thr Leu Arg Asn Thr Ala Ile Glu Ala Arg Asp Leu Ser Asn Pro
435 440 445
Ile Pro Lys Asn Met Val Trp Glu Ala Val Lys Asn Val Gly
450 455 460
<210>6
<211>24
<212>DNA
<213>人工的
<220>
<223>引物
<400>6
atmagyatyg gttggtggaa rtgg 24
<210>7
<211>23
<212>DNA
<213>人工的
<220>
<223>引物
<400>7
aatccaccrt graaccartc cat 23
<210>8
<211>27
<212>DNA
<213>人工的
<220>
<223>引物
<400>8
cacacatgac cggataaaac gaccagt 27
<210>9
<211>27
<212>DNA
<213>人工的
<220>
<223>引物
<400>9
gggaatgtac tggaggtcag ggtcgta 27
<210>10
<211>27
<212>DNA
<213>人工的
<220>
<223>引物
<400>10
cgtgcagttc agcttgaacc atttctc 27
<210>11
<211>27
<212>DNA
<213>人工的
<220>
<223>引物
<400>11
tgcagggaca ctcaacatat cgtgccc 27
<210>12
<211>27
<212>DNA
<213>人工的
<220>
<223>引物
<400>12
gtaggttggt ggagaaggga gggagga 27
<210>13
<211>27
<212>DNA
<213>人工的
<220>
<223>引物
<400>13
ggaaggggga tggtaagcga ggaaagc 27
<210>14
<211>33
<212>DNA
<213>人工的
<220>
<223>引物
<400>14
gtcgacatgg aaaacacatt ttcaccacca cct 33
<210>15
<211>33
<212>DNA
<213>人工的
<220>
<223>引物
<400>15
gtcgacatga ctaagaccatt tacataacc agc 33
<210>16
<211>34
<212>DNA
<213>人工的
<220>
<223>引物
<400>16
cctgcaggtc acccgacatt tttaacagcc tccc 34
<210>17
<211>1290
<212>DNA
<213>粗糙链孢霉
<400>17
atggctgtca ctactaggtc acacaaagcc gccgctgcca ccgaacctga agttgtgtct 60
acaggagtgg atgcagtcag cgctgccgca ccaagcagta gtagctcctc atcctcccaa 120
aagtcagctg agcctatcga atatccagac atcaagacaa ttcgtgacgc tataccagac 180
cactgcttta gacctcgcgt ttggatatcc atggcgtact ttattcgcga ttttgcaatg 240
gctttcggcc tcggatactt ggcatggcaa tacatccctt tgattgcaag taccccattg 300
agatacggag cttgggcttt gtacggttac ctccagggac tcgtctgtac tggaatttgg 360
atcttggctc acgaatgcgg tcacggagcc ttttctagac acacctggtt caacaacgtt 420
atgggttgga ttggtcactc tttcctacta gtcccatatt ttagctggaa attttcccat 480
caccgtcatc ataggttcac cggacatatg gaaaaagata tggcgttcgt tccagccacg 540
gaggcggaca gaaatcagag aaaactagct aatctctata tggacaaaga gactgcggag 600
atgttcgagg atgttcctat tgtgcagttg gttaaactaa ttgctcacca actcgccggt 660
tggcagatgt atctcttgtt caacgttagt gccggaaaag gctccaaaca gtgggaaacc 720
ggcaaaggtg gaatgggatg gctccgcgtg agccatttcg aaccaagttc agccgttttc 780
agaaacagcg aagcaattta catagctcta agcgatctcg gacttatgat tatgggatac 840
attctctacc aggcagccca agttgttgga tggcaaatgg ttggtctctt gtattttcaa 900
cagtacttct gggttcacca ttggctcgtt gccatcactt accttcatca cacacacgaa 960
gaagttcacc actttgatgc agattcttgg acatttgtta agggtgccct cgctaccgtg 1020
gacagagact tcggtttcat cggcaagcac ctcttccata acatcattga ccatcatgtt 1080
gttcatcacc tcttcccaag aatccctttc tactacgctg aagaagctac caattcaata 1140
agacctatgc tcggacctct ttaccacaga gatgaccgtt ctttcatggg gcaactctgg 1200
tacaacttca cacactgcaa atgggttgtc cctgatcctc aagtgccagg tgctctaatc 1260
tgggctcaca ccgttcagag tactcagtaa 1290
<210>18
<211>1209
<212>DNA
<213>构巢曲霉
<400>18
atggccgcaa ccgcgaccac tctcgctgaa atagaaaaga agaaggaaga gattacacta 60
cagacaatca agaatgccat accaaagcac tgttttaacc gtagtttgct tatttcaagt 120
gcctacgtcg tcagagacct cctctacgca tcagttttgt tctattttgc acttcatatt 180
gatacgctct tctcatccca gctccttagg atcttggcat ggacagctta cggtttcatg 240
caaggctgcg tgggaacggg tatatggata ttggcacatg aatgcggaca cggagctttt 300
agcccttacc aaacctggaa cgacgttgtt gggtggaccc ttcattctct tctcatggtc 360
ccttacttct cttggaaaat aacccacgca aggcaccaca gatatacgaa caataccgag 420
agggacacag ccttcgttcc ctggaccgag aaggaatacg acaccagacc tcgttacttc 480
cctgcatggt tcgagatgtt tgaagacaca ccagtgtata acttgatttc attgctcgcc 540
catcagatcg ccggctggca aatgtacctc tgcttctacg tctcagccgg agccaaaagt 600
aagcctgttc cacaaggcaa gcagtccgga tggtttggag gtcaacaatc tgcatcacac 660
tttgacccag gaagctctct atggaccgaa aaccagcgcc atctaatcgc aatctccgac 720
cttggactcc ttctcgtggc cgccgcgaat tggtacttgg ctcaacaagt tggtgttcta 780
agaatggtgc tcatttacgt cgtcccctac ttttgggtcc accactggct agtcgccatc 840
acgtacctcc accacactca cccatccata ccacactaca ccgactctac ctggacattc 900
actaaaggag cactctcaac agtggatcgt gacttcggat ttataggaag gcacttcttt 960
caccacatca ttgatcacca cgtcgttcat cacttgttca ataggatacc attctatcac 1020
gcagaggaag ctactaacgc aataatacca gttctcggtg atatgtacca tagagaagaa 1080
accggattcc tctggagtct tatggaaact tataaaaact gtcgctttgt tggcgtggag 1140
aacgatgtgg gtaaggaggg agttctccat tgggttttcg aagaaaagaa aggcgctaaa 1200
gctgaatag 1209
<210>19
<211>1290
<212>DNA
<213>粗糙链孢霉
<400>19
atgacggtca ccacccgcag ccacaaggcc gcggccgcca ccgagcccga ggttgtcagc 60
accggcgttg acgccgtctc tgctgctgct ccctcctcct cctcctcctc ttccagccaa 120
aagtcggccg agcccatcga ataccccgac atcaagacca tccgcgacgc catccccgac 180
cactgcttcc gcccgcgcgt ctggatctcc atggcctact tcatccgcga cttcgccatg 240
gcctttggcc tcggctacct cgcctggcag tacatccccc tgatcgcctc caccccgctc 300
cgctacggcg cctgggctct gtacggctac ctccagggtc tcgtctgcac gggcatctgg 360
attctggcgc acgagtgcgg ccacggcgcc ttctcgaggc acacgtggtt caacaacgtc 420
atggggtgga ttggccactc cttcctcttg gtcccttact tcagctggaa gttcagccac 480
catcgccacc atcgcttcac cggccacatg gagaaggaca tggcgtttgt gcctgccacc 540
gaggctgatc gcaaccagag gaagctggcc aacttgtaca tggacaagga gacggccgag 600
atgtttgagg atgtgcccat tgtccagctc gtcaagctca tcgcccacca gctggccggc 660
tggcagatgt acctcctctt caacgtctcc gccggtaagg gcagcaagca gtgggagact 720
ggcaagggcg gcatgggctg gttgagggtt agccactttg agccttcctc tgctgtgttc 780
cgcaactccg aggccatcta cattgccctg tccgatcttg gtctcatgat catgggctat 840
atcctctacc aggccgcgca ggttgttggc tggcagatgg taggtctgct gtacttccag 900
cagtacttct gggttcacca ttggttggtc gccatcactt acctccacca cacccacgag 960
gaagtccacc actttgacgc cgactcgtgg accttcgtca agggcgctct cgccaccgtc 1020
gaccgcgatt ttggcttcat tggcaagcac ctcttccaca acattatcga ccaccacgtc 1080
gtccaccact tgttccctcg catccccttc tactacgccg aagaagccac caactcgatc 1140
cgccccatgc tcggccccct ctaccaccgc gacgaccgct ccttcatggg ccagctgtgg 1200
tacaacttca cccactgcaa gtgggtcgtt ccggaccccc aggtccccgg cgcgcttatt 1260
tgggcgcaca ccgttcagag cacccagtaa 1290
<210>20
<211>1290
<212>DNA
<213>粗糙链孢霉
<400>20
atggcggtca ccacccgcag ccacaaggcc gcggccgcca ccgagcccga ggttgtcagc 60
accggcgttg acgccgtctc tgctgctgct ccctcctcct cctcctcctc ttccagccaa 120
aagtcggccg agcccatcga ataccccgac atcaagacca tccgcgacgc catccccgac 180
cactgcttcc gcccgcgcgt ctggatctcc atggcctact tcatccgcga cttcgccatg 240
gcctttggcc tcggctacct cgcctggcag tacatccccc tgatcgcctc caccccgctc 300
cgctacggcg cctgggctct gtacggctac ctccagggtc tcgtctgcac gggcatctgg 360
attctggcgc acgagtgcgg ccacggcgcc ttctcgaggc acacgtggtt caacaacgtc 420
atggggtgga ttggccactc cttcctcttg gtcccttact tcagctggaa gttcagccac 480
catcgccacc atcgcttcac cggccacatg gagaaggaca tggcgtttgt gcctgccacc 540
gaggctgatc gcaaccagag gaagctggcc aacttgtaca tggacaagga gacggccgag 600
atgtttgagg atgtgcccat tgtccagctc gtcaagctca tcgcccacca gctggccggc 660
tggcagatgt acctcctctt caacgtctcc gccggtaagg gcagcaagca gtgggagact 720
ggcaagggcg gcatgggctg gttgagggtt agccactttg agccttcctc tgctgtgttc 780
cgcaactccg aggccatcta cattgccctg tccgatcttg gtctcatgat catgggctac 840
atcctctacc aggccgcgca ggttgttggc tggcagatgg tgggtctgct gtacttccag 900
cagtacttct gggttcacca ttggttggtc gccatcactt acctccacca cacccacgag 960
gaagtccacc actttgacgc cgactcgtgg accttcgtca agggcgctct cgccaccgtc 1020
gaccgcgatt ttggcttcat tggcaagcac ctcttccaca acattatcga ccaccacgtc 1080
gtccaccact tgttccctcg catccccttc tactacgccg aagaagccac caactcgatc 1140
cgccccatgc tcggccccct ctaccaccgc gacgaccgct ccttcatggg ccagctgtgg 1200
tacaacttca cccactgcaa gtgggtcgtt ccggaccccc aggtccccgg cgcgcttatt 1260
tgggcgcaca ccgttcagag cacccagtaa 1290
<210>21
<211>1341
<212>DNA
<213>Primula alpicola
<400>21
atggccaaca ctagttacat ttccagctca gacctcaaaa ctcataataa ggctgacgac 60
ctttggatat ccattcacgg ccaagtgtac gatgtctccg cctgggccac ccaccacccc 120
ggaggtgcct ctctcctcct cgcccttgca ggcaatgatg tcactgatgc cttcctcgcc 180
taccaccctc cctccacctg ccacctcctc cctcctcttt ctaccaacat cctcctcgaa 240
aactactccg tctcccacat ctcctccaat taccgcaacc tcctcaatca tttccacaag 300
ctcggcctat tccgtaccag ggcccacacc gctttcacta cattcttcat catgatactt 360
atgttcttta ttagtgtaac cggaatattt tgcagtgata gtttatgggt ccatttggcg 420
tgcggtggct tgatggggtt tgcatggatt caatgcggat ggatagcgca cgactctggg 480
cattaccgga taacatcaag taggaaatgg aatagattcg ctcagatcct taccggaaat 540
tgcctccagg ggttgagtat tgggtggtgg aagtggaacc ataacgccca ccacatcgct 600
tgcaatagcc tagactacga tccggacctc cagtatattc ctttattggt cgtgtccccg 660
aagtttttca actccatcac ttctcgtttt tatgataaga agctgaactt cgatggtgtg 720
tcgaggtttt tagtcagcta ccaacactgg acgttttatc cggtcatgtg tgttgctagg 780
tttaacatga ttgcacagtc ggttatacat ctcttctcga atagaaacgt gactgatagg 840
gtcctagaga ttttcggact aggggtgttc tgggtttggt attcgctcct actttcgtgc 900
cttcctgatt ggggtgagcg aataatgttt gtgattgcgt gctatttcgt tactgggata 960
caacacgtac agttcagtgt aaaccatttt tcttcggacg tatacatggg ccctccagta 1020
ggtaacgatt ggtttaaaaa acagacttcg gggacactga acatatcgtg cccaccctgg 1080
atggattggt tccacggtgg gttgcagttt caagtggagc accacttgtt cccgcggatg 1140
ccgaggggtc aattcaggaa gatctctcct tttgtaaagg atctgtgtaa taaacacaat 1200
ctgccttaca atatcgcgtc ttttaccatg gcaaacgtgt tgacgcttag gaccctaaga 1260
aatacggcca tcgaggctcg ggacctttct aatccgattc caaagaatat ggtctgggaa 1320
gctgttaaca ctctggggtg a 1341
<210>22
<211>446
<212>PRT
<213>Primula alpicola
<400>22
Met Ala Asn Thr Ser Tyr Ile Ser Ser Ser Asp Leu Lys Thr His Asn
1 5 10 15
Lys Ala Asp Asp Leu Trp Ile Ser Ile His Gly Gln Val Tyr Asp Val
20 25 30
Ser Ala Trp Ala Thr His His Pro Gly Gly Ala Ser Leu Leu Leu Ala
35 40 45
Leu Ala Gly Asn Asp Val Thr Asp Ala Phe Leu Ala Tyr His Pro Pro
50 55 60
Ser Thr Cys His Leu Leu Pro Pro Leu Ser Thr Asn Ile Leu Leu Glu
65 70 75 80
Asn Tyr Ser Val Ser His Ile Ser Ser Asn Tyr Arg Asn Leu Leu Asn
85 90 95
His Phe His Lys Leu Gly Leu Phe Arg Thr Arg Ala His Thr Ala Phe
100 105 110
Thr Thr Phe Phe Ile Met Ile Leu Met Phe Phe Ile Ser Val Thr Gly
115 120 125
Ile Phe Cys Ser Asp Ser Leu Trp Val His Leu Ala Cys Gly Gly Leu
130 135 140
Met Gly Phe Ala Trp Ile Gln Cys Gly Trp Ile Ala His Asp Ser Gly
145 150 155 160
His Tyr Arg Ile Thr Ser Ser Arg Lys Trp Asn Arg Phe Ala Gln Ile
165 170 175
Leu Thr Gly Asn Cys Leu Gln Gly Leu Ser Ile Gly Trp Trp Lys Trp
180 185 190
Asn His Asn Ala His His Ile Ala Cys Asn Ser Leu Asp Tyr Asp Pro
195 200 205
Asp Leu Gln Tyr Ile Pro Leu Leu Val Val Ser Pro Lys Phe Phe Asn
210 215 220
Ser Ile Thr Ser Arg Phe Tyr Asp Lys Lys Leu Asn Phe Asp Gly Val
225 230 235 240
Ser Arg Phe Leu Val Ser Tyr Gln His Trp Thr Phe Tyr Pro Val Met
245 250 255
Cys Val Ala Arg Phe Asn Met Ile Ala Gln Ser Val Ile His Leu Phe
260 265 270
Ser Asn Arg Asn Val Thr Asp Arg Val Leu Glu Ile Phe Gly Leu Gly
275 280 285
Val Phe Trp Val Trp Tyr Ser Leu Leu Leu Ser Cys Leu Pro Asp Trp
290 295 300
Gly Glu Arg Ile Met Phe Val Ile Ala Cys Tyr Phe Val Thr Gly Ile
305 310 315 320
Gln His Val Gln Phe Ser Val Asn His Phe Ser Ser Asp Val Tyr Met
325 330 335
Gly Pro Pro Val Gly Asn Asp Trp Phe Lys Lys Gln Thr Ser Gly Thr
340 345 350
Leu Asn Ile Ser Cys Pro Pro Trp Met Asp Trp Phe His Gly Gly Leu
355 360 365
Gln Phe Gln Val Glu His His Leu Phe Pro Arg Met Pro Arg Gly Gln
370 375 380
Phe Arg Lys Ile Ser Pro Phe Val Lys Asp Leu Cys Asn Lys His Asn
385 390 395 400
Leu Pro Tyr Asn Ile Ala Ser Phe Thr Met Ala Asn Val Leu Thr Leu
405 410 415
Arg Thr Leu Arg Asn Thr Ala Ile Glu Ala Arg Asp Leu Ser Asn Pro
420 425 430
Ile Pro Lys Asn Met Val Trp Glu Ala Val Asn Thr Leu Gly
435 440 445
<210>23
<211>1551
<212>DNA
<213>Primula alpicola
<400>23
atggctaaca aatctcaaac aggttacata accagctcag acctgaaagg tcacaataag 60
gcaggtgatc tatggatatc aatccacggg caggtctacg acgtgtcctc gtgggccagc 120
cttcacccgg ggggcagtgc ccccctcctg gccctcgcag gacacgacgt gaccgacgct 180
ttcctcgctt accatccccc ttccaccgcc cgcctcctcc ctcccctctc cgctaacctc 240
cttctgcaac accattccgt ctcccccacc tcctccgatt accgctccct cctcaacaac 300
tttcataaac ttggtctgtt ccgcgccagg ggccacaccg cttacgcaac cttcgtcttc 360
atgatagcga tgtttgtaat gagcgtaacc ggagtgcttt ttagcgacga tgcgtggatc 420
catctggctt gcgccggagc aatggggatt gcctggatcc agtgcggatg gataggtcgg 480
tagactaatt tattagtaca tatttaatat ttaaaatacc atattcttaa atcttattta 540
aattttggta tgtatcaata tagcttaaaa caaaaagtaa aaataagtag aatgtatact 600
taaaatagat attaactatg agtttttgat aattaagatt caaattatgt actaaatgat 660
catttttcct ggataggtca cgactctggg cattaccgga tgatgtctga caggaaatgg 720
aaccggtttg cgcaaatcct gagcgcaaac tgcctccagg ggattagcat cgggtggtgg 780
aagtggaacc acaacgcaca ccacatcgct tgcaatagcc tggagtacga ccccgacctc 840
cagtatatcc ctttgctcgt tgtctccccc aagtttttca actcccttac ttctcgtttc 900
tacaacaaga aactgaactt cgacggtgtg gcgaggttct tggtttgcta ccagcactgg 960
acgttttatc cggtcatgtg tgtcgctagg ctgaacatga tcgtgcagtc gtttataacg 1020
ctttttttga atagggaggt ggcgcatagg gcgcaagaga ttttgggact tgctgtgttt 1080
tgggtttggt ttccgctttt actttcttgc ttacctaatt ggggtgagag gataatgttt 1140
ctgcttgtga gctattccgt tacggggata caacacgtgc agttcagctt gaaccatttt 1200
tcttcggacg tctacgtggg tccgccagta ggtaacgact ggttcaagaa acagactgca 1260
gggacactta acatatcgtg cccagcgtgg atggattggt tccatggtgg gttgcaattt 1320
caggtcgagc accacttgtt cccgcggatg cctaggagtc agtttaggaa gatttctcct 1380
tttgtgaaag atttgtgtaa gaaacacaat ttgccttaca acatcgcgtc ttttactaaa 1440
gcgaatgtgt taacgcttaa gacgctgaga aatacggccg ttgaggctcg ggacctctct 1500
aatccgatcc caaagaatat ggtgtgggag gctgttaaaa ctctcgggtg a 1551
<210>24
<211>449
<212>PRT
<213>Primula alpicola
<400>24
Met Ala Asn Lys Ser Gln Thr Gly Tyr Ile Thr Ser Ser Asp Leu Lys
1 5 10 15
Gly His Asn Lys Ala Gly Asp Leu Trp Ile Ser Ile His Gly Gln Val
20 25 30
Tyr Asp Val Ser Ser Trp Ala Ser Leu His Pro Gly Gly Ser Ala Pro
35 40 45
Leu Leu Ala Leu Ala Gly His Asp Val Thr Asp Ala Phe Leu Ala Tyr
50 55 60
His Pro Pro Ser Thr Ala Arg Leu Leu Pro Pro Leu Ser Ala Asn Leu
65 70 75 80
Leu Leu Gln His His Ser Val Ser Pro Thr Ser Ser Asp Tyr Arg Ser
85 90 95
Leu Leu Asn Asn Phe His Lys Leu Gly Leu Phe Arg Ala Arg Gly His
100 105 110
Thr Ala Tyr Ala Thr Phe Val Phe Met Ile Ala Met Phe Val Met Ser
115 120 125
Val Thr Gly Val Leu Phe Ser Asp Asp Ala Trp Ile His Leu Ala Cys
130 135 140
Ala Gly Ala Met Gly Ile Ala Trp Ile Gln Cys Gly Trp Ile Gly His
145 150 155 160
Asp Ser Gly His Tyr Arg Met Met Ser Asp Arg Lys Trp Asn Arg Phe
165 170 175
Ala Gln Ile Leu Ser Ala Asn Cys Leu Gln Gly Ile Ser Ile Gly Trp
180 185 190
Trp Lys Trp Asn His Asn Ala His His Ile Ala Cys Asn Ser Leu Glu
195 200 205
Tyr Asp Pro Asp Leu Gln Tyr Ile Pro Leu Leu Val Val Ser Pro Lys
210 215 220
Phe Phe Asn Ser Leu Thr Ser Arg Phe Tyr Asn Lys Lys Leu Asn Phe
225 230 235 240
Asp Gly Val Ala Arg Phe Leu Val Cys Tyr Gln His Trp Thr Phe Tyr
245 250 255
Pro Val Met Cys Val Ala Arg Leu Asn Met Ile Val Gln Ser Phe Ile
260 265 270
Thr Leu Phe Leu Asn Arg Glu Val Ala His Arg Ala Gln Glu Ile Leu
275 280 285
Gly Leu Ala Val Phe Trp Val Trp Phe Pro Leu Leu Leu Ser Cys Leu
290 295 300
Pro Asn Trp Gly Glu Arg Ile Met Phe Leu Leu Val Ser Tyr Ser Val
305 310 315 320
Thr Gly Ile Gln His Val Gln Phe Ser Leu Asn His Phe Ser Ser Asp
325 330 335
Val Tyr Val Gly Pro Pro Val Gly Asn Asp Trp Phe Lys Lys Gln Thr
340 345 350
Ala Gly Thr Leu Asn Ile Ser Cys Pro Ala Trp Met Asp Trp Phe His
355 360 365
Gly Gly Leu Gln Phe Gln Val Glu His His Leu Phe Pro Arg Met Pro
370 375 380
Arg Ser Gln Phe Arg Lys Ile Ser Pro Phe Val Arg Asp Leu Cys Lys
385 390 395 400
Lys His Asn Leu Pro Tyr Asn Ile Ala Ser Phe Thr Lys Ala Asn Val
405 410 415
Leu Thr Leu Lys Thr Leu Arg Asn Thr Ala Val Glu Ala Arg Asp Leu
420 425 430
Ser Asn Pro Ile Pro Lys Asn Met Val Trp Glu Ala Val Lys Thr Leu
435 440 445
Gly
<210>25
<211>1526
<212>DNA
<213>Primula waltonii
<400>25
atggctaaca aatctcaaac aggttacata accagctcag acctgaaaag ccacaataag 60
gcaggtgatc tatggatatc aatccacggg caggtctacg acgtgtcctc gtgggccagt 120
cttcacccgg ggggctctgc cccccttctg gccctcgcag gacacgacgt taccgacgct 180
ttcctcgctt accatcctcc ttccaccgcc cgcctcctcc ctcccctctc cgctaacctc 240
cttctacaac accactccgt ctcccccaca tcctccgatt accgatccct cctcaacaac 300
tttcacaaac ttggcctgtt ccgctccagg ggccacaccg cttacgccac cttcgtcttc 360
atgataacga tgtttgtaat gagcgtaacc ggagtgctct tcagcgacga tgcgtgggtc 420
catctggctt gcggcggagc aatggggatt gcctggatcc agtgcggatg gataggtcgg 480
tagagtaatt tattagtaca tatttaaaat accatattct taaatcttat ttaaattttg 540
gtatgtatca atctttttat gtactaatat atacttaaaa tagatattaa ctatgagttt 600
ttgataatta agtttcaaat tatgtactaa atgatcaatt ttcctggata ggtcacgact 660
ctgggcatta ccggatgatg tctgacagga aatggaaccg gttcgcgcaa atcctgagcg 720
caaactgcct ccaggggatt agcatcgggt ggtggaagtg gaaccacaac gcgcaccaca 780
tcgcttgcaa tagcctggaa tacgaccccg acctccagta tatccctttg ctcgtcgtct 840
cccccaaatt tttcaactcc cttacttctc gtttctacaa caagaaactg aacttcgacg 900
gtgtgtcgag gttcttggtt tgctaccagc actggacgtt ttatccggtc atgtgtgtcg 960
ctaggctgaa catgctcgtg cagtcgttta taacgctttt ttcgaatagg gaggtggcgc 1020
atagggcgca agagattttg ggacttgctg tgttttgggt ttggtttccg cttttagttt 1080
cttgcttacc taattggggt gagaggataa tgtttctgct tgtgagctat tccgttacgg 1140
ggatacaaca cgtgcagttc agcttgaacc atttttcttc ggacgtctac gtgggtccgc 1200
cagtaggtaa cgactggttc aagaaacaga ctgcagggac actgaacata tcgtgcccgg 1260
cgtggatgga ttggttccat ggtgggttgc aatttcaggt ggagcaccac ttgttcccgc 1320
ggatgcctag gagtcagttt aggaagattt ctccttttgt gagggatttg tgtaagaaac 1380
acaatttgcc ttacaacatc gcgtctttta ctaaagcgaa tgtgttgacg cttaagacgc 1440
tgagaaatac ggccgttgag gctcgggacc tctctaatcc gatcccaaag aacatggtgt 1500
gggaggctgt taaaactctc gggtga 1526
<210>26
<211>449
<212>PRT
<213>Primula waltonii
<400>26
Met Ala Asn Lys Ser Gln Thr Gly Tyr Ile Thr Ser Ser Asp Leu Lys
1 5 10 15
Ser His Asn Lys Ala Gly Asp Leu Trp Ile Ser Ile His Gly Gln Val
20 25 30
Tyr Asp Val Ser Ser Trp Ala Ser Leu His Pro Gly Gly Ser Ala Pro
35 40 45
Leu Leu Ala Leu Ala Gly His Asp Val Thr Asp Ala Phe Leu Ala Tyr
50 55 60
His Pro Pro Ser Thr Ala Arg Leu Leu Pro Pro Leu Ser Ala Asn Leu
65 70 75 80
Leu Leu Gln His His Ser Val Ser Pro Thr Ser Ser Asp Tyr Arg Ser
85 90 95
Leu Leu Asn Asn Phe His Lys Leu Gly Leu Phe Arg Ser Arg Gly His
100 105 110
Thr Ala Tyr Ala Thr Phe Val Phe Met Ile Thr Met Phe Val Met Ser
115 120 125
Val Thr Gly Val Leu Phe Ser Asp Asp Ala Trp Val His Leu Ala Cys
130 135 140
Gly Gly Ala Met Gly Ile Ala Trp Ile Gln Cys Gly Trp Ile Gly His
145 150 155 160
Asp Ser Gly His Tyr Arg Met Met Ser Asp Arg Lys Trp Asn Arg Phe
165 170 175
Ala Gln Ile Leu Ser Ala Asn Cys Leu Gln Gly Ile Ser Ile Gly Trp
180 185 190
Trp Lys Trp Asn His Asn Ala His His Ile Ala Cys Asn Ser Leu Glu
195 200 205
Tyr Asp Pro Asp Leu Gln Tyr Ile Pro Leu Leu Val Val Ser Pro Lys
210 215 220
Phe Phe Asn Ser Leu Thr Ser Arg Phe Tyr Asn Lys Lys Leu Asn Phe
225 230 235 240
Asp Gly Val Ser Arg Phe Leu Val Cys Tyr Gln His Trp Thr Phe Tyr
245 250 255
Pro Val Met Cys Val Ala Arg Leu Asn Met Leu Val Gln Ser Phe Ile
260 265 270
Thr Leu Phe Ser Asn Arg Glu Val Ala His Arg Ala Gln Glu Ile Leu
275 280 285
Gly Leu Ala Val Phe Trp Val Trp Phe Pro Leu Leu Val Ser Cys Leu
290 295 300
Pro Asn Trp Gly Glu Arg Ile Met Phe Leu Leu Val Ser Tyr Ser Val
305 310 315 320
Thr Gly Ile Gln His Val Gln Phe Ser Leu Asn His Phe Ser Ser Asp
325 330 335
Val Tyr Val Gly Pro Pro Val Gly Asn Asp Trp Phe Lys Lys Gln Thr
340 345 350
Ala Gly Thr Leu Asn Ile Ser Cys Pro Ala Trp Met Asp Trp Phe His
355 360 365
Gly Gly Leu Gln Phe Gln Val Glu His His Leu Phe Pro Arg Met Pro
370 375 380
Arg Ser Gln Phe Arg Lys Ile Ser Pro Phe Val Arg Asp Leu Cys Lys
385 390 395 400
Lys His Asn Leu Pro Tyr Asn Ile Ala Ser Phe Thr Lys Ala Asn Val
405 410 415
Leu Thr Leu Lys Thr Leu Arg Asn Thr Ala Val Glu Ala Arg Asp Leu
420 425 430
Ser Asn Pro Ile Pro Lys Asn Met Val Trp Glu Ala Val Lys Thr Leu
435 440 445
Gly
<210>27
<211>27
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成引物
<400>27
cacacatgac cggataaaac gtccagt 27
<210>28
<211>27
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成引物
<400>28
agggatatac tggaggtcgg ggtcgta 27
<210>29
<211>27
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成引物
<400>29
gagctattcc gttacgggga tacaaca 27
<210>30
<211>27
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成引物
<400>30
tgcagggaca cttaacatat cgtgccc 27
<210>31
<211>27
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成引物
<400>31
gtgaaagttg ttgaggaggg atcggta 27
<210>32
<211>27
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成引物
<400>32
gtggaaggag gatggtaagc gaggaaa 27
<210>33
<211>33
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成引物
<400>33
gtcgacatgg ctaacaaatc tcaaacaggt tac 33
<210>34
<211>33
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成引物
<400>34
cctgcaggtc acccgagagt tttaacagcc tcc 33
<210>35
<211>27
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成引物
<400>35
cacacattac cggataaaac gtccagt 27
<210>36
<211>27
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成引物
<400>36
aggaatatac tggaggtctg ggtcgta 27
<210>37
<211>27
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成引物
<400>37
atttttcttc ggacgtatac atgggcc 27
<210>38
<211>27
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成引物
<400>38
ttcggggaca ctgaacatat cgtgccc 27
<210>39
<211>34
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成引物
<400>39
gtcgacatgg ccaacactag ttacatttcc agct 34
<210>40
<211>32
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成引物
<400>40
gatatcaccc cagagtgtta acagcttccc ag 32
<210>41
<211>20
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成引物
<400>41
tggaggtctg ggtcgtaatc 20
<210>42
<211>20
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成引物
<400>42
cttcggacgt atacatgggc 20
<210>43
<211>20
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成引物
<400>43
tcgtaatcca ggctattgca 20
<210>44
<211>20
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成引物
<400>44
ttttcttcgg acgtccatgt 20
<210>45
<211>1365
<212>DNA
<213>有粉报春
<400>45
atgtccaaca catatccacc aaatcccaaa actagtcatt acatttccag ctcagacctc 60
aaaacccata ataaacctga agacctttgg atatccattc acggccacgt gtacgatgtc 120
tccgcctggg ccccccacca cctcggaggt gcctctctcc tcctcgccct cgcaggcaat 180
gatgtcaccg acaccttcct cgcctaccac cctccctcca cctgccgcct tctccctcct 240
ttctctacca acatcctcct cgaaaactac tccgtctccc acacctcctc tgactaccgc 300
aacctcctca atcatttcca caagctcggc ctattccgta caaaggccca tacgactttc 360
actactttct tcatcatgat acttatgttc tttctcagtg taaccggaat attttgcagt 420
gatagttttt gggtccattt ggcgtgcggt ggcttgatgg ggttcgcatg gatccaatgt 480
ggatggatag cgcacgactc tgggcattac cggataacat caagcaggaa atggaataga 540
ttcgctcaga tccttatcgg aaattgcctc caggggttga gtattgggtg gtggaagtgg 600
aaccataacg ctcaccacat cgcttgcaat agcctagatt acgacccgga tctccagtat 660
attcctttat tggtagtttc ttcaaagttt ttcaactcca tcacttctgg tttctatgat 720
aagaagctga acttcgacgg tgtgtcgagg ttcttagtta gctaccaaca ctggacgttt 780
tatccggtaa tgtgtgttgc taggtttaac atggttgcac agtcggttat acatgttttc 840
tcaaatagaa aggtgcccaa tagggttcta gaggttttcg gactaggcgt gttctgggtt 900
tggtattcgc tcctactttc gtgccttcct gattggggcg agcgaataat gtttgtgatt 960
gcatgctact ttgttacggg gatacaacac gtacagttca gtgtaaacca tttttcttcg 1020
gacgtataca tgggccctcc cgtaggtaac gattggttta aaaaacagac tgcagggaca 1080
ctgaacatat cgtgcccgcc ctggatggat tggttccacg gtgggttgca gtttcaaatc 1140
gagcaccact tgttcccacg gatgccgagg ggtcaattta ggaagatctc tccttttgta 1200
aaggatttgt gtaataaaca caatctgcct tacaatatcg catcttttac tatggctaac 1260
gtgttgacgc ttaggaccct gagaaatacg gccattgagg cttgggacct ttctaatccg 1320
atcccaaaga atatggtctg ggaagctgtt aacactctgg ggtga 1365
<210>46
<211>454
<212>PRT
<213>有粉报春
<400>46
Met Ser Asn Thr Tyr Pro Pro Asn Pro Lys Thr Ser His Tyr Ile Ser
1 5 10 15
Ser Ser Asp Leu Lys Thr His Asn Lys Pro Glu Asp Leu Trp Ile Ser
20 25 30
Ile His Gly His Val Tyr Asp Val Ser Ala Trp Ala Pro His His Leu
35 40 45
Gly Gly Ala Ser Leu Leu Leu Ala Leu Ala Gly Asn Asp Val Thr Asp
50 55 60
Thr Phe Leu Ala Tyr His Pro Pro Ser Thr Cys Arg Leu Leu Pro Pro
65 70 75 80
Phe Ser Thr Asn Ile Leu Leu Glu Asn Tyr Ser Val Ser His Thr Ser
85 90 95
Ser Asp Tyr Arg Asn Leu Leu Asn His Phe His Lys Leu Gly Leu Phe
100 105 110
Arg Thr Lys Ala His Thr Thr Phe Thr Thr Phe Phe Ile Met Ile Leu
115 120 125
Met Phe Phe Leu Ser Val Thr Gly Ile Phe Cys Ser Asp Ser Phe Trp
130 135 140
Val His Leu Ala Cys Gly Gly Leu Met Gly Phe Ala Trp Ile Gln Cys
145 150 155 160
Gly Trp Ile Ala His Asp Ser Gly His Tyr Arg Ile Thr Ser Ser Arg
165 170 175
Lys Trp Asn Arg Phe Ala Gln Ile Leu Ile Gly Asn Cys Leu Gln Gly
180 185 190
Leu Ser Ile Gly Trp Trp Lys Trp Asn His Asn Ala His His Ile Ala
195 200 205
Cys Asn Ser Leu Asp Tyr Asp Pro Asp Leu Gln Tyr Ile Pro Leu Leu
210 215 220
Val Val Ser Ser Lys Phe Phe Asn Ser Ile Thr Ser Gly Phe Tyr Asp
225 230 235 240
Lys Lys Leu Asn Phe Asp Gly Val Ser Arg Phe Leu Val Ser Tyr Gln
245 250 255
His Trp Thr Phe Tyr Pro Val Met Cys Val Ala Arg Phe Asn Met Val
260 265 270
Ala Gln Ser Val Ile His Val Phe Ser Asn Arg Lys Val Pro Asn Arg
275 280 285
Val Leu Glu Val Phe Gly Leu Gly Val Phe Trp Val Trp Tyr Ser Leu
290 295 300
Leu Leu Ser Cys Leu Pro Asp Trp Gly Glu Arg Ile Met Phe Val Ile
305 310 315 320
Ala Cys Tyr Phe Val Thr Gly Ile Gln His Val Gln Phe Ser Val Asn
325 330 335
His Phe Ser Ser Asp Val Tyr Met Gly Pro Pro Val Gly Asn Asp Trp
340 345 350
Phe Lys Lys Gln Thr Ala Gly Thr Leu Asn Ile Ser Cys Pro Pro Trp
355 360 365
Met Asp Trp Phe His Gly Gly Leu Gln Phe Gln Ile Glu His His Leu
370 375 380
Phe Pro Arg Met Pro Arg Gly Gln Phe Arg Lys Ile Ser Pro Phe Val
385 390 395 400
Lys Asp Leu Cys Asn Lys His Asn Leu Pro Tyr Asn Ile Ala Ser Phe
405 410 415
Thr Met Ala Asn Val Leu Thr Leu Arg Thr Leu Arg Asn Thr Ala Ile
420 425 430
Glu Ala Trp Asp Leu Ser Asn Pro Ile Pro Lys Asn Met Val Trp Glu
435 440 445
Ala Val Asn Thr Leu Gly
450
<210>47
<211>1559
<212>DNA
<213>Primula florindae
<400>47
atggctaaca aatctcaaac aggttacata accagctcag acctgaaagg ccacaataag 60
gcaggtgatc tatggatatc aatccacggt caggtctacg acgtgtcctc gtgggccagc 120
cttcacccgg ggggcagtgc ccccctcctg gccctcgcag gacacgacgt gaccgacgct 180
ttcctcgctt accatccccc ttccaccgcc cgccttctcc ctcccctctc cgctaacctc 240
cttctacaac accactccgt ctcccccacc tcctctgatt accgctccct cctcaacaac 300
tttcataaac ttggcctgtt ccgcaccagg ggccacaccg cttacgcaac cttcgtcttc 360
atgatagcga tgtttgtaat gagcgtgacc ggagtgcttt ttagcgacga tgcgtgggtc 420
catctggctt gcgccggagc aatggggatt gcctggatcc aatgcggatg gataggtcgg 480
tagactaatc tattagtaca taaaaacata tttaatattt aaaataccat attcttaaat 540
cttatttaaa ttttggtatg tatcaatatg ggttaaaaca aaaagtaaaa ataagtagaa 600
tgtatactta aaatagatat taactatgag tttttgataa ttaagattca aattatgtac 660
taaatgatca tttttcctgg ataggtcacg actctgggca ttaccggatg atgtctgaca 720
ggaaatggaa ccggtttgcg caaatcctga gcgcaaactg cctccagggg attagcatcg 780
ggtggtggaa gtggaaccac aacgcgcacc acatcgcttg caatagcctg gattacgacc 840
ccgacctcca gtatatccct ttgctcgtcg tctcccccaa gtttttcaac tcccttactt 900
ctcgtttcta caacaagaaa ctgaacttcg acggtgtgtc gaggttcttg gtttgctacc 960
agcactggac gttttatccg gtcatgtgtg tcgctaggct gaacatgctc gtgcagtcgt 1020
ttataacgct tttttcgaat agggaggtgg cgcatagggc gcaagagatt ttgggacttg 1080
ctgtgttttg ggtttggttt ccgcttttac tttcttgctt acctaattgg ggtgagagga 1140
taatgtttct gcttgtgagc tattccatta cggggataca acacgtgcag tttagcttga 1200
accatttttc ttcggacgtc tatgtgggtc cgccagtagg taacgactgg ttcaagaaac 1260
agactgcagg gacacttaac atatcgtgcc cggcgtggat ggattggttc catggtgggt 1320
tgcagtttca ggtcgagcac cacttgttcc cgcggatgcc taggagtcag tttaggaaga 1380
tttctccttt tgtgagggat ttgtgtaaga aacacaattt gccttacaac atcgcgtctt 1440
ttactaaagc gaatgtgttg acgcttaaga cgctgagaaa tacagccgtt gaggctcggg 1500
acctctctaa tccgatccca aagaatatgg tgtgggaggc tgttaaaact ctcgggtga 1559
<210>48
<211>449
<212>PRT
<213>Primula florindae
<400>48
Met Ala Asn Lys Ser Gln Thr Gly Tyr Ile Thr Ser Ser Asp Leu Lys
1 5 10 15
Gly His Asn Lys Ala Gly Asp Leu Trp Ile Ser Ile His Gly Gln Val
20 25 30
Tyr Asp Val Ser Ser Trp Ala Ser Leu His Pro Gly Gly Ser Ala Pro
35 40 45
Leu Leu Ala Leu Ala Gly His Asp Val Thr Asp Ala Phe Leu Ala Tyr
50 55 60
His Pro Pro Ser Thr Ala Arg Leu Leu Pro Pro Leu Ser Ala Asn Leu
65 70 75 80
Leu Leu Gln His His Ser Val Ser Pro Thr Ser Ser Asp Tyr Arg Ser
85 90 95
Leu Leu Asn Asn Phe His Lys Leu Gly Leu Phe Arg Thr Arg Gly His
100 105 110
Thr Ala Tyr Ala Thr Phe Val Phe Met Ile Ala Met Phe Val Met Ser
115 120 125
Val Thr Gly Val Leu Phe Ser Asp Asp Ala Trp Val His Leu Ala Cys
130 135 140
Ala Gly Ala Met Gly Ile Ala Trp Ile Gln Cys Gly Trp Ile Gly His
145 150 155 160
Asp Ser Gly His Tyr Arg Met Met Ser Asp Arg Lys Trp Asn Arg Phe
165 170 175
Ala Gln Ile Leu Ser Ala Asn Cys Leu Gln Gly Ile Ser Ile Gly Trp
180 185 190
Trp Lys Trp Asn His Asn Ala His His Ile Ala Cys Asn Ser Leu Asp
195 200 205
Tyr Asp Pro Asp Leu Gln Tyr Ile Pro Leu Leu Val Val Ser Pro Lys
210 215 220
Phe Phe Asn Ser Leu Thr Ser Arg Phe Tyr Asn Lys Lys Leu Asn Phe
225 230 235 240
Asp Gly Val Ser Arg Phe Leu Val Cys Tyr Gln His Trp Thr Phe Tyr
245 250 255
Pro Val Met Cys Val Ala Arg Leu Asn Met Leu Val Gln Ser Phe Ile
260 265 270
Thr Leu Phe Ser Asn Arg Glu Val Ala His Arg Ala Gln Glu Ile Leu
275 280 285
Gly Leu Ala Val Phe Trp Val Trp Phe Pro Leu Leu Leu Ser Cys Leu
290 295 300
Pro Asn Trp Gly Glu Arg Ile Met Phe Leu Leu Val Ser Tyr Ser Ile
305 310 315 320
Thr Gly Ile Gln His Val Gln Phe Ser Leu Asn His Phe Ser Ser Asp
325 330 335
Val Tyr Val Gly Pro Pro Val Gly Asn Asp Trp Phe Lys Lys Gln Thr
340 345 350
Ala Gly Thr Leu Asn Ile Ser Cys Pro Ala Trp Met Asp Trp Phe His
355 360 365
Gly 6ly Leu Gln Phe Gln Val Glu His His Leu Phe Pro Arg Met Pro
370 375 380
Arg Ser Gln Phe Arg Lys Ile Ser Pro Phe Val Arg Asp Leu Cys Lys
385 390 395 400
Lys His Asn Leu Pro Tyr Asn Ile Ala Ser Phe Thr Lys Ala Asn Val
405 410 415
Leu Thr Leu Lys Thr Leu Arg Asn Thr Ala Val Glu Ala Arg Asp Leu
420 425 430
Ser Asn Pro Ile Pro Lys Asn Met Val Trp Glu Ala Val Lys Thr Leu
435 440 445
Gly
<210>49
<211>38
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成引物
<400>49
gacgattttt gagtgagagt taatttgagt caataata 38
<210>50
<211>30
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成引物
<400>50
cgacatcata gacaatcatc aagacaccgt 30
<210>51
<211>25
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成引物
<400>51
ataccccctc aaaacacccc caaat 25
<210>52
<211>30
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成引物
<400>52
ctcaatatca cccgagagtt ttaacagcct 30
<210>53
<211>35
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成引物
<400>53
gtcgacaaca atgtccaaca catatccacc aaatc 35
<210>54
<211>32
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成引物
<400>54
cctgcaggtc accccagagt gttaacagct tc 32
<210>55
<211>26
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成引物
<400>55
gtcgacatgg ctaacaaatc tcaaac 26
<210>56
<211>20
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成引物
<400>56
cctgcaggtc acccgagagt 20
<210>57
<211>41
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成引物
<400>57
gtaatgccca gagtcgtgac ctatccatcc gcactggatc c 41
<210>58
<211>42
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:合成引物
<400>58
gatccagtgc ggatggatag gtcacgactc tgggcattac cg 42
<210>59
<211>448
<212>PRT
<213>Borago officinalis
<400>59
Met Ala Ala Gln Ile Lys Lys Tyr Ile Thr Ser Asp Glu Leu Lys Asn
1 5 10 15
His Asp Lys Pro Gly Asp Leu Trp Ile Ser Ile Gln Gly Lys Ala Tyr
20 25 30
Asp Val Ser Asp Trp Val Lys Asp His Pro Gly Gly Ser Phe Pro Leu
35 40 45
Lys Ser Leu Ala Gly Gln Glu Val Thr Asp Ala Phe Val Ala Phe His
50 55 60
Pro Ala Ser Thr Trp Lys Asn Leu Asp Lys Phe Phe Thr Gly Tyr Tyr
65 70 75 80
Leu Lys Asp Tyr Ser Val Ser Glu Val Ser Lys Asp Tyr Arg Lys Leu
85 90 95
Val Phe Glu Phe Ser Lys Met Gly Leu Tyr Asp Lys Lys Gly His Ile
100 105 110
Met Phe Ala Thr Leu Cys Phe Ile Ala Met Leu Phe Ala Met Ser Val
115 120 125
Tyr Gly Val Leu Phe Cys Glu Gly Val Leu Val His Leu Phe Ser Gly
130 135 140
Cys Leu Met Gly Phe Leu Trp Ile Gln Ser Gly Trp Ile Gly His Asp
145 150 155 160
Ala Gly His Tyr Met Val Val Ser Asp Ser Arg Leu Asn Lys Phe Met
165 170 175
Gly Ile Phe Ala Ala Asn Cys Leu Ser Gly Ile Ser Ile Gly Trp Trp
180 185 190
Lys Trp Asn His Asn Ala His His Ile Ala Cys Asn Ser Leu Glu Tyr
195 200 205
Asp Pro Asp Leu Gln Tyr Ile Pro Phe Leu Val Val Ser Ser Lys Phe
210 215 220
Phe Gly Ser Leu Thr Ser His Phe Tyr Glu Lys Arg Leu Thr Phe Asp
225 230 235 240
Ser Leu Ser Arg Phe Phe Val Ser Tyr Gln His Trp Thr Phe Tyr Pro
245 250 255
Ile Met Cys Ala Ala Arg Leu Asn Met Tyr Val Gln Ser Leu Ile Met
260 265 270
Leu Leu Thr Lys Arg Asn Val Ser Tyr Arg Ala His Glu Leu Leu Gly
275 280 285
Cys Leu Val Phe Ser Ile Trp Tyr Pro Leu Leu Val Ser Cys Leu Pro
290 295 300
Asn Trp Gly Glu Arg Ile Met Phe Val Ile Ala Ser Leu Ser Val Thr
305 310 315 320
Gly Met Gln Gln Val Gln Phe Ser Leu Asn His Phe Ser Ser Ser Val
325 330 335
Tyr Val Gly Lys Pro Lys Gly Asn Asn Trp Phe Glu Lys Gln Thr Asp
340 345 350
Gly Thr Leu Asp Ile Ser Cys Pro Pro Trp Met Asp Trp Phe His Gly
355 360 365
Gly Leu Gln Phe Gln Ile Glu His His Leu Phe Pro Lys Met Pro Arg
370 375 380
Cys Asn Leu Arg Lys Ile Ser Pro Tyr Val Ile Glu Leu Cys Lys Lys
385 390 395 400
His Asn Leu Pro Tyr Asn Tyr Ala Ser Phe Ser Lys Ala Asn Glu Met
405 410 415
Thr Leu Arg Thr Leu Arg Asn Thr Ala Leu Gln Ala Arg Asp Ile Thr
420 425 430
Lys Pro Leu Pro Lys Asn Leu Val Trp Glu Ala Leu His Thr His Gly
435 440 445
<210>60
<211>448
<212>PRT
<213>Echium gentianoides
<400>60
Met Ala Asn Ala Ile Lys Lys Tyr Ile Thr Ala Glu Glu Leu Lys Lys
1 5 l0 15
His Asp Lys G1u Gly Asp Leu Trp Ile Ser Ile Gln Gly Lys Val Tyr
20 25 30
Asp Val Ser Asp Trp Leu Lys Asp His Pro Gly Gly Lys Phe Pro Leu
35 40 45
Leu Ser Leu Ala Gly Gln Glu Val Thr Asp Ala Phe Val Ala Phe His
50 55 60
Ser Gly Ser Thr Trp Lys Phe Leu Asp Ser Phe Phe Thr Gly Tyr Tyr
65 70 75 80
Leu Lys Asp Tyr Ser Val Ser Glu Val Ser Lys Asp Tyr Arg Lys Leu
85 90 95
Val Phe Glu Phe Asn Lys Met Gly Leu Phe Asp Lys Lys Gly His Ile
100 105 110
Val Leu Val Thr Val Leu Phe Ile Ala Met Met Phe Ala Met Ser Val
115 120 125
Tyr Gly Val Leu Phe Cys Glu Gly Val Leu Val His Leu Leu Ala Gly
130 135 140
Gly Leu Met Gly Phe Val Trp Ile Gln Ser Gly Trp Ile Gly His Asp
145 150 155 160
Ala Gly His Tyr Ile Val Met Pro Asn Pro Arg Leu Asn Lys Leu Met
165 170 175
Gly Ile Val Ala Gly Asn Cys Leu Ser Gly Ile Ser Ile Gly Trp Trp
180 185 190
Lys Trp Asn His Asn Ala His His Ile Ala Cys Asn Ser Leu Asp Tyr
195 200 205
Asp Pro Asp Leu Gln Tyr Ile Pro Phe Leu Val Val Ser Ser Lys Leu
210 215 220
Phe Ser Ser Leu Thr Ser His Phe Tyr Glu Lys Lys Leu Thr Phe Asp
225 230 235 240
Ser Leu Ser Arg Phe Phe Val Ser His Gln His Trp Thr Phe Tyr Pro
245 250 255
Val Met Cys Ser Ala Arg Val Asn Met Phe Val Gln Ser Leu Ile Met
260 265 270
Leu Leu Thr Lys Arg Asn Val Phe Tyr Arg Ser Gln Glu Leu Leu Gly
275 280 285
Leu Val Val Phe Trp Ile Trp Tyr Pro Leu Leu Val Ser Cys Leu Pro
290 295 300
Asn Trp Gly Glu Arg Ile Met Phe Val Val Ala Ser Leu Ser Val Thr
305 310 315 320
Gly Met Gln Gln Val Gln Phe Ser Leu Asn His Phe Ser Ala Ser Val
325 330 335
Tyr Val Gly Gln Pro Lys Gly Asn Asp Trp Phe Glu Lys Gln Thr Cys
340 345 350
Gly Thr Leu Asp Ile Ser Cys Pro Ser Trp Met Asp Trp Phe His Gly
355 360 365
Gly Leu Gln Phe Gln Val Glu His His Leu Phe Pro Lys Leu Pro Arg
370 375 380
Cys His Leu Arg Lys Ile Ser Pro Phe Val Met Glu Leu Cys Lys Lys
385 390 395 400
His Asn Leu Ser Tyr Asn Cys Ala Ser Phe Ser Glu Ala Asn Glu Met
405 410 415
Thr Leu Arg Thr Leu Arg Asp Thr Ala Leu Gln Ala Arg Asp Leu Thr
420 425 430
Lys Pro Leu Pro Lys Asn Leu Val Trp Glu Ala Leu Asn Thr His Gly
435 440 445
Claims (52)
1.分离的多核苷酸,编码具有在碳6上使脂肪酸分子去饱和的去饱和酶活性的多肽其中所述多核苷酸选自:
(a)编码SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:22、SEQ IDNO:24、SEQ ID NO:26、SEQ ID NO:46或SEQ ID NO:48的多肽序列的多核苷酸;
(b)包含SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:21、SEQ IDNO:23、SEQ ID NO:25、SEQ ID NO:45或SEQ ID NO:47的核酸序列的多核苷酸;
(c)在5X SSC、50%甲酰胺和42℃条件下与SEQ ID NO:2、SEQID NO:3、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ IDNO:45或SEQ ID NO:47或者其互补序列杂交的多核苷酸;
(d)编码与SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:22、SEQID NO:24、SEQ ID NO:26、SEQ ID NO:46或SEQ ID NO:48的多肽序列具有至少90%序列同一性的多肽的多核苷酸。
2.权利要求1的分离的多核苷酸,其中多核苷酸编码SEQ IDNO:4、SEQ ID NO:5、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:46或SEQ ID NO:48的多肽。
3.权利要求1的分离的多核苷酸,进一步限定为编码与SEQ IDNO:4、SEQ ID NO:5、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:46或SEQ ID NO:48的多肽序列具有至少95%序列同一性的多肽。
4.权利要求1的分离的多核苷酸,进一步限定为可操作性连接到异源启动子上。
5.包含SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:22、SEQ IDNO:24、SEQ ID NO:26、SEQ ID NO:46或SEQ ID NO:48的多肽序列的分离的多肽,或者其具有在碳6上使脂肪酸分子去饱和的去饱和酶活性的片段。
6.包含权利要求1的分离的多核苷酸序列的重组载体。
7.权利要求6的重组载体,进一步包含至少一种附加序列,选自:
(a)可操作性连接到多核苷酸上的调节序列;
(b)可操作性连接到多核苷酸上的选择标记;
(c)可操作性连接到多核苷酸上的标记序列;
(d)可操作性连接到多核苷酸上的纯化部分;以及
(e)可操作性连接到多核苷酸上的导向序列。
8.权利要求6的重组载体,进一步限定为包含可操作性连接到所述分离的多核苷酸上的启动子。
9.权利要求8的重组载体,其中启动子是受发育调节的、细胞器特异性的、组织特异性的、组成型的或者细胞特异性的启动子。
10.权利要求8的重组载体,其中所述启动子选自35S CaMV、34SFMV、Napin、7Sα、7Sα’、Glob和Lec。
11.权利要求6的重组载体,限定为分离的表达盒。
12.用权利要求6的重组载体转化的转基因植物。
13.权利要求12的转基因植物,进一步限定为用编码具有在碳12上使脂肪酸分子去饱和的去饱和酶活性的多肽的核酸序列转化。
14.权利要求12的转基因植物,进一步限定为用编码具有在碳15上使脂肪酸分子去饱和的去饱和酶活性的多肽的核酸序列转化。
15.用权利要求6的重组载体转化的宿主细胞。
16.权利要求15的宿主细胞,其中所述宿主细胞表达由所述载体编码的蛋白质。
17.权利要求15的宿主细胞,其中细胞具有从细胞祖先遗传而来的所述重组载体。
18.权利要求15的宿主细胞,其中细胞用所述重组载体转化。
19.权利要求15的宿主细胞,限定为植物细胞。
20.权利要求12的植物的种子,其中种子包含重组载体。
21.生产含有来自植物种子的ω-3脂肪酸的种子油的方法,包括步骤:
(a)获得根据权利要求12的植物的种子;以及
(b)从所述种子提取油。
22.生产包含含有改变水平的ω-3脂肪酸的种子油的植物的方法,包括将权利要求6的重组载体引入产油植物。
23.权利要求22的方法,其中引入重组载体包括植物育种。
24.权利要求22的方法,其中引入重组载体包括遗传转化。
25.权利要求22的方法,其中植物是选自下组的植物:拟南芥、油籽芸苔、油菜籽、向日葵、红花、canola、玉米、大豆、棉花、亚麻、西蒙德木、乌桕、烟草、可可、花生、水果类植物、柑桔类植物以及产生坚果和浆果的植物。
26.权利要求22的方法,其中植物进一步限定为用编码具有在碳15上对脂肪酸分子去饱和的去饱和酶活性的多肽的核酸序列转化。
27.权利要求26的方法,其中增加了十八碳四烯酸。
28.权利要求22的方法,进一步限定为包括将权利要求6的重组载体引入多种产油植物中,筛选具有从具有期望的ω-3脂肪酸谱的植物遗传而来的重组载体的所述植物或其后代。
29.具有大约5%到大约50%十八碳四烯酸含量和少于10%γ-亚油酸含量的内源性大豆种子油。
30.权利要求29的大豆种子油,进一步限定为包含少于10%的组合的α-亚油酸、亚油酸和γ-亚油酸。
31.权利要求29的大豆种子油,其中十八碳四烯酸含量进一步限定为大约15%到大约35%。
32.权利要求29的大豆种子油,其中十八碳四烯酸含量进一步限定为大约22%到大约30%。
33.权利要求29的大豆种子油,其中γ-亚油酸进一步限定为少于5%。
34.权利要求29的大豆种子油,其中十八碳四烯酸含量进一步被限定为大约15%到大约35%,γ-亚油酸含量进一步限定为少于5%。
35.权利要求29的大豆种子油,其中油中的ω-3与ω-6脂肪酸的比值为大约0.35∶1到大约3.5∶1。
36.权利要求29的大豆种子油,其中油中的ω-3与ω-6脂肪酸的比值为大约1∶1到大约3.5∶1。
37.增加用于人或非人动物消耗的可食用产品的营养价值的方法,包括将权利要求29的大豆种子油加入可食用产品中。
38.权利要求37的方法,其中可食用产品选自人的食物、动物饲料以及食物补充剂。
39.权利要求37的方法,其中大豆种子油增加了可食用产品中十八碳四烯酸的含量。
40.权利要求37的方法,其中大豆种子油增加了可食用产品中ω-3与ω-6脂肪酸的比值。
41.权利要求37的方法,其中可食用产品在加入大豆种子油之前没有十八碳四烯酸。
42.制造食物和/或饲料的方法,包括将权利要求29的大豆种子油加入起始食物和/或饲料成分中以生产食物和/或饲料。
43.由权利要求42的方法制造的食物或饲料。
44.向人或非人动物提供十八碳四烯酸的方法,包括将权利要求29的大豆种子油给予所述的人或非人动物。
45.权利要求44的方法,其中大豆种子油在可食用组合物中被给予。
46.权利要求45的方法,其中可食用组合物是食物或饲料。
47.权利要求46的方法,其中食物包括饮料、浸制食物、酱油、调味品、色拉调料、果汁、糖浆、甜点、糖霜和馅、软冻产品、糖果或半成品食物。
48.权利要求45的方法,其中可食用组合物基本上是液体或固体。
49.权利要求45的方法,其中可食用组合物是食物补充剂和/或保健食品。
50.权利要求44的方法,其中大豆种子油被给予人。
51.权利要求44的方法,其中大豆种子油被给予非人动物。
52.权利要求51的方法,其中大豆种子油被给予家畜或家禽。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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US49675103P | 2003-08-21 | 2003-08-21 | |
US60/496,751 | 2003-08-21 | ||
PCT/US2004/026944 WO2005021761A1 (en) | 2003-08-21 | 2004-08-20 | Fatty acid desaturases from primula |
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CN1871353A true CN1871353A (zh) | 2006-11-29 |
CN1871353B CN1871353B (zh) | 2013-04-24 |
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EP (1) | EP1656449B1 (zh) |
CN (1) | CN1871353B (zh) |
AR (1) | AR045290A1 (zh) |
AT (1) | ATE430802T1 (zh) |
AU (1) | AU2004268196B2 (zh) |
BR (1) | BRPI0413786B1 (zh) |
CA (2) | CA2881252C (zh) |
DE (1) | DE602004021001D1 (zh) |
DK (1) | DK1656449T3 (zh) |
ES (1) | ES2323644T3 (zh) |
MX (1) | MXPA06002028A (zh) |
PL (1) | PL1656449T3 (zh) |
PT (1) | PT1656449E (zh) |
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2004
- 2004-08-20 EP EP04781601A patent/EP1656449B1/en not_active Expired - Lifetime
- 2004-08-20 CN CN2004800310731A patent/CN1871353B/zh not_active Expired - Lifetime
- 2004-08-20 BR BRPI0413786A patent/BRPI0413786B1/pt active IP Right Grant
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- 2004-08-20 CA CA2881252A patent/CA2881252C/en not_active Expired - Lifetime
- 2004-08-20 US US10/569,387 patent/US8173870B2/en active Active
- 2004-08-20 WO PCT/US2004/026944 patent/WO2005021761A1/en active Application Filing
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- 2004-08-20 DE DE602004021001T patent/DE602004021001D1/de not_active Expired - Lifetime
- 2004-08-20 AU AU2004268196A patent/AU2004268196B2/en not_active Expired
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- 2008-06-12 US US11/891,426 patent/US8221819B2/en not_active Expired - Fee Related
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8692076B2 (en) | 2008-02-15 | 2014-04-08 | Monsanto Technology Llc | Soybean plant and seed corresponding to transgenic event MON87769 and methods for detection thereof |
US8999411B2 (en) | 2008-02-15 | 2015-04-07 | Monsanto Technology Llc | Soybean plant and seed corresponding to transgenic event MON87769 and methods for detection thereof |
CN104651355A (zh) * | 2008-02-15 | 2015-05-27 | 孟山都技术公司 | 对应于转基因事件mon87769的大豆植物和种子及其检测方法 |
CN102469820A (zh) * | 2009-06-30 | 2012-05-23 | 孟山都技术公司 | 富含ω-3的坚果黄油及其相关产物 |
CN101914148A (zh) * | 2010-08-20 | 2010-12-15 | 中国科学院遗传与发育生物学研究所 | 与脂肪酸合成相关的蛋白GmLEC1A及其编码基因与应用 |
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CA2881252A1 (en) | 2005-03-10 |
BRPI0413786A (pt) | 2006-11-07 |
ZA200601438B (en) | 2007-05-30 |
US10174297B2 (en) | 2019-01-08 |
US20080063691A1 (en) | 2008-03-13 |
US20090176879A1 (en) | 2009-07-09 |
BRPI0413786B1 (pt) | 2019-12-24 |
CA2881252C (en) | 2017-02-28 |
ES2323644T3 (es) | 2009-07-22 |
AU2004268196A1 (en) | 2005-03-10 |
EP1656449A1 (en) | 2006-05-17 |
PT1656449E (pt) | 2009-07-27 |
PL1656449T3 (pl) | 2009-08-31 |
US20130041032A1 (en) | 2013-02-14 |
US20170283780A1 (en) | 2017-10-05 |
US11041148B2 (en) | 2021-06-22 |
CA2535310A1 (en) | 2005-03-10 |
US9701947B2 (en) | 2017-07-11 |
EP1656449B1 (en) | 2009-05-06 |
CA2535310C (en) | 2015-06-09 |
WO2005021761A1 (en) | 2005-03-10 |
US20130041031A1 (en) | 2013-02-14 |
ATE430802T1 (de) | 2009-05-15 |
DK1656449T3 (da) | 2009-06-02 |
DE602004021001D1 (de) | 2009-06-18 |
US8173870B2 (en) | 2012-05-08 |
MXPA06002028A (es) | 2006-05-17 |
CN1871353B (zh) | 2013-04-24 |
US8221819B2 (en) | 2012-07-17 |
AR045290A1 (es) | 2005-10-19 |
AU2004268196B2 (en) | 2010-03-04 |
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