CN1252684A - 编码光敏剂抗性的分离基因和蛋白质 - Google Patents
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Abstract
本发明提供了可为光敏剂敏感型细胞和生物体提供光敏剂抗性的方法和化合物。特别描述了经表达可提供光敏剂抗性的一种分离的核酸分子。还描述了用该分离核酸分子转化细胞和生物体的方法,使经过这样转化的细胞和生物体可增加或获得光敏剂抗性。
Description
联邦政府资助的研究或开发
本发明是受到政府支持的、国家科学基金授予号MCB-9205578和MCB-9631375,及USDA的NRI竞争授予号9601197。
发明背景
尾孢属(Cercospora)是一类强致病性真菌,在多种多样的宿主植物中致病,这些植物包括玉米、甜菜、烟草、咖啡、大豆和香蕉,以及许多观赏植物和种子植物。如尾孢属中烟草尾孢、落花生尾孢、玉米尾孢(C.zeae-maydis)、菊池尾孢、稻尾孢和甜菜生尾孢(C.beticol)分别对烟草、花生、玉米、大豆、大米和甜菜致病。尾孢菌均为好气性病原菌。通常,这些真菌产生的孢子在叶表面发芽,随后进入叶内(即穿过气孔)。真菌菌丝体在叶片组织的细胞间隙中扩散,杀死叶片细胞,导致叶片组织严重枯萎。见M.E.Daub,美国化学学会专题论丛339号,271-80页(J.R.Heitz和K.R.Downum编,华盛顿特区,1987)。除破坏叶组织之外,尾孢菌还可能破坏其它植物组织,如大豆的种皮。尾孢菌会导致主要经济问题不仅仅是由于它们的广泛分布和广泛的宿主范围,还因为许多宿主中未发现有对该疾病的天然抗性。
这类病原菌具有很强致病性的原因之一是它们会产生尾孢菌素(cercosporin),这是一种芘醌植物毒素,也是一种光敏剂。尾孢属的很多个种均产生尾孢菌素,它对植物有几乎普遍的毒性。尾孢菌素还对小鼠、细菌和许多真菌有毒性。见R.B.Batcharova等,植物病理学,82卷,642-46页(1992);C.Balis和M.G.Payne,植物病理学,61卷1477-84页(1971);M.E.Daub植物病理学77卷,1515-20页(1987);A.O.Fajola,Physiol.Plant.Pathol.,13卷,157-64页(1978);S.Yamazaki等,农业生物化学,39卷287-88页(1975)。尾孢菌素还显示可灭活蛋白激酶C,对人类肿瘤细胞有细胞毒性。见T.Tamaoki和H.Nakano,生物/技术,8,732-35页(1990)。这些发现说明尾孢菌素对细胞有几乎普遍的毒性,对它的抗性归结于极少数抗性机体内存在的活性防御机制。尾孢菌素的产生似乎是成功致病的关键,尾孢菌素合成有缺陷的真菌突变体不能寄生于它们的宿主植物内(R.G.Upchurch等,应用环境生物学(Appl.Envir.Biol.),57卷,2940-45页(1991))。
对尾孢菌感染比较敏感的宿主而言,光线的存在对病症的发展极为重要,高光强度下感染植物的症状发展受促进。见,如M.E.Daub和M.Ehrenshaft,植物生理学,89卷,227-36页(1993);L.Calpouzos,植物病理学年鉴,4卷,369至390页(1967)。故尾孢菌素作为光敏剂的作用与其在细胞内致毒性和损伤的能力有关。尽管尾孢菌素是第一个由植物病原体合成的被认为是光敏剂的毒素,但很多其他植物致病性真菌也产生芘醌毒素及其它光敏剂化合物。如此众多的植物病原体产生光活化芘醌,这说明光敏作用可能是比以往所认识到的更为常见的植物致病因子。
事实上,唯一显示抗尾孢菌素的生物是尾孢菌自身及产生相似毒素的一些相关真菌。经诱变及对培养的细胞进行选择以期获得抗性植物和真菌的尝试尚未成功。M.Ehrenshaft等(植物病理学,86卷,S11(摘要93A)(1996增刊))曾报道用野生型烟草尾孢的基因组文库分离出与对尾孢菌素敏感的两类烟草尾孢突变体互补的粘粒克隆。用其中一个克隆转化突变体恢复了它们对尾孢菌素及其它光敏剂的野生型抗性。但该报道中并未描述抗光敏剂的具体基因的分离及测序。
故大家极希望能对编码抗诸如尾孢菌素之类光敏剂的基因进行分离。而且还希望能提供对由尾孢菌及其它产光敏剂的致病性真菌引起的疾病具有抗性的植物和其他生物。
发明概要
本发明的第一方面是一种分离的核酸分子,它经表达后可提供或增强细胞对光敏剂的抗性。
本发明的更进一方面是一种分离的核酸分子,它经表达后可提供或增加细胞对尾孢菌素的抗性。
本发明的更进一方面是一种含有嵌合基因的转化细胞,该基因包含与启动子可操作连接的本发明核酸分子。
本发明更进一方面是增加细胞对光敏剂抗性的方法。包括用本发明的核酸分子转化该细胞。
本发明更进一方面是增加细胞对病原体抗性的方法,包括用本发明的核酸转化该细胞。
本发明更进一方面是增加细胞对单线态氧的抗性的方法,包括用本发明的核酸转化该细胞。
本发明更进一方面是包含一嵌合基因的表达盒,该基因包含与启动子可操作连接的本发明核酸分子。
本发明更进一方面是一种转化植物,其中包含本发明的表达盒或上述嵌合基因。
本发明更进一方面是增加植物对光敏剂的抗性的方法,包括用本发明的表达盒转化该植物。
本发明更进一方面是增加植物对真菌病原体所致感染的抗性的方法,包括用本发明的表达盒转化该植物。
本发明更进一方面是用包含本发明核酸分子的转化构建体选择转化的真菌或植物细胞的方法。
附图简述
图1示编码尾孢菌素抗性的烟草尾孢基因sor 1的核苷酸序列(SEQID NO:1)。
图2示由烟草尾孢基因sor 1编码、可提供尾孢菌素抗性的多肽的氨基酸序列(SEQ ID NO:2)。
图3A示分离自链格孢的与烟草尾孢基因sor 1同源的部分核苷酸序列(SEQ ID NO:3)。
图3B示由图3A的链格孢核酸序列所编码的蛋白质的预计部分氨基酸序列(SEQ ID NO:4)。该预计氨基酸序列表示的是链格孢基因编码的蛋白质的C末端。
图4A示链格孢基因的部分核苷酸序列(SEQ ID NO:3)与烟草尾孢基因sor 1片段(SEQ ID NO:5)之间的对比。“AA1T7”指链格孢基因序列,而“CSG”指烟草尾孢sor 1基因片段序列。两序列间的符号“=”指并列残基相同。
图4B示链格孢基因的预计C末端氨基酸序列(SEQ ID NO:6)与烟草尾孢sor 1基因片段(SEQ ID NO:7)之间的对比。“AAHOMOLOG”表示链格孢基因某部分的氨基酸序列;“MAYBE2”表示烟草尾孢基因sor 1片段的氨基酸序列。“=”号指并列残基相同,而“-”指并列残基相似。“相似”的残基如下:A、S和T相互间视为相似;D与E相似;N与Q相似;R与K相似;I、L、 M和V相似;F、Y和W相似。
图4C示链格孢基因产物的预计氨基酸序列(SEQ ID NO:11)与烟草尾孢sor 1基因产物(SEQ ID NO:2)间的对比。“=”号指并列残基相同,“-”号指并列残基相似。“相似的”残基如上述。
发明详述
以下将参考附图对本发明作更详尽描述,附图中显示了本发明的某些实施方案。但本发明的实施可采用多种不同形式,并不应为本文所示的实施方案所限;提供这些实施方案只是为了使本文更全面和完整,并向本领域的技术人员全面显示本发明的范围。
本文只给出单链的核苷酸序列,从左至右为5’至3’方向。本文的氨基酸序列,除非另有说明,从左至右均为氨基至羧基方向。序列中并不给出氨基和羧基。本文的核苷酸和氨基酸均按IUPAC-IUB生化命名委员会所建议的方式表示。
本文中,术语“蛋白质”和“多肽”可互换使用,指以肽键相连的氨基酸多聚体(二肽或更大)。故“多肽”包括蛋白质、寡肽、蛋白质片段、蛋白质类似物等。术语“多肽”包括上文所限定的、由核酸编码的、重组产生的、分离自适当来源的或合成的多肽。
本文术语“光敏剂”指光活化的化合物,当有光时,它与氧反应产生能毁坏或毒害细胞的化合物(如活性氧之类)。光敏剂是一组结构多样的化合物,共同具有使细胞对光敏感的能力。更具体地说,光敏剂吸收光能,转变成活性态,在该活性态可通过还原性底物的基团反应(I型反应)或直接经能量转移机制(II型反应)而与氧反应。J.D.Spikes,光生物学,第二版,79-110页(K.C.Smith编,Plenum出版,纽约1989)。I型反应导致产生各种反应性种类,包括多种氧基如超氧化物(O2 -)、过氧化氢(H2O2),和氢氧基(OH-)。II型反应导致产生最具反应性和毒性的氧种类之一,即氧的活性单线态(单线态氧或1O2)。细胞暴露于光敏剂和光线可导致对关键细胞成分如脂、蛋白质和DNA造成破坏,并经常会导致细胞死亡。在本发明范围之内,术语“光敏剂”特包括可与氧通过I型和II型途径发生反应的化合物,及在暴露于光并与氧反应时会产生氧的基团形式和氧的活性单线态形式的化合物。
本发明所限定的光敏剂化合物可以是或不是由致病性真菌产生的。由致病性真菌产生的产单线态氧的光敏剂一般是芘醌毒素,包括但不限于尾孢菌素(由各类尾孢菌如烟草尾孢、菊池尾孢、稻尾孢产生的);痂囊腔菌色素(elsinochromes)(如由痂囊腔菌和痂圆孢产生的痂囊腔菌色素);phleichrome(分离自感染有瓜枝孢的黄瓜,及分离自Cl.phlei和扁豆枝孢);竹黄色素(shiraiachromes)(分离自竹子的病原体竹黄);亚肉座菌素(hypocrellin)[分离自如竹子的病原体竹亚肉座菌(Hypocrellabambusae)];格孢壁菌素、格孢毒素(altertoxin)I、II和III,及alterlosinI和II(分离自链格孢属各个种);匍柄毒素III(分离自链格孢属各个种及匍柄霉)以及由单隔孢属产生的芘醌毒素。见如J.C.Overseem和A.K.Sijpesteijn,植物化学,卷6,99-105页(1967);D.J.Robeson和M.A.F.Jalal,生物科学、生物技术与生物化学,56卷,949-52页(1992);H.Wu等,国产产品杂志(J.Nat.Products),5卷,948-51页(1989);V.M.Davis和M.E.Stack,应用与环境微生物学(Appl.Environ.Microbiol),55卷,7-14页(1991)。本发明所限定的其它光敏剂包括玫瑰红、血卟啉、伊红Y、亚甲蓝和甲苯胺蓝等化合物。
本发明描绘了提供或增强光敏剂敏感型生物对光敏剂的细胞耐受性的组合物和方法。组合物为蛋白质及其编码基因,可提供或增强这类生物对光敏剂的细胞耐受性。这些抗光敏剂的蛋白质及其编码基因、本文所述应用这些化合物的方法,均可用于提供针对产光敏剂的病原体的细胞抗性,使这些细胞存活,尤其是在病原体攻击后。这些同样组合物和方法还可提供或增强对单线态氧自身的抗性。
本发明的一个方面所描绘的蛋白质涉及在细胞或生物体内提供对光敏剂的抗性。本发明的光敏剂抗性蛋白质包括新一类真菌蛋白。分离自烟草尾孢的光敏剂抗性蛋白的氨基酸序列示于图2,也示于SEQ ID NO:2中。然而,这些蛋白质在真菌和其它生物中是保守的。故还如下文所讨论,有一些方法可用于从任意生物鉴定并分离基因和蛋白质。同样,序列相似性可用于鉴定和分离编码光敏剂抗性的其它基因和蛋白质。这些蛋白质的功能是抑制由编码光敏剂的致病性真菌所致感染的扩散,并控制许多生物体(包括植物、细菌、昆虫和动物体)对这类化合物的抗性。因此,这些蛋白质应用广泛,包括在植物及其它生物中控制疾病和毒性抗性。
本发明亦包括对这类蛋白质的修饰。这些修饰包括氨基酸残基的取代、缺失、添加等。相应的,本发明的蛋白质包括天然存在的真菌蛋白及其修饰物。
本发明还提供了编码该新型蛋白质的核苷酸序列。烟草尾孢的sor 1基因列于图1,也示于SEQ ID NO:1。该基因编码烟草尾孢新蛋白,它能赋予针对多种光敏剂(包括尾孢菌素)的抗性。烟草尾孢的sor 1基因可用于从其它生物分离同源基因。例如,图2提供了与烟草尾孢sor 1有显著序列同源性的链格孢基因核苷酸序列,该链格孢基因就是用sor 1基因分离的。
本发明的核酸和蛋白质可用于防止或增加细胞和生物对光敏剂的抗性。蛋白质尤其可用于保护生物体抵抗病原体的感染。在此方式中,该生物体转化有编码该蛋白质的核苷酸序列。根据本发明,如此转化的生物可以是植物、细菌、真菌和动物,较优选植物。该蛋白质在生物体内的表达可防止由光敏剂引起的毒性和损伤,并提供或增加对真菌性病原体感染的抗性。
尽管本发明的化合物和方法均可用于提供针对任何产生光敏剂的真菌性病原体感染的抗性,但本发明特别可用于提供针对由超过2000种的尾孢菌所致感染及由它们所产生的光敏剂的抗性。这组真菌病原体包括但不限于:落花生尾孢(感染花生),C.ariminiensis,石刁柏尾孢(感染芦笋),C.bertoreae,甜菜生尾孢(感染甜菜),C.bizzozeriana,芸苔生尾孢(感染芸苔,如卷心菜、油菜),变灰尾孢(感染如大豆、西红柿),胡萝卜尾孢(感染胡萝卜),C.chenopodii,C.cistineareum,芽孢状尾孢,C.diazu,C.erysimi,C.hayli,菊池尾孢(感染大豆),C.longpipes(感染甘蔗),锦葵生尾孢,苜蓿尾孢(C.medicaginus)(感染三叶草),烟草尾孢(感染烟草),稻尾孢(感染大米),球座尾孢,C.plantaginis,葛麻尾孢,粟尾孢,密梗尾孢,堇菜尾孢和玉米尾孢(感染玉米)。
本领域有简易可行的核酸序列杂交方法。来自较大范围物种的光敏剂编码序列可根据已知技术基于它们与本文所列编码序列(如sor 1)的序列同源性而分离。在这些技术中,利用已知编码序列的所有或部分作为探针,它可与所选生物的克隆基因组DNA片段或cDNA片段群体(即基因组或cDNA文库中)存在的其它光敏剂抗性编码序列选择性杂交。
例如,SEQ ID NO:1的完整序列或其部分有可能用作能与相应编码序列和信使RNA特异性杂交的探针。为在各种条件下获得特异性杂交,这类探针中包含编码光敏剂抗性的序列(下文“抗性编码序列”)中的独特序列,它们较优选至少长约10个核苷酸,最优选至少长约20个核苷酸。这类探针可用于经众所周知的聚合酶链式反应(PCR)程序从所选生物体中扩增抗性编码序列。该技术可用于从目的生物体中分离另外的抗性编码序列,或作为诊断试验来测定生物体中抗性编码序列的存在。
这些技术包括杂交筛选平板上的DNA文库(噬斑或菌落;见,如,Sambrook等,分子克隆,编辑,冷泉港实验室出版社(1989)),以及用相应于氨基酸序列内保守序列区的寡核苷酸引物经PCR的扩增(见,如,Innis等,PCR方案,方法和应用指南,编辑,Academic Press(1990))。
例如,这些序列可在弱严紧度、中度严紧度、甚至高严紧度条件下(如,洗涤严紧度分别为下述的几种条件:35%-40%甲酰胺加5×Denhardt’s溶液,0.5% SDS及1×SSPE,于37℃;40-45%甲酰胺加5×Denhardt’s溶液,0.5%SDS,1×SSPE,于42℃;50%甲酰胺加5×Denhardt’s溶液,0.5%SDS,1×SSPE,于42℃),对本文所公开的编码光敏剂抗性的DNA在标准杂交实验中进行杂交。见J.Sambrook等,分子克隆,实验室手册第二版(1989)冷泉港实验室。通常,编码光敏剂抗性蛋白、并可与本文示为SEQ ID NO:1的烟草尾孢基因杂交的序列与烟草尾孢序列应至少有50%、70%、甚至85%或更多同源。即序列间相似性可能至少约50%、约70%、以及甚至约85%、90%、95%或更多相似。
本发明还提供了烟草尾孢光敏剂抗性基因的突变形式,及它们编码的蛋白质。诱变及核苷酸转换之方法为本领域所熟知。见,如,Kunkel,T.(1985)美国国家科学院院报,82卷488-492页;Kunkel等(1987)酶学方法154卷367-382页;美国专利号4,873,192;Walker和Gaastra(编)分子生物学技术,MacMillan出版公司,NY(1983)及其中所引参考文献。故本发明的基因和核苷酸序列既包括天然存在的序列,又包括突变形式。同样,本发明的蛋白质既包括天然蛋白质,又包括其变异和修饰形式。
编码本发明蛋白质或多肽的核苷酸序列可用于对包括细菌、真菌、植物和动物在内的各种生物体进行遗传操作。本发明的这一方面在本文中针对植物的遗传操作进行了说明。在此方式中,本发明的核苷酸序列提供于表达盒中,以便在感兴趣的植物中表达。此盒包括与目的基因可操作连接的5’和3’调节序列。术语“可操作连接”在此指单个DNA分子上的各DNA序列连接在一起,使得其中之一的功能被另一个影响。这样,当启动子能影响本发明基因的表达(即该基因受该启动子的转录控制)时,它即与本发明基因可操作连接。该启动子在该基因“上游”,反过来说,该基因在该启动子“下游”。
按转录的5’至3’方向,本发明的表达盒包括上述启动子、与启动子可操作连接的本发明基因,并可选择地包括含RNA聚合酶终止信号和多聚腺苷酸酶的多聚腺苷酸化信号的终止序列(如nos终止子)。所有这些调节区应能在将被转化组织的细胞中起作用。3’终止区可衍生自与转录起始区来源相同的基因或衍自不同基因。
该盒中可另含至少一个其它基因,以共转化入生物体中。或,这个(些)目的基因可提供在另一个表达盒上。适当时优化这个(些)基因,使其在转化植物中的表达增加。
表达盒构建体中,表达盒可另含5’前导序列。这类前导序列可增强翻译。翻译前导区为本领域所熟知,包括小RNA病毒前导区,如EMCV前导区(脑心肌炎病毒5’非编码区)(Elroy-Stein,O.,Fuerst,T.R.,和Moss,B.(1989)美国国家科学院院刊PNAS USA,86卷6126-6130页)马铃薯丫病毒组前导区,如TEV[烟草蚀刻病毒(Tobacco Etch Virus)]前导区(Allison等(1986);MDMV[玉米矮缩花叶病毒(Maize DwarfMosaic Virus)]前导区(病毒学154卷9-20页);及人免疫球蛋白重链结合蛋白(Bip)(Macejak,D.G.,和P.Sarnow(1991)自然,353卷90-94页);苜蓿花叶病毒衣壳蛋白mRNA的非翻译前导区(AMV RNA 4)(Jobling,S.A.和Gehrke,L.,(1987)自然,325卷:622至625页);烟草花叶病毒(TMV)前导区(Gallie,D.R.等(1989)RNA的分子生物学,237-256页),玉米褪绿斑驳病毒(maize chlorotic mottle Virus,MCMV)前导区(Lommel,S.A.等(1991)病毒学,81卷382-385页)。亦见Della-lioppa等(1987)植物生理学,84卷,965-968页。也可利用其他已知能增强翻译的方法,如内含子等。
制备表达盒时,可对各个DNA片段进行操作,以使DNA序列按正确方向排列,适当时以正确阅读框排列。为此,需用衔接子或接头连接各DNA片段,或可经其它操作提供方便的限制性位点,去除多余DNA,去除限制性位点等。为此,可应用体外诱变、引物修复、限制酶切退火、切除、连接、PCR等等,其中可能涉及插入、缺失或替代,如转换和颠换。
本发明的组合物和方法可用于转化任何植物或其任意部分。这样可获得遗传修饰的植物、植物细胞、植物组织(如植物叶、茎、根)、种子、种皮等。转化程序根据要转化的植物或植物细胞类型(即单子叶植物或双子叶植物)变化。适于转化植物细胞的方法包括显微注射[Crossway等(1986)生物技术(Biotechniques),4卷:320-334页],电穿孔[Riggs等(1986)美国国家科学院院报83卷:5602-5606页],农杆菌介导的转化[Hinchee等(1988)生物技术学(Biotechnology),6卷915-921页],直接基因转换[Paszkowski等(1984)EMBOJ.,3卷2717-2722页];弹道粒子加速法(ballistic particle acceleration)[见,如,Sanford等,美国专利号4,945,050;WO 91/10725和McCabe等(1988)生物技术学,6卷923-926页]。亦见Weissinger等(1988)遗传年鉴,22卷421-477页;Sanford等(1987)粒子科学与技术(Particulate Science andTechnology),5卷,27-37页(洋葱);Christou等(1988)植物生理学,87卷671-674页(大豆);McCabe等(1988)生物/技术(Bio/Technology),6卷923-926页(大豆);Datta等(1990)生物技术学,8卷:736-740页(大米);Klein等(1988)美国国家科学院院报,85卷:4305-4309(玉米);Klein等(1988)生物技术学,6卷;559-563页(玉米);WO91/10725(玉米);Klein等(1988)植物生理学,91卷:440-444页(玉米);Fromm等(1990)生物技术学,8卷:833-839页;Gordon-Kamm等(1990)植物细胞,2卷:603-618页(玉米);Hooydaas-Van Slogteren & Hooykaas(1984)自然(伦敦),311卷;763-764页;Bytebier等(1987)美国国家科学院院报,84卷:5345-5349页(百合科);De Wet等(1985)“胚珠组织的实验操作”一书中,G.P.Chapman等编,197-209页,Longman出版,NY(花粉);Kaeppler等(1990),植物细胞报道,9卷415-418页;Kaeppler等(1992)理论及应用遗传学,84卷:560-566页(颈须介导的转化);D.Halluin等(1992)植物细胞,4卷:1495-1505(电穿孔);Li等(1993)植物细胞报道,12卷:250-255页和Christou和Ford(1995)植物学年鉴(Annalsof Botany),75卷:407-413(大米);Osjoda等(1996)自然生物技术学,14卷:745-750页(玉米经根瘤农杆菌感染);以上所有资料均引入本文作为参考。
可用本发明的DNA构建体,按本领域熟知的程序,通过DNA介导的植物细胞原生质体转化及随后从转化的原生质体使植物再生,从而对植物进行转化。
能经器官生成或经胚胎发生进行后面的无性繁殖的任何植物组织均可用本发明的载体转化。本文中“器官生成”指从分生组织中心依次发育出茎干和根的过程;“胚胎发生”指茎干和根以协同形式(非依次)一起发育时的过程,不管是从体细胞发育还是从配子发育。所选具体组织根据所用无性繁殖不同而不同,并应适合于被转化的特定物种。组织靶的实例有叶盘、花粉、胚芽、子叶、下胚轴、大配子体、愈伤组织、已存在的分生组织(如顶端分生组织,腋芽,根分生组织)及诱导的分生组织(如子叶分生组织和下胚轴分生组织)。
本发明的植物可能形式多样,可以是转化细胞与未转化细胞的嵌合体;可以是无性转化体(如所有细胞均转化有表达盒);可包含转化和未转化组织的移植物(如转化的根状茎移植至柑桔的未转化幼芽上)。
有可能用于实践本发明的植物包括(但不限于)烟草(Nicotianatabacum),马铃薯(Solanum tuberosum),大豆(glycine max),花生(Arachis hypogaea),芸苔属各种(如油菜,canola),高粱(Sorghumbicolor),棉花(Gossypium hirsutum),甘薯(Ipomoea batatus),木薯(Manihot esculenta),咖啡(咖啡属各种),椰子(Cocos nucifera),菠萝(Ananas comosus),柑橘树(柑橘属各种),可可树(Theobroma cacao),茶树(Camellia sinensis),香蕉(芭蕉属各种),鳄梨(Persea americana),无花果(Ficus casica),番石榴(Psidium guajava),芒果(Mangifera indica),橄榄(Olea europaea),番木瓜(Carica papaya),腰果(Anacardiumoccidentale),澳大利亚坚果(Macadamia integrifolia),杏树(Prunusamygdalus),甜菜(Beta vulgaris),玉米(Zea mays),小麦,燕麦,黑麦,大麦,大米,蔬菜,观赏植物和针叶树。蔬菜包括西红柿(Lycopersiconesculentum),胡萝卜,芦笋,莴苣(如Lactuea sativa),青豆(Phaseolusvulgaris),赖马豆(Phaseolus limensis),豌豆(香豌豆属各种),以及黄瓜属的成员,如黄瓜(C.sativus),香瓜(C.cantalupensis),和甜瓜(C.melo)。装饰植物包括杜鹃花(杜鹃花属各种),八仙花(Macrophylla hydrangea),木槿(朱槿),蔷薇(蔷薇属各种),郁金香(郁金香属各种),黄水仙(水仙属各种),牵牛花(Petunia hybrida),康乃馨(dianthus caryophyllus),一品红(Euphorbia pulcherima)和菊花(chyrsanthemum)。可用于实施本发明的针叶树包括例如松树,如火炬松(Pinus taeda),湿地松(Pinus elliotii),西黄松(Pinus ponderosa),美国黑松(Pinus contorta),和辐射松(Pinusradiata);花旗松(Pseudotsuga menziesii);西部铁杉(加拿大铁杉);北美云杉(白云杉);红木(红杉);冷杉,如银杉(温哥华冷杉)和香脂冷杉(Abiesbalsamea);以及雪松,如崖柏(北美香柏)和阿拉斯加柏木(黄扁柏)。
转化细胞可按常规方式培养成植株。见,如,McCormick等(1986)植物细胞报道,5卷:81-84页。然后可培养这些植物,并与同转化植物相同品系或不同品系的植株授粉,所得杂合体有预期的表型鉴定特征。可培养两代或更多代,以确保所要表型特征稳定维持并遗传,然后收获种子以确保获得所要表型或其它特性。
当编码光敏剂抗性的基因包含在表达盒内时,该基因可与能用于一个或多个宿主的一个标记基因或用于不同宿主个体的不同标记结合使用。即,一个标记可用于在原核细胞宿主中进行选择,另一个标记可用于在真核宿主,尤其植物宿主中进行选择。这些标记可以是对生物杀伤剂(如抗生素、毒素、重金属等)的保护作用;可通过赋予异养型宿主原养型特征而提供的互补性;或通过在植物中产生新化合物而提供可见表型。可利用的基因实例有新霉素磷酸转移酶(NPTII),潮霉素磷酸转移酶(HPT),氯霉素乙酰转移酶(CAT),腈水解酶和庆大霉素抗性基因。选择植物宿主时,适宜标记的非限制性例子有β-葡糖醛酸糖苷酶(产生靛蓝),荧光素酶(产生可见光),NPTII(提供卡那霉素抗性或G418抗性),HPT(提供潮霉素抗性),突变的aroA基因(提供草甘磷抗性)。可选择标记基因及报道基因为本领域熟知。总见,G.T.Yarranton(1992),当代生物技术观点(Curr.Opin,Biotech)3卷;506-511页;Christopherson等(1992),美国国家科学院院报,89卷,6314-6318页;Yao等(1992)细胞,71卷:63-72页;W.S.Reznikoff(1992)分子微生物学,6卷:2419-2422页:Barkley等(1980)操纵子,177-220页;Hu等(1987)细胞,48卷:555-566页;Brown等(1987)细胞,49卷:603-612页;Figge等(1988)细胞,52卷:713-722页;Deuschle等(1989)美国国家科学院院报86卷:5400-5404页。还包括其它感兴趣的基因。上述各基因可包含于单个表达盒中,或包含于不同盒中。盒的构建方法和转化方法已在上文有述。
正如所讨论的,可对本发明的基因进行操作以增强植物的抗病性。该方式中,光敏剂抗性编码基因的表达或活性受到改变。改变基因的这类方法包括协同抑制、反义、诱变、改变蛋白质的亚细胞定位等。某些例子中,从诱导型启动子、尤其是从病原体诱导型启动子表达基因比较有利。这些启动子包括来自致病性相关蛋白(PR蛋白)的启动子,它们在受到病原体感染后被诱导;如PR蛋白、SAR蛋白、β-1,3-葡聚糖酶,壳多糖酶等。参见如Refolfi等(1983)荷兰植物病理学杂志(Neth.J.Plant Pathol.)89卷:245-254页;Uknes等(1992)植物细胞,4卷:645-656页;Van Loon(1985)植物分子病毒学,4卷;111-116页。
本发明的核苷酸序列也可用作转化植物或真菌细胞的可选择标记。吡哆醇缺陷型黄曲霉转化有SOR1后发现能在缺吡哆醇的基本培养基上生长。相应地,当需要用一个基因转化植物时,可将SOR 1基因用作标记基因与该基因组合。转化成功的标志是转化体可在缺乏吡哆醇的培养基上生长,而初始(未转化)植物不能生长。可选择标记的应用方法和构建为本领域所熟知。
在从经过了转化操作的许多细胞中只选择出已成功发生了转化的细胞的方法中使用本发明核苷酸序列时,应包括对细胞进行转化,所述细胞来自在缺乏外源性吡哆醇时(例如在不含吡哆醇的培养基中)不能生长(生长很差)的生物体。多种适用的转化过程为本领域所熟知;合适转化过程的选择取决于被转化的细胞和所希望得到的效果。合适转化过程的选择为本领域技术人员所显而易见。用含有目的异源DNA序列和根据本发明的另一DNA序列(如SEQ ID NO:1或其开放阅读框)的构建体对细胞进行转化。然后将细胞置于缺乏吡哆醇的培养基上;只有能在培养基上生长的细胞已成功发生了转化。
以下实施例将更全面阐述本发明,但并非限制本发明。
实施例1
尾孢菌素敏感型突变体的分离
依据描述于A.E.Jenns等,光化学与光生物学,61卷:488-493页(1995)和A.E.Jenns及M.E.Daub.植物病理学,85卷,96-912页(1995)的程序,分离出对尾孢菌素敏感的烟草尾孢突变体。突变体从紫外线诱变的菌丝体原生质体中分离,通过将集落影印到含尾孢菌素的培养基上而筛选尾孢菌素敏感性。所有分离及筛选过程均在抑制内源尾孢菌素合成的条件下完成[A.E.Jenns等,植物病理学,79卷,213-219页(1989))。分离到6个尾孢菌素敏感(CS)突变体,可分为两种表型类。5个突变体(CS2,CS6,CS7,CS8和CS9,命名为1类)在含低至1μM浓度的尾孢菌素培养基上生长受到完全抑制。第六个突变体(CS10,命名为2类)在尾孢菌素为10μM时部分受抑,但更低浓度下不被抑制。用荧光显微镜分析这些突变体时,发现1类突变体不能还原尾孢菌素。而部分敏感型CS10的尾孢菌素还原能力正常。
根据Jenns和Daub(文献同上)的方法进一步鉴定表型。所有突变体若在可诱导尾孢菌素合成的条件下生长时均能合成尾孢菌素。1类突变体在有尾孢菌素产生时完全不生长,而令人惊讶的是,内源尾孢菌素的产生似乎对CS10的生长几乎没影响。尾孢菌素敏感性不是由于任一突变体对光的一般敏感性。相对于其它敏感性真菌而言,突变体能通过加还原剂(如抗坏血酸、半胱氨酸和还原了的谷胱甘肽)而抵抗尾孢菌素的毒性。然而,无一突变体在这些化合物的产生方面或在总可溶性的或蛋白质硫醇基水平方面有改动,说明抗性与这些物质的内源生产无关。
还检验了这些突变体对另5种单线态氧产生型光敏剂的抗性:亚甲蓝,甲苯胺蓝,玫瑰红,伊红Y和血卟啉。烟草尾孢野生型对除玫瑰红(其对这些真菌有些毒性)以外的所有这些光敏剂有高抗性。令人惊讶的是,1类敏感型突变体受到所有这些光敏剂的彻底抑制。这一敏感性水平出乎意料,因为即使是对尾孢菌素最敏感的天然真菌在有这些化合物时也至少有一定程度的生长。故1类突变体似乎是在介导对一群单线态氧产生型光敏剂的抗性的基因内发生了突变,这种突变使得光敏剂敏感性达到了自然界真菌中并不存在的水平。相反,突变体CS10的敏感性只特异性针对尾孢菌素;CS10对其它光敏剂的反应与野生型完全相同。
实施例2
尾孢菌素抗性编码基因的分离
在已通过添加COS位点使得能克隆大的45kb插入片段而受到修饰、可赋予bialaphos抗性的质粒-pBAR3质粒(Straubinger等,真菌遗传学通讯(Fungal Genetics Newsletter),39卷,82-83页(1992)中构建烟草尾孢野生型菌株(ATCC#18366)的基因组文库。分离到约4,000个粘粒克隆,各个存放于微滴板的孔中。估计烟草尾孢单倍体基因组的大小等同于粗糙脉孢菌的大小(4×107),因此预计此文库代表作为完整片段的全部基因组的概率有99%。
用于转化的DNA制备方法如下,将每个粘粒克隆培养为5ml培养物,然后收集一个微滴板上的所有培养物。然后将所集中的DNA转化CS10和CS8。选CS10是因为它是唯一对尾孢菌素部分敏感、不受尾孢菌素还原的影响、并且不受其它光敏剂影响的突变体。CS8代表5个1类突变体(均对尾孢菌素和其它光敏剂完全敏感,亦不能还原尾孢菌素)。选CS8是因为缺尾孢菌素时它生长良好,孢子形成良好,并且是1类突变体中最易转化的。选择转化体对bialaphos的抗性,然后将每板DNA转化所得的300-400个转化体培养于含10μM尾孢菌素的培养基上进行尾孢菌素抗性筛选。
鉴定出弥补两个突变的克隆。从用所选某平板的DNA转化的尾孢菌素部分敏感型CS10中回收4个显示野生型抗性水平的菌落。用该平板的各单个克隆的DNA转化CS10,鉴定出了一个特异性的可弥补突变的粘粒克隆,命名为30Hz。用30Hz克隆转化的CS10菌落中42%表现的尾孢菌素抗性表型与野生型的无差别。
从另选DNA转化所得的11个CS8克隆表现野生型水平的尾孢菌素抗性。鉴定出一个特异性的可弥补突变的粘粒克隆,命名为18E1。用18E1转化的CS8克隆中78%显示野生型水平的尾孢菌素抗性。18E1克隆还恢复了CS8对其它5种单线态氧产生型光敏剂(甲苯胺蓝、亚甲蓝、伊红丫,血卟啉和玫瑰红)的野生型抗性水平。
所有的1类突变体(CS2,CS6,CS7,CS8和CS9)有相同表型。1类突变体的表型说明能弥补突变的抗性基因只特异性赋予对1O2的抗性。由于CS10的表型不同于1类突变体,故认为它们的突变在不同位点。这一假说受到转化实验的支持。用克隆30Hz转化CS8不能恢复任何水平的尾孢菌素抗性。相似地,18E1克隆不能弥补CS10的突变。
实施例3
尾孢菌素抗性编码基因的序列分析
由于弥补CS8和CS10的粘粒每个均含有超过40kb的烟草尾孢DNA,故对重叠的限制性片段亚克隆以鉴别赋予尾孢菌素抗性的较短DNA序列(5-10kb)。对来自每个粘粒的互补亚克隆进行制图,并利用一段公共限制性片段作为探针进行Northern分析,以确定它们与一个信号杂交。这些探针随后用于鉴定相应于18E1和30Hz中提供光敏剂抗性的基因的全长cDNA。
对基因组片段和cDNA均进行测序。采用引物步行技术,通过以前用于烟草尾孢八氢番茄红素脱氢酶基因测序的方法[M.Ehrenshaft和M.E.Daub.应用与环境微生物学,60卷,2766-2771页(1994)],产生系列缺失体以供测序)。缺失克隆的测序在佐治亚大学分子遗传学研究所(Athens,Georgia,USA)完成。
对位于18E1克隆内、为烟草尾孢提供尾孢菌素抗性的基因(该基因命名为sor1)进行了测序。sor 1的测序分析揭示其中有一个编码343个氨基酸残基的蛋白质的开放读码框。该基因的核苷酸序列按从5’至3’的方向在本文中示为SEQ ID NO:1(见图1)。SEQ ID NO:1的开放读码框为其内部825-1853位核苷酸。
由该分离sor 1基因表达的、编码尾孢菌素抗性的蛋白质,其预期氨基酸序列按氨基末端至羧基末端的方向示为如下:MASNGTSVSP FRSQKNAAMA VNDTPANGHA EPSTITAASK TNTTKITSQN DPQSSFAVKV GLAQMLKGGVIMDVVNAEQA RIAEEAGACA VMALERVPAD IRKDGGVARM SDPQMIKDIM NAVTIPVMAK SRIGHFVECQILQAIGVDYI DESEVLTPAD PVNHIDKSVY NVPFVCGCKN LGEALRRISE GAAMIRTKGE AGTGDVVEAVRHMQTVNAEI AKASSASDAD LRMMARELQC DYNLLKQTAQ LKRLPVVNFA AGGIATPADA ALMMQMGCDGVFVGSGIFKS GDAAKRAKAI VQATTHYNDP KVLAEVSSGL GEAMVGINCD KLPETQKLAT RGW(SEQ ID NO:2)
实施例3A
对位于30Hz克隆内、也为烟草尾孢提供尾孢菌素抗性的基因(命名为crg1)进行了测序;该基因的核苷酸序列按5’至3’方向提供为本文的SEQ ID NO:9。对crg1的测序分析揭示出一个编码550个氨基酸残基的蛋白质的开放读码框(SEQ ID NO:9内的742-2391位核苷酸)。由该分离crg 1基因所表达的编码尾孢菌素抗性的蛋白质,其预期氨基酸序列按氨基端至羧基端方向提供在SEQ ID NO:10中。
实施例4
尾孢菌素抗性编码基因特性的确证
生成缺失SOR1基因的尾孢突变体(无效突变体)。这些突变体与初始突变体一样对尾孢菌素和其他光敏剂均敏感,这样就确证了SOR1的功能。
对cDNA和基因组克隆经制图并测序鉴定出特异性尾孢菌素抗性基因后,在基因破裂实验中应用互补基因以确证它们的同一性。之前用这种方法产生了烟草尾孢的类胡萝卜素缺陷突变体(Ehrenshaft等,Molec.Plant.Microb.Interact.,8卷569-575页(1995))。野生型烟草尾孢菌株ATCC#18366用sor 1抗性基因的破裂形式转化。Southern分析鉴别出两种转化体,一种内破裂基因取代了野生型的相应基因,另一种既含有野生型基因又含有破裂基因。筛选丧失或降低了尾孢菌素抗性的转化体,及丧失或降低了对前面Jenns等(1995,文献同上),Ehrenshaft等(1995,文献同上)所述其它单线态氧产生型光敏剂的抗性的转化体。
上述方法产生的无效突变体对尾孢菌素和对其它光敏剂完全敏感。这些结果证实SOR1功能是提供对尾孢菌素及其它单线态氧产生型光敏剂的抗性。
利用与上文所述相同的方法证实CRG 1基因的功能。
实施例5
抗性基因在黄曲霉中的表达
1类突变体的表型说明抗性基因互补赋予了单线态氧抗性。若果真如此,该基因(有或无CS10互补基因)可用于对其它生物进行基因加工,使其具有光敏剂或单线态氧抗性。
用对尾孢菌素敏感的真菌黄曲霉检验CS8和CS10互补基因(单个或一起)为异源生物赋予抗性的能力。
故将本发明的尾孢菌素抗性基因在黄曲霉中表达。选择黄曲霉来表达本发明的基因是因为它对尾孢菌素高度敏感。M.E.Daub等,美国国家科学院院报,89卷,9588-9592(1992)。黄曲霉菌株656-2是由GaryPayne博士慷慨提供(北卡罗来纳州立大学植物病理学系,Raleigh,NorthCarolina)的,用该基因按C.P.Woloshuk等[应用与环境微生物学,55卷,86-90页(1989)]的方法转化该菌株。简述为,将黄曲霉菌株656-2(一种需尿苷的突变体)的菌丝体温育在酶溶液(10mM NaPO4,pH5.8,20mM CaCl2,105U/ml β-glucorinidase,Novozym 234,1.2M NaCl)中分离出原生质体。将尾孢菌素抗性基因sor 1克隆至质粒pUC19pyr4中。该质粒含有粗糙脉孢菌基因pyr4,可恢复其在无尿苷情况下的生长能力。将原生质体与质粒DNA混合,铺板于缺尿苷的再生培养基(MLS)上。将平板上长出来的转化体再接种于含μM尾孢菌素的马铃薯葡萄糖琼脂上,于光下培养,3天后测菌落直径。
另一方案是,将sor 1开放读码框克隆进质粒pBargpe1中曲霉gpdA基因启动子的下游。该启动子在黄曲霉中为组成型表达。作为转化进烟草尾孢的标记,该质粒包含bar基因,它编码对bialaphos的抗性。该质粒转化进入上述CS8突变体中,显示可与尾孢菌素敏感性突变互补。然后,如上述,将pyr4基因克隆入该质粒构建体中,以提供转化至曲霉菌株656-2中的标记。然后如上述检测出有尾孢菌素抗性的转化体。
实施例5A
抗性基因在黄曲霉中的表达
按实施例5的方法使SOR 1在黄曲霉中表达(从gpdA启动子的下游表达SOR 1 ORF)。表1提供了有关的gpdA:SOR 1转化体和三个只转化了载体的对照菌落的数据,其中说明黄曲霉的尾孢菌素抗性经转化SOR1基因构建体后可得到改善。还观察到有一种生长效应;SOR1转化体在无尾孢菌素时生长更旺盛。此效应可能归因于SOR1具有与吡哆醇突变互补的能力。所用的特殊黄曲霉为吡哆醇突变体,然而,所有菌落均在含吡哆醇的复合培养基上生长。
表1
黄曲霉gpdA:SOR1转化体的生长
菌株 | 5天时菌落直径毫米数(+10μm尾孢菌素) | 5天时菌落直径毫米数(未加尾孢菌素) |
SOR转化体#1 | 9 | 59 |
SOR转化体#2 | 7 | 54 |
SOR转化体#3 | 8 | 60 |
载体转化体#1 | 0 | 42 |
载体转化体#2 | 0 | 51 |
载体转化体#3 | 0 | 40 |
实施例6
分离链格孢中的sor1同系物
以烟草尾孢sor1基因为探针,在上述较低严紧条件下探查链格孢的基因组文库。分离出烟草尾孢sor1基因的链格孢同系物的全长基因,用已知技术对该基因测序(为SEQ ID NO:10)。该链格孢基因的部分DNA序列(310个碱基对)提供在图3A(SEQ ID NO:3)中;相应于该核苷酸序列所编码蛋白质的C末端的预期氨基酸序列提供在图3B(SEQ ID NO:4)中。将链格孢基因的该部分DNA序列与烟草尾孢sor1基因(SEQ ID NO:5)的相应片段在图4A中进行对比。图中,“=”号说明并列的残基相同。将链格孢基因的完整DNA序列(SEQ ID NO:10)与烟草尾孢sor 1基因(SEQ ID NO:1)以相似的方式(未显示)进行对比,可见序列间有78.2%相同。
图4B中显示两个基因的预计C末端氨基酸序列(SEQ ID NO:6为链格孢基因,SEQ ID NO:7为SOR1)的对比;每个基因的完整氨基酸序列(SEQ ID NO:2和SEQ ID NO:11)的对比显示于图4C中。图4B和4C中,“=”号表示并列的残基相同,而“-”号说明并列的残基相似。“相似”的残基如下:A、S和T视为互相相似;D和E相似;N和Q相似;R和K相似;I、L、M和V相似;F、Y和W相似。
对SOR 1的链格孢同系物的完整核苷酸序列进行测序(SEQ IDNO:10);该链格孢同系物的预计氨基酸序列提供在SEQ ID NO:11中。
实施例7
与其它已知基因和基因产物同源性的测定
利用生物技术研究所国家中心的BLAST网络服务器,分析衍生自上述核苷酸序列的蛋白质序列与其它蛋白质序列的同源性。(S.F.Altshul等,分子生物学杂志,215卷,403-410页(1990))。对数据资料进行检索,显示不同生物界中好几种蛋白质序列与SEQ ID NO:2的预期多肽有55-75%同源,见表2。这些已鉴定的同系物无一具有明确的功能。一种酵母同系物编码的蛋白质据报道在稳定期增多;说明该蛋白质可能是一种细胞停滞蛋白(Braun等,细菌学杂志,178卷:6865页(1996))。橡胶树内的该基因当有乙烯和水杨酸时上调(Sivasubramaniam等,植物分子生物学,29卷,173页(1995))。
表2
SOR1的同系物
生物 | 界 | 基因组序列* | SOR1? |
詹氏甲烷球菌 | 古细菌 | 完全 | 是 |
万尼氏甲烷球菌 | ″ | 是 | |
激烈热球菌 | ″ | est | 是 |
热自养甲烷杆菌 | ″ | 完全 | 是 |
闪烁古生球菌 | ″ | 完全 | 是 |
流感嗜血杆菌 | 真细菌 | 完全 | 是 |
枯草芽孢杆菌 | ″ | 完全 | 是 |
结核分枝杆菌 | ″ | 不完全 | 是 |
麻风分枝杆菌 | ″ | 是 | |
海栖热袍菌 | ″ | 不完全 | 是 |
耐放射异常球菌 | ″ | 不完全 | 是 |
酿酒酵母 | 真菌 | 完全 | 是/3个拷贝 |
粟酒裂殖酵母 | ″ | 是 | |
构巢曲霉 | ″ | est | 是 |
秀丽新杆线虫 | 动物 | 不完全 | 是 |
拟南芥属 | 植物 | est | 是 |
大米 | 植物 | est | 是 |
Stellaria longpipes | 植物 | 是 | |
橡胶树 | 植物 | 表达文库(乙烯) | 是 |
*完全=完整基因组均已测序
不完全=基因组测序工作正在进行中,尚未完成
est=表达的序列标记
也鉴定出了真细菌界中缺乏SOR1同系物的一些生物:大肠杆菌;集胞蓝细菌(Synechocysits);幽门螺杆菌;布氏疏螺旋体;肺炎枝原体;生殖道枝原体。这些生物和整个基因组均已测序。
本发明的很多修改方案和其它实施方案对于本发明所属领域的技术人员是很容易想到的,借助于前文所述及相关附图,它们也在本发明范围内。因此,应明白本发明不限于所公开的这些特殊实施方案。尽管用到专有名词,但只取其一般性及描述性涵意,不是为了作出限制。本发明的修饰方案和其它实施方案均包含在所附权利要求书的范围内。
实施例8
SOR1作为选择性标记的用途
发现SOR1突变体不能生长于基本培养基上,但当基本培养基中加有吡哆醇时可生长。SOR1与迄今鉴别出的在吡哆醇生物合成途径中起作用的任何基因未见有明显的序列相似性。SOR1突变体对尾孢菌素敏感,甚至在含有吡哆醇的培养基上也如此,说明吡哆醇并非尾孢菌素抗性所必需。
用上述实施例5中的程序转化黄曲霉(ATCC #60045)的吡哆醇缺陷型突变体。实施例5所述转化和选择用到pyr4基因,且依据在无尿苷情况下的生长能力筛选转化体。本实施方案中,用处于构巢曲霉甘油醛-3-磷酸脱氢酶基因启动子(gpdA启动子)控制之下的SOR1 ORF的构建体转化原生质体。已知gpdA启动子在黄曲霉中能提供较高的组成型表达。在缺乏吡哆醇的基本培养基上选择转化体。未转化的原生质体和转化有无SOR1的载体质粒的原生质体未见生长;表达SOR1的原生质体有生长。尽管未做并列比较,但转化频率似乎近似于用pyr4标记得到的转化频率。
序列表(1)一般资料:
(i)申请人:Daub,Margaret E.
Ehrenshaft,Marilyn
Jenns,Ann E.
(ii)发明题目:编码光敏剂抗性的分离基因和蛋白质
(iii)序列数:11
(iv)通讯地址:
(A)收件人:Virginia C.Bennett
(B)街名:PO Box 37428
(C)城市:Raleigh
(D)州名:North Carolina
(E)国家:US
(F)邮编:27627
(v)计算机可读形式:
(A)介质类型:软盘
(B)计算机:IBM PC可兼容机
(C)操作系统:PC-DOS/MS-DOS
(D)软件:PatentIn Release #1.0,Version #1.30
(vi)当前申请资料:
(A)申请号:
(B)申请日:
(C)分类:
(viii)代理人/代理机构资料:
(A)姓名:Bennett,Virginia C.
(B)登记号:37,092
(C)参考/文档号:5405.333
(ix)电信资料:
(A)电话:919-854-1400
(B)电传:919-854-1401(2)SEQ ID NO:1的资料
(i)序列特征
(A)长度:2010个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(ix)特征:
(A)名称/关键词:CDS
(B)位置:825..1853
(xi)序列描述:SEQ ID NO:1GGATCCAAAA TTGGGCCATG TTGTGCAGAG TGCGGTCTGG GAGGGTAGAG TGTTTTCGGT60GTCAAGCTTC ACTCACGCAG GAGGTCAAAG TCTACACAGA AGACCTCATC GAGCAGCTGA120GCTCTCTTAT TGCGATTACA GCTCAGCACT CGGCCGAGGA ACGCGTGGAT TCCTGGTTCC180CCTCATGGGC ATTGTAGCTT CAGCAGACTC GTCCGCCGCG ACTCTGCTCG CGTATGAGTA240GCTCGTGCTG ACCAGCAATC GCGGAGCTGA GCTGCAGTGC TCGCATCGCA GTCACAGAAG300GATGACCACC GTCTTGTAGC GCGGCGCACG CGGGCCAGCA CCTGAGGTCG GAGGGTTGGC360ACATGGACGG ACGAGCTGGT GGTTGTCACA TGCCGGCTCA GTCGCGGCAC TCAGCAGGCA420GGGAAGGCGC CGTCGGACCC TGCAGCACGT CTCGGCTCCT TGGCCGAAGC ACGATCTTCC480CCGCGATCGC AGGCGCAAAT GACTCTTCGA ACATTTTCTC GCCGCATCTG GCCGCTGTCA540GAGGCAAGTC TCGCACCGTG TCGCGCCCTA CCAGACACAA GCACCCTCCT GTTCCAGTGC600TCGCCAAAGC ATTGCCGCAT CGAGCTTCCT TTCGCGACCA TTGCCTGCCC TCCGAGCCCA660GCATATAGAC TTTCCTAGTT CCGCCGATTT TCTTTCCAAG TGCCACCACC TCAATATCGC720CTTCGACTTT CTTTCACTGC TGCCCCGCCC TGCATCTGCA CGCCCCACCG CCATTGACCA780AATATAACAT CCTCCTACCC TCCGTCTCCA GCACCAGCTA GCAC ATG GCC TCT AAC836
Met Ala Ser Asn
1GGA ACT TCT GTA TCA CCT TTC CGA TCT CAA AAG AAC GCC GCA ATG GCT884Gly Thr Ser Val Ser Pro Phe Arg Ser Gln Lys Asn Ala Ala Met Ala5 10 15 20GTC AAC GAC ACC CCC GCC AAC GGC CAC GCC GAG CCC TCC ACC ATC ACC932Val Asn Asp Thr Pro Ala Asn Gly His Ala Glu Pro Ser Thr Ile Thr
25 30 35GCC GCC TCG AAG ACC AAC ACC ACG AAG ATC ACA TCT CAG AAT GAT CCT980Ala Ala Ser Lys Thr Asn Thr Thr Lys Ile Thr Ser Gln Asn Asp Pro
40 45 50CAG TCA TCC TTC GCC GTC AAG GTC GGC TTG GCC CAG ATG CTC AAG GGT1028Gln Ser Ser Phe Ala Val Lys Val Gly Leu Ala Gln Met Leu Lys Gly
55 60 65GGC GTG ATC ATG GAT GTG GTC AAC GCA GAG CAA GCA CGC ATT GCT GAA1076Gly Val Ile Met Asp Val Val Asn Ala Glu Gln Ala Arg Ile Ala Glu
70 75 80GAG GCG GGT GCA TGT GCC GTC ATG GCC CTC GAG CGT GTG CCA GCA GAT1124Glu Ala Gly Ala Cys Ala Val Met Ala Leu Glu Arg Val Pro Ala Asp85 90 95 100ATT CGA AAG GAC GGT GGC GTC GCT CGC ATG AGC GAC CCA CAA ATG ATC1172Ile Arg Lys Asp Gly Gly Val Ala Arg Met Ser Asp Pro Gln Met Ile
105 110 115AAG GAC ATC ATG AAT GCT GTG ACC ATC CCT GTC ATG GCG AAG TCG AGG1220Lys Asp Ile Met Asn Ala Val Thr Ile Pro Val Met Ala Lys Ser Arg
120 125 130ATT GGT CAC TTC GTG GAA TGT CAG ATT CTC CAA GCC ATT GGC GTG GAC1268Ile Gly His Phe Val Glu Cys Gln Ile Leu Gln Ala Ile Gly Val Asp
135 140 145TAC ATC GAT GAG TCC GAG GTG CTC ACA CCT GCC GAT CCA GTC AAC CAC1316Tyr Ile Asp Glu Ser Glu Val Leu Thr Pro Ala Asp Pro Val Asn His
150 155 160ATC GAC AAG AGC GTT TAC AAT GTT CCA TTC GTG TGT GGA TGC AAG AAC1364Ile Asp Lys Ser Val Tyr Asn Val Pro Phe Val Cys Gly Cys Lys Asn165 170 175 180TTG GGT GAG GCC CTT CGA AGA ATA TCA GAG GGC GCT GCC ATG ATC CGG1412Leu Gly Glu Ala Leu Arg Arg Ile Ser Glu Gly Ala Ala Met Ile Arg
185 190 195ACA AAG GGT GAA GCA GGA ACG GGA GAT GTC GTC GAG GCC GTG AGA CAC1460Thr Lys Gly Glu Ala Gly Thr Gly Asp Val Val Glu Ala Val Arg His
200 205 210ATG CAG ACT GTC AAT GCT GAG ATC GCA AAG GCC AGC TCA GCA TCT GAC1508Met Gln Thr Val Asn Ala Glu Ile Ala Lys Ala Ser Ser Ala Ser Asp
215 220 225GCT GAT CTT CGC ATG ATG GCA CGA GAG CTG CAG TGC GAC TAC AAC CTG1556Ala Asp Leu Arg Met Met Ala Arg Glu Leu Gln Cys Asp Tyr Asn Leu
230 235 240
CTC AAG CAG ACC GCA CAG CTC AAG AGA CTG CCA GTG GTC AAC TTC GCT
1604
Leu Lys Gln Thr Ala Gln Leu Lys Arg Leu Pro Val Val Asn Phe Ala
245 250 255 260
GCA GGA GGT ATC GCC ACG CCG GCC GAC GCT GCC TTG ATG ATG CAA ATG
1652
Ala Gly Gly Ile Ala Thr Pro Ala Asp Ala Ala Leu Met Met Gln Met
265 270 275
GGT TGC GAT GGT GTC TTC GTT GGA TCT GGT ATC TTC AAG TCA GGC GAC
1700
Gly Cys Asp Gly Val Phe Val Gly Ser Gly Ile Phe Lys Ser Gly Asp
280 285 290
GCG GCG AAG CGA GCA AAG GCC ATT GTG CAG GCC ACC ACA CAC TAC AAC
1748
Ala Ala Lys Arg Ala Lys Ala Ile Val Gln Ala Thr Thr His Tyr Asn
295 300 305
GAC CCC AAG GTC CTG GCT GAG GTC AGC TCG GGT CTT GGT GAG GCA ATG
1796
Asp Pro Lys Val Leu Ala Glu Val Ser Ser Gly Leu Gly Glu Ala Met
310 315 320
GTG GGC ATC AAC TGC GAC AAG CTG CCA GAG ACA CAG AAG CTG GCG ACC
1844
Val Gly Ile Asn Cys Asp Lys Leu Pro Glu Thr Gln Lys Leu Ala Thr
325 330 335 340
CGT GGC TGG TAGATGCTGC AAATTCGAAA AAGAAAACGG GAACATGACT
1893
Arg Gly Trp
GTAGGCATAG CAGCGGGCGC TTGGGTATGG GTGTGATTGC AATCAAAAGA AAAGCGAGCG
1953
AGTTAGAGAG CACATCTGGG CGTGTTAGAT TCTGTATCGC GCCTCACCGC GCCTAGG
2010(2)SEQ ID NO:2的资料
(i)序列特征:
(A)长度:343个氨基酸
(B)类型:氨基酸
(D)拓朴结构:线性
(ii)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:2
Met Ala Ser Asn Gly Thr Ser Val Ser Pro Phe Arg Ser Gln Lys Asn
1 5 10 15
Ala Ala Met Ala Val Asn Asp Thr Pro Ala Asn Gly His Ala Glu Pro
20 25 30
Ser Thr Ile Thr Ala Ala Ser Lys Thr Asn Thr Thr Lys Ile Thr Ser
35 40 45
Gln Asn Asp Pro Gln Ser Ser Phe Ala Val Lys Val Gly Leu Ala Gln
50 55 60
Met Leu Lys Gly Gly Val Ile Met Asp Val Val Asn Ala Glu Gln Ala
65 70 75 80
Arg Ile Ala Glu Glu Ala Gly Ala Cys Ala Val Met Ala Leu Glu Arg
85 90 95
Val Pro Ala Asp Ile Arg Lys Asp Gly Gly Val Ala Arg Met Ser Asp
100 105 110
Pro Gln Met Ile Lys Asp Ile Met Asn Ala Val Thr Ile Pro Val Met
115 120 125
Ala Lys Ser Arg Ile Gly His Phe Val Glu Cys Gln Ile Leu Gln Ala
130 135 140
Ile Gly Val Asp Tyr Ile Asp Glu Ser Glu Val Leu Thr Pro Ala Asp
145 150 155 160
Pro Val Asn His Ile Asp Lys Ser Val Tyr Asn Val Pro Phe Val Cys
165 170 175
Gly Cys Lys Asn Leu Gly Glu Ala Leu Arg Arg Ile Ser Glu Gly Ala
180 185 190
Ala Met Ile Arg Thr Lys Gly Glu Ala Gly Thr Gly Asp Val Val Glu
195 200 205
Ala Val Arg His Met Gln Thr Val Asn Ala Glu Ile Ala Lys Ala Ser
210 215 220
Ser Ala Ser Asp Ala Asp Leu Arg Met Met Ala Arg Glu Leu Gln Cys
225 230 235 240
Asp Tyr Asn Leu Leu Lys Gln Thr Ala Gln Leu Lys Arg Leu Pro Val
245 250 255
Val Asn Phe Ala Ala Gly Gly Ile Ala Thr Pro Ala Asp Ala Ala Leu
260 265 270
Met Met Gln Met Gly Cys Asp Gly Val Phe Val Gly Ser Gly Ile Phe
275 280 285
Lys Ser Gly Asp Ala Ala Lys Arg Ala Lys Ala Ile Val Gln Ala Thr
290 295 300
Thr His Tyr Asn Asp Pro Lys Val Leu Ala Glu Val Ser Ser Gly Leu
305 310 315 320
Gly Glu Ala Met Val Gly Ile Asn Cys Asp Lys Leu Pro Glu Thr Gln
325 330 335
Lys Leu Ala Thr Arg Gly Trp
340(2)SEQ ID NO:3的资料
(i)序列特征
(A)长度:310个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓朴结构:线性
(ii)分子类型:cDNA
(xi)序列描述:SEQ ID NO:3TATGCGCTGC TCAAGGAGAC GGCTAAGCTT GGTCGTCTGC CTGTTGTCAA CTTTGCGGCG60GGTGGTGTCG CAACACCCGC TGATGCTGCG TTGATGATGC AGTTGGGTTG CGATGGTGTC120TTTGTTGGTA GCGGTATCTT CAAGTCTGGA GACGCAGCCA AGAGGGCCAA GGCCATCGTA180CAGGCTGTTA CTCACTACAA AGACCCCAAG GTGCTCATGG AAGTCAGCAT GGATTTGGGT240GAGGCCATGG TTGGTATCAA CTGCGGTACA ATGGGCGAGG AGGAGAAGCT TGCTAAGAGG300GGATGGTAGA310(2)SEQ ID NO:4的资料
(i)序列特征:
(A)长度:102个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓朴结构:线性
(ii)分子类型:cDNA
(xi)序列描述:SEQ ID NO:4
Tyr Ala Leu Leu Lys Glu Thr Ala Lys Leu Gly Arg Leu Pro Val Val
1 5 10 15
Asn Phe Ala Ala Gly Gly Val Ala Thr Pro Ala Asp Ala Ala Leu Met
20 25 30
Met Gln Leu Gly Cys Asp Gly Val Phe Val Gly Ser Gly Ile Phe Lys
35 40 45
Ser Gly Asp Ala Ala Lys Arg Ala Lys Ala Ile Val Gln Ala Val Thr
50 55 60
His Tyr Lys Asp Pro Lys Val Leu Met Glu Val Ser Met Asp Leu Gly
65 70 75 80
Glu Ala Met Val Gly Ile Asn Cys Gly Thr Met Gly Glu Glu Glu Lys
85 90 95
Leu Ala Lys Arg Gly Trp
100(2)SEQ ID NO:5的资料
(i)序列特征:
(A)长度:311个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓朴结构:线性
(ii)分子类型:cDNA
(xi)序列描述:SEQ ID NO:5
TACAACCTGC TCAAGCAGAC CGCACAGCTC AAGAGACTGC CAGTGGTCAA CTTCGCTGCA
60
GGAGGTATCG CCACGCCGGC CGACGCTGCC TTGATGATGC AAATGGGTTG CGATGGTGTC
120
TTCGTTGGAT CTGGTATCTT CAAGTCAGGC GACGCGGCGA AGCGAGCAAA GGCCATTGTG
180
CAGGCCACCA CACACTACAA CGACCCCAAG GTCCTGGCTG AGGTCAGCTC GGGTCTTGGT
240
GAGGCAATGG TGGGCATCAA CTGCGACAAG CTGCCAGAGA CACAGAAGCT GGCGACCCGT
300
GGCTGGTAGA T
311(2)SEQ ID NO:6的资料
(i)序列特征:
(A)长度:100个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓朴结构:线性
(ii)分子类型:肽
(xi)序列描述:SEQ ID NO:6
Leu Leu Lys Glu Thr Ala Lys Leu Gly Arg Leu Pro Val Val Asn Phe
1 5 10 15
Ala Ala Gly Gly Val Ala Thr Pro Ala Asp Ala Ala Leu Met Met Gln
20 25 30
Leu Gly Cys Asp Gly Val Phe Val Gly Ser Gly Ile Phe Lys Ser Gly
35 40 45
Asp Ala Ala Lys Arg Ala Lys Ala Ile Val Gln Ala Val Thr His Tyr
50 55 60
Lys Asp Pro Lys Val Leu Met Glu Val Ser Met Asp Leu Gly Glu Ala
65 70 75 80
Met Val Gly Ile Asn Cys Gly Thr Met Gly Glu Glu Glu Lys Leu Ala
85 90 95
Lys Arg Gly Trp
100(2)SEQ ID NO:7的资料
(i)序列特征:
(A)长度:143个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓朴结构:线性
(ii)分子类型:肽
(xi)序列描述:SEQ ID NO:7
Ala Gly Thr Gly Asp Val Val Glu Ala Val Arg His Met Gln Thr Val
1 5 10 15
Asn Ala Glu Ile Ala Lys Ala Ser Ser Ala Ser Asp Ala Asp Leu Arg
20 25 30
Met Met Ala Arg Glu Leu Gln Cys Asp Tyr Asn Leu Leu Lys Gln Thr
35 40 45
Ala Gln Leu Lys Arg Leu Pro Val Val Asn Phe Ala Ala Gly Gly Ile
50 55 60
Ala Thr Pro Ala Asp Ala Ala Leu Met Met Gln Met Gly Cys Asp Gly
65 70 75 80
Val Phe Val Gly Ser Gly Ile Phe Lys Ser Gly Asp Ala Ala Lys Arg
85 90 95
Ala Lys Ala Ile Val Gln Ala Thr Thr His Tyr Asn Asp Pro Lys Val
100 105 110
Leu Ala Glu Val Ser Ser Gly Leu Gly Glu Ala Met Val Gly Ile Asn
115 120 125
Cys Asp Lys Leu Pro Glu Thr Gln Lys Leu Ala Thr Arg Gly Trp
130 135 140(2)SEQ ID NO:8的资料
(i)序列特征:
(A)长度:3420个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓朴结构:线性
(ii)分子类型:cDNA
(ix)特征:
(A)名称/关键词:CDS
(B)位置:742…2391(xi)序列描述:SEQ ID NO:8GGATCCCCGA TCCAGCGGGA GTATTTGACA TGATTAGGTT CCTTCGGCGA CTACATTGTG60AAGTGGTATG CTCGAAGGTC GCATTCCATT GCCATGTCTC TGCAGCGTAG CGTCGAATAC120AGCACTTGTT GGGAGAATTG CAGATAGGAG TACAGTCAAC TCAACTTCGA AACAAGTCGT180ACTGTACTGC TCAAAGAACC TCAGAGAAAG GTTTCCCACA AGTCTACAGG GATGATTACC240GGCATCTGAT TCGCTACCCA ATCGCGCTAT TCCACGTCTG AGCCTTAGCA TCAACTCACC300TCTCTCTCAC CCAAGACATT CTGTCACAGC CTCGCGGTGC TTTTTCCGTC ATGCCACATC360GCACTTTTCG ACGGCCATGT CACCACGAAT GCCGCAGAAA CGGGGCAGAG CCTGTGAGGC420TTGCTCAAAG ATCAAAATCC GGTGCGTGGT CATAACTTCC CGATCAATAG TGGTGCCTGC480CACGCGCTGA TGTGATCTGC ATCAAGGTGT TCGCTGGGCC AGGCATCAGA GGATGCAGCA540CCGCCTTGCG AGAGATGCGT GCGATTGAAC AAGGAATGCA TTTTGAGCGC TCCAAAGCGT600CAGAAAGACC GCGTCGCCGA ACTTGAAGCA CAAGTGGCAC AGTTGACACG ACTGCTTGAG660AGTCAGCATA TCCAAGTACC TTCCGTTTCT CCGGCTACGG CCTCACAAAG CAATCAAGAT720GAATCACCTA CACCCGCGCA G ATG GTA AGC GCG TCT GGA ACA GCG ACG AAG771
Met Val Ser Ala Ser Gly Thr Ala Thr Lys
1 5 10AAG CGA CGA CTA GAC TCC GAT GGA GAA ACG CCG CAA TCG AGC GTA TCT819Lys Arg Arg Leu Asp Ser Asp Gly Glu Thr Pro Gln Ser Ser Val Ser
15 20 25TCA CCA GGC ACC CAG AAT CCA GAC ATC TCT GAC ATC CAA CGA CTT GAT867Ser Pro Gly Thr Gln Asn Pro Asp Ile Ser Asp Ile Gln Arg Leu Asp
30 35 40CGG GTA CTC TCC TAT GAG CTG CAA CAG CGA ACG CTG ACT CGC TAT GTC915Arg Val Leu Ser Tyr Glu Leu Gln Gln Arg Thr Leu Thr Arg Tyr Val
45 50 55ACC GAG ATA GCA CCA CTC TTC CCA GCA GTG CCA GCG CCA GCG GAT TGC963Thr Glu Ile Ala Pro Leu Phe Pro Ala Val Pro Ala Pro Ala Asp Cys
60 65 70TCG TTG CCC GAA ATG AGA GCG AAT CGA CCC ACG TTG CTT ATG GCT TTC1011Ser Leu Pro Glu Met Arg Ala Asn Arg Pro Thr Leu Leu Met Ala Phe75 80 85 90TTA TAT GCT GCC AGT TGC GGC TTT CTT TCG CTT GAT ACT CAA GAA GAT1059Leu Tyr Ala Ala Ser Cys Gly Phe Leu Ser Leu Asp Thr Gln Glu Asp
95 100 105GTA GCT CAA ATT CTG CTC AAT ACC CTC TCT GCA AGA GCA ATC ACG CAC1107Val Ala Gln Ile Leu Leu Asn Thr Leu Ser Ala Arg Ala Ile Thr His
110 115 120GGA GAG GAG ACG CTT GAA TTG ATA CAA GCT ATT CAG ATT GCC TGC TTG1155Gly Glu Glu Thr Leu Glu Leu Ile Gln Ala Ile Gln Ile Ala Cys Leu
125 130 135TGG TAT CGC TCA CCG AAG CAC CAT CGA CGT GCG GCC GTC TAC CAG CTC1203Trp Tyr Arg Ser Pro Lys His His Arg Arg Ala Ala Val Tyr Gln Leu
140 145 150ATT GAC ATC GCT TCT GCC ATG GCC AAT GGT CTC AGC GCA GGC GGT CCA1251Ile Asp Ile Ala Ser Ala Met Ala Asn Gly Leu Ser Ala Gly Gly Pro155 160 165 170CTC GCT CCT CCG ACC AAA GGA CTG ACT TTG GAC GAT TGC GCG GAT ACG1299Leu Ala Pro Pro Thr Lys Gly Leu Thr Leu Asp Asp Cys Ala Asp Thr
175 180 185GGG TCG TAC GAG TCG GTA GAG GGC TGG CGC GCC TGG CTT GGC TGC CAT1347Gly Ser Tyr Glu Ser Val Glu Gly Trp Arg Ala Trp Leu Gly Cys His
190 195 200GTA CTG TCT GTC TCT ATG GCC ATT TTC ATG AGG AAA TCG ATG ACT GCA1395Val Leu Ser Val Ser Met Ala Ile Phe Met Arg Lys Ser Met Thr Ala
205 210 215AGT TGG ACC GAA CAG CAC GAG CAG GCA CGT CTG ATG CTG CAG TAC TCG1443Ser Trp Thr Glu Gln His Glu Gln Ala Arg Leu Met Leu Gln Tyr Ser
220 225 230CCC TTG AAC GCA GAC TCT GAT AGG TGG CTT GCT CAG TAC ATC AGA GCC1491Pro Leu Asn Ala Asp Ser Asp Arg Trp Leu Ala Gln Tyr Ile Arg Ala235 240 245 250GAG CGA CTA TGC GAA GAG GTT TCT GAA CAG GTG GAT TTG ACT AAC ACA1539Glu Arg Leu Cys Glu Glu Val Ser Glu Gln Val Asp Leu Thr Asn Thr
255 260 265TCT TTC TAT CGC GAC GTT GCT GAT CCT GCA ACA AGA AAT CCA GTG CAG1587Ser Phe Tyr Arg Asp Val Ala Asp Pro Ala Thr Arg Asn Pro Val Gln
270 275 280ACA TGT CGA AAC AAG ATT CTG AAT TGG AAA ATG GGT GTT CCG CAA AGG1635Thr Cys Arg Asn Lys Ile Leu Asn Trp Lys Met Gly Val Pro Gln Arg
285 290 295TTA CGC TCT CCG TTG ATC ATG TTC TGG GAA CAT GTA GCA ACA GCA TAC1683Leu Arg Ser Pro Leu Ile Met Phe Trp Glu His Val Ala Thr Ala Tyr
300 305 310ATG CAT GAA CCA GTC CTG CAC ACA GCA ACG AAC AAG GAC AGC TTT ACG1731Met His Glu Pro Val Leu His Thr Ala Thr Asn Lys Asp Ser Phe Thr315 320 325 330GCA CCT TAT TTG GCA GAA AGG CTG TCA CTG ACA GAC TTT CCG ACT CCG1779Ala Pro Tyr Leu Ala Glu Arg Leu Ser Leu Thr Asp Phe Pro Thr Pro
335 340 345CTC GTC ACT CAA GAT CAC ATC ACA GCT GTG TAC GAG CTG ACT GCG GCT1827Leu Val Thr Gln Asp His Ile Thr Ala Val Tyr Glu Leu Thr Ala Ala
350 355 360GTA CAA GCC GTT CTG GAC ATC TTT ATC AAC TAC GAC ACT AAA TCT CTC1875Val Gln Ala Val Leu Asp Ile Phe Ile Asn Tyr Asp Thr Lys Ser Leu
365 370 375GTT GCC TCT CCG AGC TTG GTG TAT GCT GCC AGA GCT GCG TAT GCG CTC1923Val Ala Ser Pro Ser Leu Val Tyr Ala Ala Arg Ala Ala Tyr Ala Leu
380 385 390TAT GTT CTG GCG AAG CTA TAC ATC GCT GTC ACT GCA CCA GGA AAT ACA1971Tyr Val Leu Ala Lys Leu Tyr Ile Ala Val Thr Ala Pro Gly Asn Thr395 400 405 410CTT GGC ACA ATT CTG GAC GCC AGT ATT CTT GCC CTG CCG GAG TAC GCT2019Leu Gly Thr Ile Leu Asp Ala Ser Ile Leu Ala Leu Pro Glu Tyr Ala
415 420 425GAC AGG CTG GCA ACA TGC GGC TCA CGA ATT AGA GCG CTC GAT GAG CGT2067Asp Arg Leu Ala Thr Cys Gly Ser Arg Ile Arg Ala Leu Asp Glu Arg
430 435 440TGC GGT CCG GCT CGA ATC ATG CAT TGC GCA CCG GCG ATC AAG GAC TGG2115Cys Gly Pro Ala Arg Ile Met His Cys Ala Pro Ala Ile Lys Asp Trp
445 450 455TAT CTG AAC TAT ACT CAA TTC CTC TCC TCG AAC GCT GCA CTC GCC CAG2163Tyr Leu Asn Tyr Thr Gln Phe Leu Ser Ser Asn Ala Ala Leu Ala Gln
460 465 470TCG ATC CAG GTC TCC AAC GAC AAT GTG GCG GAG GCT CAG ATG ACT TTG2211Ser Ile Gln Val Ser Asn Asp Asn Val Ala Glu Ala Gln Met Thr Leu475 480 485 490CCG CCG CTC CAA GAC AAC ACG AAC GCA TTT AGC AAT ATT CCA CCG GAT2259Pro Pro Leu Gln Asp Asn Thr Asn Ala Phe Ser Asn Ile Pro Pro Asp
495 500 505TGG GAG AAT CTG CTC ATG TTC GGT GAT AGT TCC ACG GAC TAT GGC TTC2307Trp Glu Asn Leu Leu Met Phe Gly Asp Ser Ser Thr Asp Tyr Gly Phe
510 515 520GAT CAG CTG TTT GCT GAA CCT ATT CCT CTA CAG CTC GAG CAG CCC ATA2355Asp Gln Leu Phe Ala Glu Pro Ile Pro Leu Gln Leu Glu Gln Pro Ile
525 530 535TTT GCC AAT ACG ATA CCT ACT GCG TTT GCG ACG AAG TGATCCAACA2401Phe Ala Asn Thr Ile Pro Thr Ala Phe Ala Thr Lys
540 545 550CGCGGCAAGA CGGGATCTCT GCTGTCAACG AAGCAGCGCA TGAAGCTCCA GAATGGGGAT2461CACATACCGA CGTTACGTTC TTCTTGGGCG AAGAAGAAGA CTTGCATCAT CAGCGTACTG2521CATCGTCGAA GTCGGTGATC CACGAACAAA TCGATGGCTC GGCTCGCATG CCATCAATCC2581GAAAATTTGC GATGATTGGG CACACTCGTC TTTGCGGAGC TCTGCCATAA GTCGCGCTTG2641GAAGACTTCG TGGCAACGAT CGATGCGTCA GCTGCAGAAA GGCCGGTTCT TTGAATTGCC2701GTGTTAGCAG AGGCAGTACT GAACAAGTCC GCACCCTTAG ATGTCTGCAT CCTGCAAAAT2761GGCGAATGTC CGATCAGAGC TCGACAAAAA TTGTCAATGG GGTCTTGAGG TGTGCCCATA2821TTGAGGAGCG ATGGAAGACC GCACGTCTGC GAAGTCGTCT GTGGATGAAG AAGTCTGCAT2881TCTGCGCATG TAACCTCGTA CATGTGCACT GTCGGAAATA GCTGGAGCAA GTGGGCTAAG2941GTTACCCGAA GTGGAACATT AGCCAAGCTC CATCGGCGCG ATTGCTCGAT GTTATCGAGT3001CATGGAAACC AGGATGACAG TCCCGCGCCA GCGCCGCCCA CGTGCAACTA TCAGACTATC3061TATCTCAGTG CTATCTACTG ATAAGCACGA GGGATCACGA CGAACGGAAT CGACAGCGAT3121GACCATGAAA AGCTGCCGGA CACATGGGTT CACATCATTC ATCTGCAGTT GTGAAAACCT3181TTCGCCTGCA CATCAGTCCA TCGGTTGTGG GGTCTCGCGA CATGCAATTC TTTATAATAA3241GTACTGTTCG TCCACATGAG TGACGCGATA CAAGTGGCCA GCAGAGCCTG CTGTCAAATC3301CCTGTTTCGT CACCGGACGA TCACGGGCGC TGCTCAGAAT CAACACCTTT GCTTCAAGAC3361TCAATGTCCT CGGGTGGTCA TCGCAATATG TCGTCCAGCA TGGAGAAATT CAAGAATTC3420(2)SEQ ID NO:9的资料
(i)序列特征:
(A)长度:550个氨基酸
(B)类型:氨基酸
(D)拓朴结构:线性
(ii)分子类型:蛋白质
(ix)序列特征:SEQ ID NO:9
Met Val Ser Ala Ser Gly Thr Ala Thr Lys Lys Arg Arg Leu Asp Ser
1 5 10 15
Asp Gly Glu Thr Pro Gln Ser Ser Val Ser Ser Pro Gly Thr Gln Asn
20 25 30
Pro Asp Ile Ser Asp Ile Gln Arg Leu Asp Arg Val Leu Ser Tyr Glu
35 40 45
Leu Gln Gln Arg Thr Leu Thr Arg Tyr Val Thr Glu Ile Ala Pro Leu
50 55 60
Phe Pro Ala Val Pro Ala Pro Ala Asp Cys Ser Leu Pro Glu Met Arg
65 70 75 80
Ala Asn Arg Pro Thr Leu Leu Met Ala Phe Leu Tyr Ala Ala Ser Cys
85 90 95
Gly Phe Leu Ser Leu Asp Thr Gln Glu Asp Val Ala Gln Ile Leu Leu
100 105 110
Asn Thr Leu Ser Ala Arg Ala Ile Thr His Gly Glu Glu Thr Leu Glu
115 120 125
Leu Ile Gln Ala Ile Gln Ile Ala Cys Leu Trp Tyr Arg Ser Pro Lys
130 135 140
His His Arg Arg Ala Ala Val Tyr Gln Leu Ile Asp Ile Ala Ser Ala
145 150 155 160
Met Ala Asn Gly Leu Ser Ala Gly Gly Pro Leu Ala Pro Pro Thr Lys
165 170 175
Gly Leu Thr Leu Asp Asp Cys Ala Asp Thr Gly Ser Tyr Glu Ser Val
180 185 190
Glu Gly Trp Arg Ala Trp Leu Gly Cys His Val Leu Ser Val Ser Met
195 200 205
Ala Ile Phe Met Arg Lys Ser Met Thr Ala Ser Trp Thr Glu Gln His
210 215 220
Glu Gln Ala Arg Leu Met Leu Gln Tyr Ser Pro Leu Asn Ala Asp Ser
225 230 235 240
Asp Arg Trp Leu Ala Gln Tyr Ile Arg Ala Glu Arg Leu Cys Glu Glu
245 250 255
Val Ser Glu Gln Val Asp Leu Thr Asn Thr Ser Phe Tyr Arg Asp Val
260 265 270
Ala Asp Pro Ala Thr Arg Asn Pro Val Gln Thr Cys Arg Asn Lys Ile
275 280 285
Leu Asn Trp Lys Met Gly Val Pro Gln Arg Leu Arg Ser Pro Leu Ile
290 295 300
Met Phe Trp Glu His Val Ala Thr Ala Tyr Met His Glu Pro Val Leu
305 310 315 320
His Thr Ala Thr Asn Lys Asp Ser Phe Thr Ala Pro Tyr Leu Ala Glu
325 330 335
Arg Leu Ser Leu Thr Asp Phe Pro Thr Pro Leu Val Thr Gln Asp His
340 345 350
Ile Thr Ala Val Tyr Glu Leu Thr Ala Ala Val Gln Ala Val Leu Asp
355 360 365
Ile Phe Ile Asn Tyr Asp Thr Lys Ser Leu Val Ala Ser Pro Ser Leu
370 375 380
Val Tyr Ala Ala Arg Ala Ala Tyr Ala Leu Tyr Val Leu Ala Lys Leu
385 390 395 400
Tyr Ile Ala Val Thr Ala Pro Gly Asn Thr Leu Gly Thr Ile Leu Asp
405 410 415
Ala Ser Ile Leu Ala Leu Pro Glu Tyr Ala Asp Arg Leu Ala Thr Cys
420 425 430
Gly Ser Arg Ile Arg Ala Leu Asp Glu Arg Cys Gly Pro Ala Arg Ile
435 440 445
Met His Cys Ala Pro Ala Ile Lys Asp Trp Tyr Leu Asn Tyr Thr Gln
450 455 460
Phe Leu Ser Ser Asn Ala Ala Leu Ala Gln Ser Ile Gln Val Ser Asn
465 470 475 480
Asp Asn Val Ala Glu Ala Gln Met Thr Leu Pro Pro Leu Gln Asp Asn
485 490 495
Thr Asn Ala Phe Ser Asn Ile Pro Pro Asp Trp Glu Asn Leu Leu Met
500 505 510
Phe Gly Asp Ser Ser Thr Asp Tyr Gly Phe Asp Gln Leu Phe Ala Glu
515 520 525
Pro Ile Pro Leu Gln Leu Glu Gln Pro Ile Phe Ala Asn Thr Ile Pro
530 535 540
Thr Ala Phe Ala Thr Lys
545 550(2)SEQ ID NO:10的资料
(i)序列特征:
(A)长度:924个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓朴结构:线性
(ii)分子类型:cDNA
(ix)特征:
(A)名称/关键词:CDS
(B)位置:1..921
(xi)序列描述:SEQ ID NO:10
ATG GCA ACT GAA CTC CCC ACC ACA AAC GGC CAC AGC GCA CAG GAT GGC
48
Met Ala Thr Glu Leu Pro Thr Thr Asn Gly His Ser Ala Gln Asp Gly
1 5 10 15
GAG AAC AAC TTT GCC GTC AAG GCC GGT CTG GCA CGC ATG TTG AAG GGT
96
Glu Asn Asn Phe Ala Val Lys Ala Gly Leu Ala Arg Met Leu Lys Gly
20 25 30
GGA GTC ATC ATG GAC GTT GTC AAC GCT GAG CAA GCG CGG ATA GCA GAA
144
Gly Val Ile Met Asp Val Val Asn Ala Glu Gln Ala Arg Ile Ala Glu
35 40 45
GAA GCC GGT GCT TCA GCC GTC ATG GCC CTC GAG CGC GTG CCC GCA GAC
192
Glu Ala Gly Ala Ser Ala Val Met Ala Leu Glu Arg Val Pro Ala Asp
50 55 60
ATT CGA TCC CAA GGT GGT GTC GCA CGT ATG AGC GAC CCC AAG ATG ATC
240
Ile Arg Ser Gln Gly Gly Val Ala Arg Met Ser Asp Pro Lys Met Ile
65 70 75 80
AAG GAG ATC ATG GAC ACA GTC ACA ATC CCC GTC ATG GCC AAG GCG CGA
288
Lys Glu Ile Met Asp Thr Val Thr Ile Pro Val Met Ala Lys Ala Arg
85 90 95
ATT GGA CAC TTT GTC GAA TGC CAG ATC CTC GAA GCC CTA GGC GTA GAC
336
Ile Gly His Phe Val Glu Cys Gln Ile Leu Glu Ala Leu Gly Val Asp
100 105 110
TAC ATT GAC GAA TCC GAA GTC CTC ACC CCC GCC GAC GCT ATT CAC CAC
384
Tyr Ile Asp Glu Ser Glu Val Leu Thr Pro Ala Asp Ala Ile His His
115 120 125
GTC TCC AAG CAC CCC TTC CGC ATT CCC TTC GTC TGC GGC TGC CGG GGC
432
Val Ser Lys His Pro Phe Arg Ile Pro Phe Val Cys Gly Cys Arg Gly
130 135 140
CTC GGC GAA GCC CTT CGC CGC ATC TCG GAA GGT GCA GCC ATC ATC CGC
480
Leu Gly Glu Ala Leu Arg Arg Ile Ser Glu Gly Ala Ala Ile Ile Arg
145 150 155 160
ACA AAG GGC GAA GCC GGA ACC GGC GAC GTC ATT GAG GCT GTC CGC CAC
528
Thr Lys Gly Glu Ala Gly Thr Gly Asp Val Ile Glu Ala Val Arg His
165 170 175
ATG CGT ACC GTA AAC AGC GAG ATT GCC CGC GCA AAG AGC ATG TCA GAG
576
Met Arg Thr Val Asn Ser Glu Ile Ala Arg Ala Lys Ser Met Ser Glu
180 185 190
GAG GAG CTC CGT GTC TAC GCA AAG GAG CTT CAG GTC GAC TAT GCG CTG
624
Glu Glu Leu Arg Val Tyr Ala Lys Glu Leu Gln Val Asp Tyr Ala Leu
195 200 205
CTC AAG GAG ACG GCT AAG CTT GGT CGT CTG CCT GTT GTC AAC TTT GCG
672
Leu Lys Glu Thr Ala Lys Leu Gly Arg Leu Pro Val Val Asn Phe Ala
210 215 220
GCG GGT GGT GTC GCA ACA CCC GCT GAT GCT GCG TTG ATG ATG CAG TTG
720
Ala Gly Gly Val Ala Thr Pro Ala Asp Ala Ala Leu Met Met Gln Leu
225 230 235 240
GGT TGC GAT GGT GTC TTT GTT GGT AGC GGT ATC TTC AAG TCT GGA GAC
768
Gly Cys Asp Gly Val Phe Val Gly Ser Gly Ile Phe Lys Ser Gly Asp
245 250 255
GCA GCC AAG AGG GCC AAG GCC ATC GTA CAG GCT GTT ACT CAC TAC AAA
816
Ala Ala Lys Arg Ala Lys Ala Ile Val Gln Ala Val Thr His Tyr Lys
260 265 270
GAC CCC AAG GTG CTC ATG GAA GTC AGC ATG GAT TTG GGT GAG GCC ATG
864
Asp Pro Lys Val Leu Met Glu Val Ser Met Asp Leu Gly Glu Ala Met
275 280 285
GTT GGT ATC AAC TGC GGT ACA ATG GGC GAG GAG GAG AAG CTT GCT AAG
912
Val Gly Ile Asn Cys Gly Thr Met Gly Glu Glu Glu Lys Leu Ala Lys
290 295 300
AGG GGA TGG TAG
924
Arg Gly Trp
305(2)SEQ ID NO:11的资料
(i)序列特征:
(A)长度:307个氨基酸
(B)类型:氨基酸
(D)拓朴结构:线性
(ii)分子类型:蛋白质
(ix)序列特征:SEQ ID NO:11
Met Ala Thr Glu Leu Pro Thr Thr Asn Gly His Ser Ala Gln Asp Gly
1 5 10 15
Glu Asn Asn Phe Ala Val Lys Ala Gly Leu Ala Arg Met Leu Lys Gly
20 25 30Gly Val Ile Met Asp Val Val Asn Ala Glu Gln Ala Arg Ile Ala Glu
35 40 45Glu Ala Gly Ala Ser Ala Val Met Ala Leu Glu Arg Val Pro Ala Asp
50 55 60Ile Arg Ser Gln Gly Gly Val Ala Arg Met Ser Asp Pro Lys Met Ile65 70 75 80Lys Glu Ile Met Asp Thr Val Thr Ile Pro Val Met Ala Lys Ala Arg
85 90 95Ile Gly His Phe Val Glu Cys Gln Ile Leu Glu Ala Leu Gly Val Asp
100 105 110Tyr Ile Asp Glu Ser Glu Val Leu Thr Pro Ala Asp Ala Ile His His
115 120 125Val Ser Lys His Pro Phe Arg Ile Pro Phe Val Cys Gly Cys Arg Gly
130 135 140Leu Gly Glu Ala Leu Arg Arg Ile Ser Glu Gly Ala Ala Ile Ile Arg145 150 155 160Thr Lys Gly Glu Ala Gly Thr Gly Asp Val Ile Glu Ala Val Arg His
165 170 175Met Arg Thr Val Asn Ser Glu Ile Ala Arg Ala Lys Ser Met Ser Glu
180 185 190Glu Glu Leu Arg Val Tyr Ala Lys Glu Leu Gln Val Asp Tyr Ala Leu
195 200 205Leu Lys Glu Thr Ala Lys Leu Gly Arg Leu Pro Val Val Asn Phe Ala
210 215 220Ala Gly Gly Val Ala Thr Pro Ala Asp Ala Ala Leu Met Met Gln Leu225 230 235 240Gly Cys Asp Gly Val Phe Val Gly Ser Gly Ile Phe Lys Ser Gly Asp
245 250 255Ala Ala Lys Arg Ala Lys Ala Ile Val Gln Ala Val Thr His Tyr Lys
260 265 270Asp Pro Lys Val Leu Met Glu Val Ser Met Asp Leu Gly Glu Ala Met
275 280 285Val Gly Ile Asn Cys Gly Thr Met Gly Glu Glu Glu Lys Leu Ala Lys
290 295 300Arg Gly Trp305
Claims (46)
1.一种分离的核酸分子,其通过表达能增强细胞对光敏剂的抗性。
2.权利要求1的分离的核酸分子,该分子含有选自下组的序列:
(a)SEQ ID NO:1;
(b)SEQ ID NO:1的825-1853位核苷酸;
(c)编码含有SEQ ID NO:2氨基酸序列的多肽的序列;和
(d)在严紧条件下与上述(a),(b)或(c)的序列可杂交的序列。
3.权利要求1的分离的核酸分子,其中该光敏剂通过与光和氧反应可产生单线态氧。
4.权利要求1的分离的核酸分子,其中该光敏剂为尾孢菌素。
5.权利要求1的分离的核酸分子,其中该细胞为一种真菌细胞。
6.权利要求1的分离的核酸分子,其中该细胞为植物细胞。
7.一种含嵌合基因的转化细胞,该基因含有与启动子可操作连接的权利要求1的核苷酸序列。
8.权利要求7的转化细胞,其中该细胞为植物细胞。
9.一种增加细胞对光敏剂的抗性的方法,其中包括用权利要求1的核酸分子转化该细胞。
10.权利要求9的增加细胞光敏剂抗性的方法,其中该光敏剂通过与光和氧反应可产生单线态氧。
11.权利要求9的增加细胞光敏剂抗性的方法,其中该光敏剂为尾孢菌素。
12.权利要求11的方法,其中该细胞为植物细胞。
13.一种增加细胞对一种病原体的抗性的方法,其中该病原体产生光敏剂,该方法包括用权利要求1的分离的核酸分子转化所述细胞。
14.权利要求13的方法,其中该光敏剂为尾孢菌素。
15.权利要求13的方法,其中该病原体为真菌。
16.权利要求15的方法,其中该病原体选自尾孢属。
17.一种增加细胞对单线态氧的抗性的方法,包括用权利要求1的分离的核酸分子转化该细胞。
18.一种含嵌合基因的表达盒,该基因含有与启动子可操作连接的权利要求1的核苷酸序列。
19.一种含嵌合基因的转化植物,该基因含有与启动子可操作连接的权利要求1的核苷酸序列。
20.一种含权利要求18的表达盒的转化植物。
21.一种增加植物对光敏剂的抗性的方法,包括用权利要求18的表达盒转化该植物。
22.权利要求21的方法,其中该光敏剂为尾孢菌素。
23.权利要求21的方法,其中该植物选自下组:油菜,canola,高粱,大豆,甜菜,玉米和烟草。
24.一种增加植物对真菌病原体所致感染的抵抗力的方法,其中该真菌病原体可产生一种光敏剂,该方法包括用权利要求21的表达盒转化该植物。
25.权利要求24的方法,其中该真菌病原体选自尾孢属。
26.权利要求24的方法,其中该植物选自下组:油菜,canola,高粱、大豆,甜菜,玉米和烟草。
27.一种分离的核酸分子,其经表达可增加细胞对光敏剂的抗性。
28.权利要求27的分离的核酸分子,该分子包含选自下组的序列:
(a)SEQ ID NO:8;
(b)SEQ ID NO:8的742-2391位核苷酸;
(c)编码含有SEQ ID NO:9氨基酸序列的多肽的序列;和
(d)在严紧条件下与上述(a),(b)或(c)的序列可杂交的序列。
29.权利要求27的分离的核酸,其中该细胞为真菌细胞或植物细胞。
30.一种含嵌合基因的转化细胞,该基因含有与启动子可操作连接的权利要求27的核苷酸序列。
31.权利要求30的转化细胞,其中该细胞为植物细胞。
32.一种增加细胞对尾孢菌素的抗性的方法,包括用权利要求27的核酸分子转化该细胞。
33.权利要求32的方法,其中该细胞为植物细胞。
34.一种增加细胞对病原体的抵抗力的方法,其中该病原体可产生尾孢菌素,该方法包括用权利要求27的分离的核酸分子转化该细胞。
35.权利要求34的方法,其中该病原体为真菌。
36.权利要求35的方法,其中该病原体选自尾孢属。
37.一种包含嵌合基因的表达盒,该基因含有与启动子可操作连接的权利要求27的核苷酸序列。
38.一种包含嵌合基因的转化植物,该基因含有与启动子可操作连接的权利要求27的核苷酸序列。
39.含有权利要求37的表达盒的转化植物。
40.一种增强植物对尾孢菌素的抗性的方法,包括用权利要求37的表达盒转化该植物。
41.权利要求40的方法,其中该植物选自下组:油菜,canola,高粱,大豆,甜菜,玉米和烟草。
42.一种增强植物对真菌病原体所致感染的抵抗力的方法,其中该真菌病原体可产生尾孢菌素,该方法包括用权利要求37的表达盒转化该植物。
43.权利要求42的方法,其中该真菌病原体选自尾孢属。
44.权利要求42的方法,其中该植物选自下组:油菜,canola,高粱,大豆,甜菜,玉米和烟草。
45.从众多细胞中筛选出能表达异源DNA序列的那些细胞的方法,包括:
(a)利用含有异源DNA序列和权利要求2的DNA序列的DNA构建体对细胞进行转化,所述细胞来自在无外源性吡哆醇时不能生长的生物体;然后
(b)在缺乏吡哆醇的培养基上培养该细胞;
其中能在该培养基上生长的细胞已成功地转化有该DNA构建体,并表达该异源DNA序列。
46.权利要求45的方法,其中该细胞选自真菌细胞和植物细胞。
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US4061597P | 1997-03-17 | 1997-03-17 | |
US60/040,615 | 1997-03-17 |
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CN98804353A Pending CN1252684A (zh) | 1997-03-17 | 1998-03-13 | 编码光敏剂抗性的分离基因和蛋白质 |
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US (1) | US6063987A (zh) |
EP (1) | EP0998190A4 (zh) |
CN (1) | CN1252684A (zh) |
AU (1) | AU6463398A (zh) |
BG (1) | BG103733A (zh) |
BR (1) | BR9808012A (zh) |
CA (1) | CA2283913A1 (zh) |
HU (1) | HUP0001809A3 (zh) |
WO (1) | WO1998041082A1 (zh) |
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EP1390468A4 (en) * | 2001-04-23 | 2004-09-22 | Elitra Pharmaceuticals Inc | IDENTIFICATION OF ESSENTIAL GENES OF ASPERGILLUS FUMIGATUS AND METHOD OF USE |
JP2008118981A (ja) * | 2006-10-18 | 2008-05-29 | Yokohama City Univ | 菌類及び/又は植物の新規有用変異株の取得方法 |
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- 1998-03-13 WO PCT/US1998/004981 patent/WO1998041082A1/en not_active Application Discontinuation
- 1998-03-13 EP EP98910380A patent/EP0998190A4/en not_active Withdrawn
- 1998-03-13 CN CN98804353A patent/CN1252684A/zh active Pending
- 1998-03-13 BR BR9808012-1A patent/BR9808012A/pt not_active IP Right Cessation
- 1998-03-16 US US09/039,859 patent/US6063987A/en not_active Expired - Fee Related
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1999
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Also Published As
Publication number | Publication date |
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WO1998041082A1 (en) | 1998-09-24 |
CA2283913A1 (en) | 1998-09-24 |
HUP0001809A2 (hu) | 2000-09-28 |
BR9808012A (pt) | 2000-03-08 |
HUP0001809A3 (en) | 2002-04-29 |
BG103733A (en) | 2000-07-31 |
AU6463398A (en) | 1998-10-12 |
EP0998190A1 (en) | 2000-05-10 |
EP0998190A4 (en) | 2002-10-23 |
US6063987A (en) | 2000-05-16 |
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