CN1329515C - 编码具有转谷氨酰胺酶活性的蛋白质的玉米核苷酸序列及其应用 - Google Patents
编码具有转谷氨酰胺酶活性的蛋白质的玉米核苷酸序列及其应用 Download PDFInfo
- Publication number
- CN1329515C CN1329515C CNB038175525A CN03817552A CN1329515C CN 1329515 C CN1329515 C CN 1329515C CN B038175525 A CNB038175525 A CN B038175525A CN 03817552 A CN03817552 A CN 03817552A CN 1329515 C CN1329515 C CN 1329515C
- Authority
- CN
- China
- Prior art keywords
- leu
- arg
- gln
- ser
- glu
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/104—Aminoacyltransferases (2.3.2)
- C12N9/1044—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y203/00—Acyltransferases (2.3)
- C12Y203/02—Aminoacyltransferases (2.3.2)
- C12Y203/02013—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
Abstract
本发明涉及来源于玉米、编码具有转谷氨酰胺酶活性的蛋白质的DNA分子以及包含所述DNA分子的基因表达载体。本发明也涉及上述DNA分子或载体的应用,以便产生能表达具有转谷氨酰胺酶活性的重组蛋白质的转化细胞以及将编码具有转谷氨酰胺酶活性的蛋白质序列导入植物细胞中。另外,本发明涉及产生的转基因植物和微生物细胞。此外,由上面提到的DNA序列表达的具有转谷氨酰胺酶活性的蛋白质可以用于例如食品处理、加工和转化。
Description
本发明涉及来自植物具有转谷氨酰胺酶活性的新蛋白质鉴定及其在食品处理、加工和转化领域以及开发具有新能力的转基因植物中的应用。
现有技术
转谷氨酰胺酶(TGase;EC2.3.13)(R-谷氨酰胺酰-肽酰胺酶-γ-谷氨酰-转移酶)依靠一个中间反应在多胺或赖氨酸的伯氨基(氨基供体)与某些蛋白质的谷氨酰γ-羧酰胺基(氨基受体)之间催化形成酰胺键,其中在蛋白质的谷氨酰残基γ-羧酰胺基与酶活性中心的半胱氨酸残基sufidryl基间反应,将酶连接到底物(Serafini-Fracassini,D.,Del Duca,S.& Beninati,S.1 995.Plant Transglutaminases.Phytochemistry 40:355-365):转谷氨酰胺酶活性的结果是:a)蛋白质自身构型修饰和b)其它更广泛的构型改变如蛋白质本身间连接和不同蛋白质间形成高分子量缀合物。
已有许多关于人和动物、植物、低等脊椎动物、某些细菌、藻类和酵母中的转谷氨酰胺酶研究(Makarova,K.S.,Aravind,L.&Koovin,E.V.1999.A superfamily of archaeal,bacterial and eukaryoticproteins homologous to animal transglutaminases Protein Science 8:1714-1719;Bergamini,C.M.,Dean,M.,Tanfani,F.Ferrari,C.& Scatturin.1999.Conformational stability of human erythrocyte transglutaminase:Pattrens of thermal unfolding at acid and alkaline pH.Eur.J. Biochem.266:575-582.;Cariello,L.Ristoratore,F.& Zanitti,L.1997.A newtransglutaminase-like from ascidian Ciona intestinalis.FEBS Lett408:171-176;Lorand,L.& Conrad.S.M.1984.Transglutaminases.Mol.Cell Biochem 58:9-35;Serafini-Fracassini,D.,Del Duca S.& Beninati S.1995.Plant Transglutaminases.Phytochemistry 40:355-365;Tokunaga,F.,Muta,T.Iwanaga,S.,Ichinose,A.,Davie,EW,Kuma,K.& Miyata,T.1993.Limulus hemocyte transglutaminases.cDNA cloning,amino acidsequence and tissue localization.J Biol Chem 268:262-268)。
知道最多的转谷氨酰胺酶是:凝血因子XIII,它是血浆蛋白和参与表皮角质层形成的转谷氨酰胺酶K。另一方面,已经克隆一些负责某些所提到的转谷氨酰胺酶的基因,并且逐渐认识了参与重要过程例如细胞分化、组织稳定性或程序性细胞死亡的转谷氨酰胺酶(Ichinose,A.,Bottenus,R.E.& Davie E.W.1990.Structure oftransglutaminases.J.of Biol.Chemistry.265(23):13411-13414;Bergamini,C.M.,Dean,M.,Tanfani,F.,Ferrari,C.& Scatturin.1999.Dean,M.,Tanfani,F.,Ferrari,C.& Scatturin.1999.Conformationalstability of human erythrocyte transglutaminase:patterns of thermalunfolding at acid and alkaline pH.Eur.J.Biochem.266:575-582;Nemes,Z.,Marekov,L.N.& Steinert,P.M.1999.Involucrin cross-linking bytransglutaminase 1.J.of Biol.Chemistry.274(16):11013-11021)。这些酶似乎也与神经变性性疾病、肿瘤、腹腔疾病等有关,因此,它们是一组临床研究中非常重要的酶。关于这些临床研究,有不同的专利涉及转谷氨酰胺酶:由Orthogene,Inc.1998年申请的美国5,736,132“Method of promoting adhesion between tissue surfaces”;由OklahomaMedical Reserch Eoundation 1998年申请的美国5,726,051“Transglutaminases gene”。
虽然数年前已公开关于植物转谷氨酰胺酶存在的首次数据,但是植物转谷氨酰胺酶的功能仍知之甚少(Icekson I.& Apelbaun,A.1987.Evidences for transglutaminase activity in plant tissue.Plant Physiol.84.972-974;Serafini-Fracassini D.,Del Duca S.,& D′Orazi D.1998.Firstevidence for polyamine conjugatation mediate by an enzyme activity inplants.Plant Physiol.87:757):对植物的研究已首先集中到涉及活性、相同作用底物和高丰度的组织等生物化学方面,但是其功能作用,其中关于其干涉,例如:生长和发育、一般形态发生、光合作用和细胞死亡等的部分数据仍然未有研究(Margosiak,S.A.,Drama,A.,Bruce-Carvcr,M.R.,Gonzalez,A.P.Louie,D.& kuehn.1990.Identification of the large subunit of ribulose 1,5-biphosphatecarboxylase/oxygenase as a substrate for transglutaminase in Medicagosativa L.(Alfafa):P1ant Physiol.92:88-96;Del Ducca,S.,Tidu,V.,Bassi,R.Exposito,C.,& Serafini-Fracassini,D.1994,Identification ofchlorophyll-a/b proteins as substrates of transglutaminase activity inisolated chloroplasts of Helianthus tuberosus L.Planta 193:283-289;DelDucca,S.,Della Mea,M.,
de Rueda,P.& Serafini-Fracassini,D.2000 Factors affecting transglutaminase activity catalyzing polyamineconjugation to endogenous substrates in the entire chloroplast.PlantPhysiol Biochem 38:429-439)。
此外,要强调的是,转谷氨酰胺酶具有生物技术用途的增殖价值。这种作为有用代谢物的新的附加方面来源于其在不同蛋白质间形成共价键的能力。这种特性已用于例如保持商品如鱼类和肉类的品质,减少使用盐的需要(surimi等)。用于不同密度的明胶配方等。用于制备含较少脂肪的预烹调食品(豆腐)。也可能在不同温度下保持产品的稠度、弹性、水分或粘度。同样,它也用于不同的乳制品:奶酪、酸乳、冰淇淋等。到这样的程度以致,近期它在许多生物加工食品中用作“添加剂”,在美国用于这种用途的推荐剂量是65ppm。
转谷氨酰胺酶的所有这些可能性产生不同的专利创造:获得方法、用途等并且已使这种物质成为商业化产品例如,Ajinomoto公司已用Activa TG名称分销的那些产品。上市产品的这些公司是东京的Ajinomoto Co.,Inc.(也遍及美国)和美国的Rohm Enzyme(
www. skidmore-sales.com/whatsnew/newsletter/summer 2001.pdf):然而,在西班牙还没有发现专门生产这种转谷氨酰胺酶工业产品的公司。
如上所提到的,用于商业用途的第一种转谷氨酰胺酶已由Ajinomoto公司用细菌(链霉轮枝苗条a菌(Streptoverticillium sp.))实现过量表达,该公司取得所述方法以及后来这个最初方案不同改进的专利(美国5,156,956“Transglutaminase”(1992))。同样,该公司已取得另一相似系统,但是依靠转化牡蛎(Crassostrea gigas)(美国5,736,356“Transglutaminase originating from Crassostrea gigas(1998))和来源于枯草芽孢杆菌(Bacillus subtilus)的专利(美国5,948,662“Bacillus-derived transglutaminase”(1999))。
同样,在最近几年,本研究小组即本发明的发明人已完成生物化学水平的前期研究。关于涉及玉米愈伤组织形态发生的转谷氨酰胺酶和它们与光的关系(Bernet,E.,Claparols,I.,Dondini,L.,Santos,M.A.,Serafini-Fracassini,D.& Torné,J.Ma.1999.Changes in polyaminecontent,arginine and ornithine decarboxylases and transglutaminaseactivities during light/dark phases(of initial differentiation)in maizecalluses and their chloroplast.Plant Physio Biochem.37(12):899-909):此外,该酶在不同玉米细胞系统中,与叶绿体的发育相关的免疫定位最近已公开(Villalobos,E.Torné,J.M.,Ollés,C.,Claparols,I.& Santos,M.A.2001,Subcellular localization of a transglutaminase related to granadevelopment in different maize cell types.Protoplasma.216:155-163)。然而,没有发现在植物转谷氨酰胺酶分子鉴定和功能活性方面的结果,因此,关于所述转谷氨酰胺酶的新知识是最高的商业化目标。
发明内容
概述
本发明面临涉及来源于植物,在食品处理和转化领域和具有新能力的转基因植物开发中所需的转谷氨酰胺酶短缺的问题。
本发明提供的解决方案是基于本发明人已鉴定某些来源于玉米具有转谷氨酰胺酶活性(TGase;EC2.3.13)的DNA序列的事实。由所述DNA序列编码的蛋白质的转谷氨酰胺酶活性在这些蛋白质提取物的实验中已变得显而易见。
因此,本发明的一个目的是所述DNA分子。
本发明另一个附加目的是一种至少包含所述DNA分子之一的载体。
本发明另一个附加目的包括所述DNA分子或所述载体的应用,用于产生能表达具有转谷氨酰胺酶活性的重组蛋白质的转化细胞,或将具有转谷氨酰胺酶活性的蛋白质的所述编码序列导入植物细胞中。微生物细胞和产生的转基因植物也包括本发明的附加目的。
本发明另一个附加目的包括由所述DNA序列表达的具有转谷氨酰胺酶活性的蛋白及其在食品处理及转化中的应用。
发明详述
本发明提供来源于植物并且编码具有转谷氨酰胺酶活性的蛋白质的DNA分子,在下文称为本发明的DNA分子,所述DNA分子包含选自下列之一的核苷酸序列:
a)已鉴定为SEQ ID NO 1、SEQ ID NO 3或其片段的核苷酸序列,和
b)与a)中定义的序列相似的核苷酸序列。
从本说明书中所用的意义上说,术语“相似的”是指包括任何在SEQ ID NO 1或SEQ ID NO 3中所示的核苷酸序列基础上分离或制备的DNA序列,例如,依靠引入保守或非保守的核苷酸取代,包括插入一个或多个核苷酸,在所述分子的任一末端添加一个或多个核苷酸或在所述序列的任一末端或内部缺失一个或多个核苷酸。
一般而言,相似DNA分子与鉴定为SEQ ID NO 1或SEQ ID NO3的核苷酸序列基本同源。从本说明书中所用的意义上说,术语“基本同源的”是指查询的核苷酸序列的同一性程度,在核苷酸水平至少为60%,优选至少为85%,或更优选至少为95%。
本发明的DNA分子来源于玉米并且可在高等植物的其它种属(其中包括水稻、小麦、拟南芥等)中发现相似的形式,它们可以是天然形式或在另外的情况下,它们也可以是基因转化加工的结果,其中所述转化的生物体复制所述DNA分子。本发明的DNA分子可以用常规技术,从含有它的任何植物DNA中,依靠使用本发明提供的所述DNA分子的核苷酸序列信息制备的探针或寡核苷酸分离。
本发明的DNA分子包括具有所述转谷氨酰胺酶活性的片段。
在一个具体的实施方案中,本发明的DNA分子是玉米的SEQ IDNO 1或SEQ ID NO 3的DNA分子。
本发明的DNA分子通常可以用于产生允许这些具有转谷氨酰胺酶活性的蛋白在各种各样的宿主细胞中表达的表达载体,在下文称为本发明的表达载体。一般而言,本发明的表达载体至少包含一种本发明的DNA序列和至少一个与其有效连接的、指导目标基因转录的启动子以及其它必需的或适于目标基因转录和适当调节时间和位置的序列,例如起始信号和终止信号、切割位点、聚腺苷酸化信号、复制源、转录增强子、转录沉默子等。合适的表达载体的实例可根据每一具体情况的条件和要求选择下列之一:质粒、酵母人工染色体(YAC)、细菌人工染色体(BAC)、基于P1噬菌体的人工染色体(PAC)、粘粒或病毒,它也可以含有可以在细菌或酵母中扩增的细菌源或酵母复制源,及用于选择除一种或多种目标基因外所转染的细胞的标记。因此,本发明也涉及包含本发明DNA分子的载体。载体的选择将取决于随后将导入所述载体的宿主细胞。例如,导入所述DNA序列的载体可以是质粒,当将其导入到宿主细胞中时,它可以整合到所述细胞的基因组并与宿主细胞的染色体一起复制。
本发明的载体可以由本领域的专家通过已知的常规方法获得(Kovesdi等1997.Curr Opin Biotech 8:583-589 Transgenic Res.10:83-103;Coffin等1998.Retroviruses,CSHLP;Robbins等1998.TrendsBiotech.16:35-40;Anderson.1998.Nature 392:25-30;Schindelhauer.1999.BioEssays 21:76-83):本发明的一个具体目的包括分别含有SEQID NO 1和SEQ ID NO3的质粒pGEMT15和质粒pGEMT21。
本发明也提供包含本发明DNA分子或表达载体的细胞。可用所述表达载体转化的宿主细胞可以是例如GRAS细菌细胞和酵母。含有本发明表达载体的细胞可以用于过量产生本发明DNA分子编码的具有转谷氨酰胺酶活性的蛋白质。本发明的一个具体目的包括具有转谷氨酰胺酶活性的蛋白质,其中具有SEQ ID NO 2和SEQ ID NO 4所描述的氨基酸序列。
这些结果可以创造新的可能性,以通过异源表达转化有用的GRAS(通常公认安全)细菌系统或酵母,以制备所提到的新的转谷氨酰胺酶蛋白。如上文已指出的,具有转谷氨酰胺酶活性的蛋白质,由于它在不同蛋白质间产生共价键的能力,可以用于多种食品处理、加工和转化过程。这一特征已用于例如保持食品如鱼类和肉类的品质、减少使用盐的需要,参见美国专利5928689“Method for treatingPSE meat with transglutaminase”,WO 0162888“Improved compositionof marine product”;有关生产不同密度的明胶;用于制备含较少脂肪的预烹调食品(豆腐),参见美国6342256“Tofu products excellent infreeze resistance and process for producing the same”,美国6042851“Process for producing packed tofu”。它也可能在不同的温度下保持产品的稠度、弹性、水分或粘度。同样,它可用于不同的乳制品加工食物:乳酪(美国6270814“Incorporation of whey into process cheese”,美国申请20010053398“Cheese whey protein having improved textureprocess for producing the same and use thereof”)、酸乳、冰淇淋、蛋黄酱、酱油,以及用于生产面条(EP 0948905“Enzyme preparationscomprising transglutaminase and process for producing noodles”,美国6106887“Process for obtaining a modified cereal flour),巧克力(美国6063408“Process for producing chocolate”),源自马玲薯的产品(美国申请20020004085“Methods for producing potato products”),糖(JP200354498“Production of sugar from cereal flour material bytransglutaminase treatment”)。其中在前述专利中描述的转谷氨酰胺酶的不同用途是本发明转谷氨酰胺酶潜在用途的实例。因此,本发明的一个具体目的是具有本发明转谷氨酰胺酶活性的蛋白质即蛋白质SEQ ID NO 2和SEQ ID NO 4或含有它们的溶液在食品处理、加工和转化中的应用。在下文Chiya Kuraishi等的综述,2001(Transglutaminase:Its utilization in the food industry Food ReviewsInternational17(2):221-246),表示具有本发明转谷氨酰胺酶活性的蛋白质应用的一个实例。
最后,还有其它的应用,所述应用不同于以上提及的具有转谷氨酰胺酶活性的本发明蛋白质的应用方面和那些作为以下专利所述应用举例说明的方面,其中“Method for enzymatic treatment of wool”美国专利申请第161824号(1998)MacDevitt等,2000年4月;“Enzymatically protein encapsulating oil particles by complexcoacervation申请第791953号(1997).Soper,Jon C.等,2000年3月;“Cross-linked gelatin gels and method of making them”申请第641463号(1996)Bishop,P.D.等,ZymoGenetics,Inc.(Seattle,EA,USA);Process for obtaining a modified cereal flour”申请第977575号Ajinomoto Co.Inc.(Tokyo,Japan).Yamazaki等,2000年8月;“Microbial transglutaminase,their production and use”申请第294565号(1999).NovoNordisk A/S(Bagsvaerd,DK)Bech等,2001年2月。
此外,本发明的DNA分子或表达载体可用于基础研究中植物的遗传转化过程以及依靠改变所述蛋白质的表达,通过对所述转谷氨酰胺酶特征功能(植物生长和发育、形态发生、光合作用和细胞死亡)的操作,可以开发具有新能力的转基因植物。
附图描述
图1.-对应于方法学部分描述的每一个噬溶菌产物、对应于阳性噬菌体f1和f2(含有玉米转谷氨酰胺酶的不同cDNA:f1=SEQ IDNO 1,f2=SEQ ID NO 3)和对应于阴性噬菌体f3(不含任何转谷氨酰胺酶的cDNA)的蛋白质提取物的转谷氨酰胺酶活性(Put公司测定,以皮摩尔计)。此外,显示影响提取物转谷氨酰胺酶活性的不同因素的效果,在其它系统中所描述的所述酶促转谷氨酰胺酶活性:钙=蛋白质提取物和无钙时。GTP=加入的lmM GTP。MDC=加入的1mMMDC。
图2.-对应于含有这两种转谷氨酰胺酶cDNA(f1=SEQ ID NO1;f2=SEQ ID NO 2)的两种独立噬菌体的两种蛋白质提取物的活性,关于不含有这些cDNA的任一种噬菌体(f3),关于所述试验的蛋白质量。如在方法学部分所描述的,包括生物素尸胺测定,所述活性以转谷氨酰胺酶毫单位(mU)计。
a=40mg蛋白质/ml。b=60mg蛋白质/ml。c=80mg蛋白质/ml。
发明实施例
实施例1.-通过免疫筛选分离和克隆两种编码玉米转谷氨酰胺酶家族的两种蛋白质的cDNA
表达库
本发明的cDNA是从温室条件下生长的两周龄Zea mays subsp.mays plantulae B73纯合子(由美国University of Oregon的Alice Barkan博士赠予)的信使RNA为原料,用EcoRI和XhoI靶制备的Lambda-ZAPIIcDNA表达库中分离。
用从菊芋(Helianthus tuberosus)叶片的叶绿体提取物纯化的58kda植物转谷氨酰胺酶作为抗原。在母鸡中获得多克隆抗体(Villalobos,E.,Torné,J.M.,Ollés,C.,Claparols,I.& Santos,MA.2001.Subcellularlocalization of a transglutaminases related to grana development indifferent maize cell types.Protoplasma.216:155-163)。通过斑点印迹技术,使用商业化的猪肝脏转谷氨酰胺酶,以及通过纯化蛋白质的蛋白质印迹,测定所述抗体的特异性(Dondini,L.1998.“Poliammine legatee transglutaminasi nelle plante.”博士论文。University of Bologana,Italy)。通过蛋白质印迹技术进行滴定(完全的方法学在我们的研究中详细说明:Villalobos,E.,Torné,J.M.,ollés,C.-,Claparols,I.& Santos,M.A.2001.Subcellular localization of a transglutaminases related tograna development in different maize cell types.Protoplasma.216:155-163)。
库的免疫筛选
一旦已知使用库的名称,就可以将XL-Blue菌株的菌落接种到含有MgSO4和20%麦芽糖的液体LB培养基中。
在细菌生长达到2.0 DO(600nm)后,从加有10mM IPTG的文库取4.5×104pfu制备细菌培养混合物。在感染和接种装有LB培养基+10mM MgSO4的培养皿后,将一张10mM IPTG饱和的圆盘状硝酸纤维素膜置于菌落之上。将加有滤膜的培养皿培养4小时后,将其冷却并且用PBS洗涤滤膜。最后,一旦用脱脂奶粉或BSA封闭膜,就可用抗体显像和标记。为检测溶菌作用,已发现了抗菊芋(H.tuberosus)转谷氨酰胺酶抗体与相互作用的阳性噬菌体,可进行所述膜的蛋白质印迹分析并且用ECL试剂在感光片上显像。
体内切除pBluescript SK-中的噬菌粒并筛选阳性菌落
一旦已分离和纯化含有分别编码与抗体相互作用的蛋白质的cDNA的两种噬菌体,然后就可通过“ExAssistTM Interference-ResistantHelper phage(Stratagene)”切除它们。在XL1-Blue菌株中进行共感染并且在XLOLR进行感染。在选择性培养基中铺皿,测定使用的载体(pBluescript)。在我们的案例中,选择转化菌落的培养基是加入氨苄青霉素(50μg/ml)、1mM IPTG和其基因由于插入片段或cDNA而间断的半乳糖苷酶底物X-Gal(40μg/ml)的LB-琼脂。
小规模质粒分离(小量制备)
对于每一次切除,分离含有目标cDNA的质粒DNA,可通过细菌裂解物的小规模小量制备技术进行,使用SDS和NaOH,用乙酸钾中和并且用苯酚∶氯仿∶异戊醇(25∶24∶1)混合物纯化和用乙醇沉淀。然后用加有RNA酶的1XTE缓冲液重悬浮。
检查pBluescript载体中存在的cDNA
在每种情况下,可通过消化质粒DNA样品,检查pBluescript中存在的插入片段,可用与制备所述库(EcoR1和Xhol)相同的内切核酸酶获得。根据每一种限制性酶的要求(缓冲液和温度),对其进行消化。一旦进行消化,所述cDNA或插入片段就从载体中释放出来。这可用常规电泳在1X TBE或1X TAE缓冲液的0.5%琼脂糖凝胶中检查。
测序(Barcelona IBMB“CSIC”的测序服务)
一旦已确定含有目标cDNA的小量制备样品,在我们的案例中是制备两个样品,就可在测序步骤前沉淀它们并用苯酚、氯仿和异戊醇混合物和纯氯仿进行纯化。用水溶解将要测序的样品。
通过RACE技术测定完全编码序列
pBluescript SK-中的两种噬菌体切除物使得有可能获得两种通过RACE技术定义的完全编码序列的部分cDNA。为此目的,可从玉米叶提取的总RNA通过多聚dT柱纯化获得用作单链DNA合成模板的信使RNA。为了按此进行,从已知的cDNA序列和逆转录酶推导出特异性寡核苷酸(寡核苷酸E1,3′-5′:GATTCTCCCTGATAAG,SEQ IDNO 5)。在通过末端脱氧核苷酸转移酶(TdT)将多聚T尾加到单链DNA后,获得第二条DNA链。可通过PCR技术用寡核苷酸5′RACEAbrigded Anchor Primer(GIBCO BRL)、多聚T尾DNA特异的(寡核苷酸锚5′-3′:GGCCAGGCGTCGACTAGTACGGGIIGGGIIGGGIIG,SEQ ID NO 6)和第二条以上说明的具有已知序列的部分cDNA的特异性寡核苷酸,以及对应于寡核苷酸E2,3′-5′:GTTCTCCAGCATCTCCAG,SEQ ID NO 7)的特异性寡核苷酸完成。
随后用PCR循环扩增所述DNA。所述循环的顺序如下:首先在94℃2分钟,然后34次循环的:对于1号寡核苷酸为94℃30秒,但对于2号寡核苷酸为60℃30秒,接着在这两种情况下72℃7秒。最后,置于5℃几小时。
用连接酶将PCR产物克隆到合适的载体(例如pGEMT)中。然后,转化大肠杆菌DH5-α型菌株并且让其在选择性培养基中生长。质粒DNA用上述的小量制备技术提取、纯化并且测序所获得的片段。在我们的案例中,对于两条需要完成所述编码序列片段的部分cDNA序列,证实仅有四种核苷酸。完全的编码序列核苷酸,包括所述四种通过RACE技术获得的核苷酸,分别在SEQ ID NO 1和SEQ ID NO3中描述。含有SEQ ID NO 1和SEQ ID NO 3序列并用于转化宿主细胞的表达载体分别是质粒pGEMT15和pGEMT21。
从核苷酸序列获得的氨基酸序列与其它非植物系统所描述的转谷氨酰胺酶类的活性中心域,在对应于氨基酸:SEQ ID NO 2蛋白质的431-474(60.97 kDa)和SEQ ID NO 4蛋白质的485-528(67KDa)的区域内具有同源性。在这两种情况下,在这些区域中发现描述为所述酶活性的必需氨基酸的半胱氨酸(Cys)(SEQ ID NO 2中的Cys439和SEQ ID NO 4中的Cys493)。数据库参考:(
www.ncbi.nlm.nih/)。此外,如SEQ ID NO 1和SEQ ID NO 3序列所表示的,观察到一些在SEQ ID NO 1和SEQ ID NO 3两种序列中串联重复的27个核苷酸的区域,虽然量不同,分别为15-21个重复并且其中具有一些核苷酸的少量变异。应该强调的是,以前在已知的转谷氨酰胺酶中并未描述过这些提到的重复区。因此,它们是本发明DNA分子的特征。
实施例2.-检查由所述cDNA表达的蛋白质的转谷氨酰胺酶活性
测定由cDNA表达的蛋白质的转谷氨酰胺酶活性
2个含有目标cDNA的噬菌体克隆,用每一个克隆在加有10mMIPTG的液体LB培养基中感染大肠杆菌(XL-Blue菌株)培养物。在37℃溶菌后,提取物的总蛋白质浓度用Lowry法定量(Lowry OH,Rosebrough NJ,Farr,AL & Randall RJ.1951.Protein measurement withthe Folin phenol reagent.J.Biol.Chem.193:265-275)并且为了测定转谷氨酰胺酶的活性,与不含有目标cDNA的噬菌体溶菌提取物形成对比,可用提取物进行在下文描述的实验。
1.通过测定氚标记的腐胺标记的蛋白质检测转谷氨酰胺酶活性
的方法。
用每一种由两种噬菌体(含SEQ ID NO 2的转谷氨酰胺酶的f1和含SEQ ID NO 4的转谷氨酰胺酶的f2)获得的溶菌提取物制备酶提取物,总蛋白质浓度600μg并且酶实验在30℃进行30分钟。酶混合物除含有蛋白质提取物以外,还含有0.6mM腐胺、185kBq氚标记腐胺(0.85TBq/nmol)、20mM Tris-HCl*pH8和3 mM CaCl2。反应用含2mM腐胺的10%三氯乙酸封闭。重复沉淀样品并且测量沉淀物的放射性(Bernet,E.,Claparols,L.,Dondini,L.,Santos,M.A.,Serafini-Fracassini,D.-& Torné,J.Ma 1999):Changes in polyamide content,arginine and ornithine decarboxylases and transglutaminase activitiesduring light/dark phase(of initial differentiation)in maize calluses andtheir chloroplast.Plant Physio Biochem.37(12):899-909)。用每小时每毫克蛋白质的腐胺皮摩尔量测量转谷氨酰胺酶活性,并且从f1和f2噬菌体获得的蛋白质提取物的转谷氨酰胺酶活性大于不含有任何这些转谷氨酰胺酶cDNA的噬菌体提取物的转谷氨酰胺酶活性。
2.通过Elisa类实验,用CBZ-Gln-Gly作第一底物而用生物素
尸胺作第二底物检测转谷氨酰胺酶活性的方法。
该实验包括由Covalab公司提供的试剂盒,该试剂盒用小量总蛋白质测定样品的转谷氨酰胺酶活性,与商业化的猪肝脏转谷氨酰胺酶比较。所述方法通过比色实验,检测由样品转谷氨酰胺酶活性以肽和多胺底物形成的谷氨酰衍生物。按转谷氨酰胺酶单位测量所述活性,以450nm处的1±0.05 OD吸光率对应于0.6mU的商业化转谷氨酰胺酶。
对应于两种溶菌产物的两种蛋白质提取物,与任何来自不含有这些cDNA的噬菌体提取物相比较,在检测上文所用和所描述的(f1和f2)所述活性的两种方法中显示转谷氨酰胺酶类活性。数据示于图1和图2。
此外,图1表示不同因素对提取物的转谷氨酰胺酶活性的影响,描述为天生的对所述转谷氨酰胺酶活性的影响。因此,已表达的蛋白质活性在a)无钙,b)存在1mM GTP,c)存在1mMdenodansylcadaverine(MDC)和d)所述溶菌提取物含有未携带目标cDNA的噬菌体(f3)时显著减少。
分别用含玉米cDNA的质粒(pBlueScript)和含编码玉米SEQ IDNO 2和SEQ ID NO 4序列蛋白质的基因的质粒的载体转化的一对源自大肠杆菌dH5α型的细菌培养物,鉴定为15TGZM02和21TGZM02,已于2002年5月7(?)日保藏于西班牙典型培养物保藏中心(Spanish Culture Type collection)(“Collección
de CultivosTipo(CECT”),University of Valencia,Research Building,BurjasotCampus,46100 Burjasot,Valencia,Spain。对应于它们的“CECT"保藏号分别是:对于15TGZM02是5705,对于21TGZM02是5706。
<110>CONSEJO SUPERIOR DE INVESTIGACIONES CIENTIFICAS
<120>编码具有转谷氨酰胺酶活性的蛋白质的玉米核苷酸序列
<130>玉米转谷氨酰胺酶
<160>7
<170>PatentIn version 3.1
<210>1
<211>1748
<212>DNA
<213>玉蜀黍(Zea mays L)
<220>
<221>重复区
<222>(823)..(1228)
<223>重复单位(823)..(849)重复数:15个重复
<220>
<221>3′UTR
<222>(1606)..(1729)
<223>
<220>
<221>聚腺苷酸位点
<222>(1730)..(1748)
<223>
<220>
<221>聚腺苷酸位点
<222>(1730)..(1748)
<223>
<220>
<221>CDS
<222>(1)..(1605)
<223>
<300>
<308>AJ421525
<309>2001-12-06保密直到2002-12-06
<400>1
atg gct cat cgt gga cat cta gat gga ctg act ggc caa gct cct gct 48
Met Ala His Arg Gly His Leu Asp Gly Leu Thr Gly Gln Ala Pro Ala
1 5 10 15
ctt atg cgc cat ggt tcc ttc gct gca ggc agc ctc tct agc cgc tca 96
Leu Met Arg His Gly Ser Phe Ala Ala Gly Ser Leu Ser Ser Arg Ser
20 25 30
cct ttg cag tct tca tcc aca ctg gag atg ctg gag aac aag ctt gcc 144
Pro Leu Gln Ser Ser Ser Thr Leu Glu Met Leu Glu Asn Lys Leu Ala
35 40 45
atg caa act aca gaa gtg gaa aag ctt atc acg gag aat cag cgg tta 192
Met Gln Thr Thr Glu Val Glu Lys Leu Ile Thr Glu Asn Gln Arg Leu
50 55 60
gca tca agc cat gtg gtc ttg agg cag gac att gtt gat acg gag aaa 240
Ala Ser Ser His Val Val Leu Arg Gln Asp Ile Val Asp Thr Glu Lys
65 70 75 80
gag atg caa atg atc cgc acc cac cta ggt gaa gtt cag aca gag act 288
Glu Met Gln Met Ile Arg Thr His Leu Gly Glu Val Gln Thr Glu Thr
85 90 95
gat ttg cag att aga gat ttg ttg gag aga atc aga tta atg gag gta 336
Asp Leu Gln Ile Arg Asp Leu Leu Glu Arg Ile Arg Leu Met Glu Val
100 105 110
gat ata cat agt ggt aat gta gtg aac aag gag ctt cac caa atg cat 384
Asp Ile His Ser Gly Asn Val Val Asn Lys Glu Leu His Gln Met His
115 120 125
atg gag gca aag aga ctt att act gaa agg cag atg cta acc ctt gag 432
Met Glu Ala Lys Arg Leu Ile Thr Glu Arg Gln Met Leu Thr Leu Glu
130 135 140
ata gag gat gtg act aaa gaa tta cag aaa ctc tct gcc tct ggg gac 480
Ile Glu Asp Val Thr Lys Glu Leu Gln Lys Leu Ser Ala Ser Gly Asp
145 150 155 160
aat aaa agc ctt cct gaa ttg ctt tct gag cta gat agg cta cgg aaa 528
Asn Lys Ser Leu Pro Glu Leu Leu Ser Glu Leu Asp Arg Leu Arg Lys
165 170 175
gag cat cat aat tta cga tct cag ttt gaa ttt gag aaa aat aca aac 576
Glu His His Asn Leu Arg Ser Gln Phe Glu Phe Glu Lys Asn Thr Asn
180 185 190
gtc aag caa gtt gag cag atg cgg aca atg gaa atg aac ttg ata acc 624
Val Lys Gln Val Glu Gln Met Arg Thr Met Glu Met Asn Leu Ile Thr
195 200 205
atg acc aaa caa gct gag aag tta cgt gtt gat gtg gca aat gct gaa 672
Met Thr Lys Gln Ala Glu Lys Leu Arg Val Asp Val Ala Asn Ala Glu
210 215 220
aga cgg gca caa gca gct gcg gct caa gca gca gca cat gca gct ggt 720
Arg Arg Ala Gln Ala Ala Ala Ala Gln Ala Ala Ala His Ala Ala Gly
225 230 235 240
gca cag gtg aca gct tcg cag cct gga cag ctc aag cta cca cgg ttt 768
Ala Gln Val Thr Ala Ser Gln Pro Gly Gln Leu Lys Leu Pro Arg Phe
245 250 255
cag cag cag cag cca cag act cat atg cag gtg cat ata cca gct acc 816
Gln Gln Gln Gln Pro Gln Thr His Met Gln Val His Ile Pro Ala Thr
260 265 270
ccc ctg cat atc agc agg gag ccc agg ctg ggg cat atc agc agg gtg 864
Pro Leu His Ile Ser Arg Glu Pro Arg Leu Gly His Ile Ser Arg Val
275 280 285
ctc agg ctg ggg tat atc agc agg gag ccc agg ctg ggg cat atc agc 912
Leu Arg Leu Gly Tyr Ile Ser Arg Glu Pro Arg Leu Gly His Ile Ser
290 295 300
agg gag ccc agg ctg ggg cat atc agc agg ggg gcc agg atg ggg cat 960
Arg Glu Pro Arg Leu Gly His Ile Ser Arg Gly Ala Arg Met Gly His
305 310 315 320
atc agc agg ggg ctc agg ctg ggg cat atc agc agg gag ccc agg ctg 1008
Ile Ser Arg Gly Leu Arg Leu Gly His Ile Ser Arg Glu Pro Arg Leu
325 330 335
ggg cat atc agc agg gag ccc agg ctg ggg cat atc agc agg gtg ctc 1056
Gly His Ile Ser Arg Glu Pro Arg Leu Gly His Ile Ser Arg Val Leu
340 345 350
agg ctg ggg cat atc agc agg gag ccc agg ctg ggg cat atc agc agg 1104
Arg Leu Gly His Ile Ser Arg Glu Pro Arg Leu Gly His Ile Ser Arg
355 360 365
ggg ccc agt ctg ggg cat atc agc agg ggg ccc agg ctg ggg cat atc 1152
Gly Pro Ser Leu Gly His Ile Ser Arg Gly Pro Arg Leu Gly His Ile
370 375 380
agc agg gag ccc agg atg ggg cat atc agc agg gag ccc agg atg ggg 1200
Ser Arg Glu Pro Arg Met Gly His Ile Ser Arg Glu Pro Arg Met Gly
385 390 395 400
cat atc agc agg gtg ctc agg ctg gag cat aca act atg ctt atg atg 1248
His Ile Ser Arg Val Leu Arg Leu Glu His Thr Thr Met Leu Met Met
405 410 415
ctg gca cgg ctt atg cat atg cag gtt act ctg gct atc cag ttg cag 1296
Leu Ala Arg Leu Met His Met Gln Val Thr Leu Ala Ile Gln Leu Gln
420 425 430
gct acg cgc aaa gtg cag tgc cca act att cct atg ctg cac ctc cgc 1344
Ala Thr Arg Lys Val Gln Cys Pro Thr Ile Pro Met Leu His Leu Arg
435 440 445
agc caa caa gca gcg gtg cag cta cga acg ccg cag gag gcc agt atg 1392
Ser Gln Gln Ala Ala Val Gln Leu Arg Thr Pro Gln Glu Ala Ser Met
450 455 460
ggg cag ttg gta gtg ctg gat atc cta ctg ggc aag ttc agc cga gca 1440
Gly Gln Leu Val Val Leu Asp Ile Leu Leu Gly Lys Phe Ser Arg Ala
465 470 475 480
gtg gca ctg caa atg cag cgc aag cac ctc ctc ctc cac cac cac cgg 1488
Val Ala Leu Gln Met Gln Arg Lys His Leu Leu Leu His His His Arg
485 490 495
cag cac cat atc ccc cca gca cat atg acc aaa cca gag gag ccc aga 1536
Gln His His Ile Pro Pro Ala His Met Thr Lys Pro Glu Glu Pro Arg
500 505 510
gat aaa atc tgg gat gta aac cag atg gat gtt tgc cat gca cat ttg 1584
Asp Lys Ile Trp Asp Val Asn Gln Met Asp Val Cys His Ala His Leu
515 520 525
ttg agc aga caa ata tgg tga aatctgggat gtaaaaccag atggctgtct 1635
Leu Ser Arg Gln Ile Trp
530
gtgcctccat cccattgact agggcgtatt ttcaccaata ttgtgcctcc agtgcaattt 1695
cttctgtgtt atatatcacc accatttgtt gagcaaaaaa aaaaaaaaaa aaa 1748
<210>2
<211>534
<212>PRT
<213>玉蜀黍(Zea mays L)
<400>2
Met Ala His Arg Gly His Leu Asp Gly Leu Thr Gly Gln Ala Pro Ala
1 5 10 15
Leu Met Arg His Gly Ser Phe Ala Ala Gly Ser Leu Ser Ser Arg Ser
20 25 30
Pro Leu Gln Ser Ser Ser Thr Leu Glu Met Leu Glu Asn Lys Leu Ala
35 40 45
Met Gln Thr Thr Glu Val Glu Lys Leu Ile Thr Glu Asn Gln Arg Leu
50 55 60
Ala Ser Ser His Val Val Leu Arg Gln Asp Ile Val Asp Thr Glu Lys
65 70 75 80
Glu Met Gln Met Ile Arg Thr His Leu Gly Glu Val Gln Thr Glu Thr
85 90 95
Asp Leu Gln Ile Arg Asp Leu Leu Glu Arg Ile Arg Leu Met Glu Val
100 105 110
Asp Ile His Ser Gly Asn Val Val Asn Lys Glu Leu His Gln Met His
115 120 125
Met Glu Ala Lys Arg Leu Ile Thr Glu Arg Gln Met Leu Thr Leu Glu
130 135 140
Ile Glu Asp Val Thr Lys Glu Leu Gln Lys Leu Ser Ala Ser Gly Asp
145 150 155 160
Asn Lys Ser Leu Pro Glu Leu Leu Ser Glu Leu Asp Arg Leu Arg Lys
165 170 175
Glu His His Asn Leu Arg Ser Gln Phe Glu Phe Glu Lys Asn Thr Asn
180 185 190
Val Lys Gln Val Glu Gln Met Arg Thr Met Glu Met Asn Leu Ile Thr
195 200 205
Met Thr Lys Gln Ala Glu Lys Leu Arg Val Asp Val Ala Asn Ala Glu
210 215 220
Arg Arg Ala Gln Ala Ala Ala Ala Gln Ala Ala Ala His Ala Ala Gly
225 230 235 240
Ala Gln Val Thr Ala Ser Gln Pro Gly Gln Leu Lys Leu Pro Arg Phe
245 250 255
Gln Gln Gln Gln Pro Gln Thr His Met Gln Val His Ile Pro Ala Thr
260 265 270
Pro Leu His Ile Ser Arg Glu Pro Arg Leu Gly His Ile Ser Arg Val
275 280 285
Leu Arg Leu Gly Tyr Ile Ser Arg Glu Pro Arg Leu Gly His Ile Ser
290 295 300
Arg Glu Pro Arg Leu Gly His Ile Ser Arg Gly Ala Arg Met Gly His
305 310 315 320
Ile Ser Arg Gly Leu Arg Leu Gly His Ile Ser Arg Glu Pro Arg Leu
325 330 335
Gly His Ile Ser Arg Glu Pro Arg Leu Gly His Ile Ser Arg Val Leu
340 345 350
Arg Leu Gly His Ile Ser Arg Glu Pro Arg Leu Gly His Ile Ser Arg
355 360 365
Gly Pro Ser Leu Gly His Ile Ser Arg Gly Pro Arg Leu Gly His Ile
370 375 380
Ser Arg Glu Pro Arg Met Gly His Ile Ser Arg Glu Pro Arg Met Gly
385 390 395 400
His Ile Ser Arg Val Leu Arg Leu Glu His Thr Thr Met Leu Met Met
405 410 415
Leu Ala Arg Leu Met His Met Gln Val Thr Leu Ala Ile Gln Leu Gln
420 425 430
Ala Thr Arg Lys Val Gln Cys Pro Thr Ile Pro Met Leu His Leu Arg
435 440 445
Ser Gln Gln Ala Ala Val Gln Leu Arg Thr Pro Gln Glu Ala Ser Met
450 455 460
Gly Gln Leu Val Val Leu Asp Ile Leu Leu Gly Lys Phe Ser Arg Ala
465 470 475 480
Val Ala Leu Gln Met Gln Arg Lys His Leu Leu Leu His His His Arg
485 490 495
Gln His His Ile Pro Pro Ala His Met Thr Lys Pro Glu Glu Pro Arg
500 505 510
Asp Lys Ile Trp Asp Val Asn Gln Met Asp Val Cys His Ala His Leu
515 520 525
Leu Ser Arg Gln Ile Trp
530
<210>3
<211>1910
<212>DNA
<213>玉蜀黍(Zea mays L)
<220>
<221>重复区
<222>823)..(138 9)
<223>重复单位(823)..(849)重复数:21个重复
<220>
<221>CDS
<222>(1)..(1764)
<223>
<220>
<221>聚腺苷酸位点
<222>(1892)..(1910)
<223>
<220>
<221>3′UTR
<222>(1765)..(1891)
<223>
<400>3
atg gct cat cgt gga cat cta gat gga ctg act ggc caa gct cct gct 48
Met Ala His Arg Gly His Leu Asp Gly Leu Thr Gly Gln Ala Pro Ala
1 5 10 15
ctt atg cgc cat ggt tcc ttc gct gca ggc agc ctc tct agc cgc tca 96
Leu Met Arg His Gly Ser Phe Ala Ala Gly Ser Leu Ser Ser Arg Ser
20 25 30
cct ttg cag tct tca tcc aca ctg gag atg ctg gag aac aag ctt gcc 144
Pro Leu Gln Ser Ser Ser Thr Leu Glu Met Leu Glu Asn Lys Leu Ala
35 40 45
atg caa act aca gaa gtg gaa aag ctt atc acg gag aat cag cgg tta 192
Met Gln Thr Thr Glu Val Glu Lys Leu Ile Thr Glu Asn Gln Arg Leu
50 55 60
gca tca agc cat gtg gtc ttg agg cag gac att gtt gat acg gag aaa 240
Ala Ser Ser His Val Val Leu Arg Gln Asp Ile Val Asp Thr Glu Lys
65 70 75 80
gag atg caa atg atc cgc acc cac cta ggt gaa gtt cag aca gag act 288
Glu Met Gln Met Ile Arg Thr His Leu Gly Glu Val Gln Thr Glu Thr
85 90 95
gat ttg cag att aga gat ttg ttg gag aga atc aga tta atg gag gta 336
Asp Leu Gln Ile Arg Asp Leu Leu Glu Arg Ile Arg Leu Met Glu Val
100 105 110
gat ata cat agt ggt aat gta gtg aac aag gag ctt cac caa atg cat 384
Asp Ile His Ser Gly Asn Val Val Asn Lys Glu Leu His Gln Met His
115 120 125
atg gag gca aag aga ctt att act gaa agg cag atg cta acc ctt gag 432
Met Glu Ala Lys Arg Leu Ile Thr Glu Arg Gln Met Leu Thr Leu Glu
130 135 140
ata gag gat gtg act aaa gaa tta cag aaa ctc tct gcc tct ggg gac 480
Ile Glu Asp Val Thr Lys Glu Leu Gln Lys Leu Ser Ala Ser Gly Asp
145 150 155 160
aat aaa agc ctt cct gaa ttg ctt tct gag cta gat agg cta cgg aaa 528
Asn Lys Ser Leu Pro Glu Leu Leu Ser Glu Leu Asp Arg Leu Arg Lys
165 170 175
gag cat cat aat tta cga tct cag ttt gaa ttt gag aaa aat aca aac 576
Glu His His Asn Leu Arg Ser Gln Phe Glu Phe Glu Lys Asn Thr Asn
180 185 190
gtc aag caa gtt gag cag atg cgg aca atg gaa atg aac ttg ata acc 624
Val Lys Gln Val Glu Gln Met Arg Thr Met Glu Met Asn Leu Ile Thr
195 200 205
atg acc aaa caa gct gag aag tta cgt gtt gat gtg gca aat gct gaa 672
Met Thr Lys Gln Ala Glu Lys Leu Arg Val Asp Val Ala Asn Ala Glu
210 215 220
aga cgg gca caa gca gct gcg gct caa gca gca gca cat gca gct ggt 720
Arg Arg Ala Gln Ala Ala Ala Ala Gln Ala Ala Ala His Ala Ala Gly
225 230 235 240
gca cag gtg aca gct tcg cag cct gga cag ctc aag cta cca cgg ttt 768
Ala Gln Val Thr Ala Ser Gln Pro Gly Gln Leu Lys Leu Pro Arg Phe
245 250 255
cag cag cag cag cca cag act cat atg cag gtg cat ata cca gct acc 816
Gln Gln Gln Gln Pro Gln Thr His Met Gln Val His Ile Pro Ala Thr
260 265 270
ccc ctg cat atc agc agg gag ccc agg ctg ggg cat atc agc agg gtg 864
Pro Leu His Ile Ser Arg Glu Pro Arg Leu Gly His Ile Ser Arg Val
275 280 285
ctc agg ctg ggg tat atc agc agg gag ccc agg ctg ggg cat atc agc 912
Leu Arg Leu Gly Tyr Ile Ser Arg Glu Pro Arg Leu Gly His Ile Ser
290 295 300
agg gag ccc agg ctg ggg cat atc agc agg ggg gcc agg atg ggg cat 960
Arg Glu Pro Arg Leu Gly His Ile Ser Arg Gly Ala Arg Met Gly His
305 310 315 320
atc agc agg ggg ctc agg ctg ggg cat atc agc agg gag ccc agg ctg 1008
Ile Ser Arg Gly Leu Arg Leu Gly His Ile Ser Arg Glu Pro Arg Leu
325 330 335
ggg cat atc agc agg gag ccc agg ctg ggg cat atc agc agg gtg ctc 1056
Gly His Ile Ser Arg Glu Pro Arg Leu Gly His Ile Ser Arg Val Leu
340 345 350
agg ctg ggg cat atc agc agg gtg ctc agg ctg ggg tat atc agc agg 1104
Arg Leu Gly His Ile Ser Arg Val Leu Arg Leu Gly Tyr Ile Ser Arg
355 360 365
gaa ccc agg ctg ggg cat atc agc agg gag ccc agg ctg ggg cat atc 1152
Glu Pro Arg Leu Gly His Ile Ser Arg Glu Pro Arg Leu Gly His Ile
370 375 380
agc agg ggg gcc agg atg ggg cat atc agc agg ggg ctc agg ctg ggg 1200
Ser Arg Gly Ala Arg Met Gly His Ile Ser Arg Gly Leu Arg Leu Gly
385 390 395 400
cat atc agc agg gag ccc agg ctg ggg cat atc agc agg gag ccc agg 1248
His Ile Ser Arg Glu Pro Arg Leu Gly His Ile Ser Arg Glu Pro Arg
405 410 415
ctg ggg cat atc agc agg ggg ccc agt ctg ggg cat atc agc agg ggg 1296
Leu Gly His Ile Ser Arg Gly Pro Ser Leu Gly His Ile Ser Arg Gly
420 425 430
ccc agg ctg ggg cat atc agc agg gag ccc agg atg ggg cat atc agc 1344
Pro Arg Leu Gly His Ile Ser Arg Glu Pro Arg Met Gly His Ile Ser
435 440 445
agg gag ccc agg atg ggg cat atc agc agg gtg ctc agg ctg gag cat 1392
Arg Glu Pro Arg Met Gly His Ile Ser Arg Val Leu Arg Leu Glu His
450 455 460
aca act atg ctt atg atg ctg gca cgg ctt atg cat atg cag gtt act 1440
Thr Thr Met Leu Met Met Leu Ala Arg Leu Met His Met Gln Val Thr
465 470 475 480
ctg gct atc cag ttg cag gct acg cgc aaa gtg cag tgc cca act att 1488
Leu Ala Ile Gln Leu Gln Ala Thr Arg Lys Val Gln Cys Pro Thr Ile
485 490 495
cct atg ctg cac ctc cgc agc caa caa gca gcg gtg cag cta cga acg 1536
Pro Met Leu His Leu Arg Ser Gln Gln Ala Ala Val Gln Leu Arg Thr
500 505 510
ccg cag gag gcc agt atg ggg cag ttg gta gtg ctg gat atc cta ctg 1584
Pro Gln Glu Ala Ser Met Gly Gln Leu Val Val Leu Asp Ile Leu Leu
515 520 525
ggc aag ttc agc cga gca gtg gca ctg caa atg cag cgc aag cac ctc 1632
Gly Lys Phe Ser Arg Ala Val Ala Leu Gln Met Gln Arg Lys His Leu
530 535 540
ctc ctc cac cac cac cgg cag cac cat atc ccc cca gca cat atg acc 1680
Leu Leu His His His Arg Gln His His Ile Pro Pro Ala His Met Thr
545 550 555 560
aaa cca gag gag ccc aga gat aaa atc tgg gat gta aac cag atg gat 1728
Lys Pro Glu Glu Pro Arg Asp Lys Ile Trp Asp Val Asn Gln Met Asp
565 570 575
gtt tgc cat gca cat ttg ttg agc aga caa ata tgg tgaaatctgg 1774
Val Cys His Ala His Leu Leu Ser Arg Gln Ile Trp
580 585
gatgtaaaac cagatggctg tctgtgcctc catcccattg actagggcgt attttcacca 1834
atattgtgcc tccagtgcaa tttcttctgt gttatatatc accaccattt gttgggcaaa 1894
aaaaaaaaaa aaaaaa 1910
<210>4
<211>588
<212>PRT
<213>玉蜀黍(Zea mays L)
<400>4
Met Ala His Arg Gly His Leu Asp Gly Leu Thr Gly Gln Ala Pro Ala
1 5 10 15
Leu Met Arg His Gly Ser Phe Ala Ala Gly Ser Leu Ser Ser Arg Ser
20 25 30
Pro Leu Gln Ser Ser Ser Thr Leu Glu Met Leu Glu Asn Lys Leu Ala
35 40 45
Met Gln Thr Thr Glu Val Glu Lys Leu Ile Thr Glu Asn Gln Arg Leu
50 55 60
Ala Ser Ser His Val Val Leu Arg Gln Asp Ile Val Asp Thr Glu Lys
65 70 75 80
Glu Met Gln Met Ile Arg Thr His Leu Gly Glu Val Gln Thr Glu Thr
85 90 95
Asp Leu Gln Ile Arg Asp Leu Leu Glu Arg Ile Arg Leu Met Glu Val
100 105 110
Asp Ile His Ser Gly Asn Val Val Asn Lys Glu Leu His Gln Met His
115 120 125
Met Glu Ala Lys Arg Leu Ile Thr Glu Arg Gln Met Leu Thr Leu Glu
130 135 140
Ile Glu Asp Val Thr Lys Glu Leu Gln Lys Leu Ser Ala Ser Gly Asp
145 150 155 160
Asn Lys Ser Leu Pro Glu Leu Leu Ser Glu Leu Asp Arg Leu Arg Lys
165 170 175
Glu His His Asn Leu Arg Ser Gln Phe Glu Phe Glu Lys Asn Thr Asn
180 185 190
Val Lys Gln Val Glu Gln Met Arg Thr Met Glu Met Asn Leu Ile Thr
195 200 205
Met Thr Lys Gln Ala Glu Lys Leu Arg Val Asp Val Ala Asn Ala Glu
210 215 220
Arg Arg Ala Gln Ala Ala Ala Ala Gln Ala Ala Ala His Ala Ala Gly
225 230 235 240
Ala Gln Val Thr Ala Ser Gln Pro Gly Gln Leu Lys Leu Pro Arg Phe
245 250 255
Gln Gln Gln Gln Pro Gln Thr His Met Gln Val His Ile Pro Ala Thr
260 265 270
Pro Leu His Ile Ser Arg Glu Pro Arg Leu Gly His Ile Ser Arg Val
275 280 285
Leu Arg Leu Gly Tyr Ile Ser Arg Glu Pro Arg Leu Gly His Ile Ser
290 295 300
Arg Glu Pro Arg Leu Gly His Ile Ser Arg Gly Ala Arg Met Gly His
305 310 315 320
Ile Ser Arg Gly Leu Arg Leu Gly His Ile Ser Arg Glu Pro Arg Leu
325 330 335
Gly His Ile Ser Arg Glu Pro Arg Leu Gly His Ile Ser Arg Val Leu
340 345 350
Arg Leu Gly His Ile Ser Arg Val Leu Arg Leu Gly Tyr Ile Ser Arg
355 360 365
Glu Pro Arg Leu Gly His Ile Ser Arg Glu Pro Arg Leu Gly His Ile
370 375 380
Ser Arg Gly Ala Arg Met Gly His Ile Ser Arg Gly Leu Arg Leu Gly
385 390 395 400
His Ile Ser Arg Glu Pro Arg Leu Gly His Ile Ser Arg Glu Pro Arg
405 410 415
Leu Gly His Ile Ser Arg Gly Pro Ser Leu Gly His Ile Ser Arg Gly
420 425 430
Pro Arg Leu Gly His Ile Ser Arg Glu Pro Arg Met Gly His Ile Ser
435 440 445
Arg Glu Pro Arg Met Gly His Ile Ser Arg Val Leu Arg Leu Glu His
450 455 460
Thr Thr Met Leu Met Met Leu Ala Arg Leu Met His Met Gln Val Thr
465 470 475 480
Leu Ala Ile Gln Leu Gln Ala Thr Arg Lys Val Gln Cys Pro Thr Ile
485 490 495
Pro Met Leu His Leu Arg Ser Gln Gln Ala Ala Val Gln Leu Arg Thr
500 505 510
Pro Gln Glu Ala Ser Met Gly Gln Leu Val Val Leu Asp Ile Leu Leu
515 520 525
Gly Lys Phe Ser Arg Ala Val Ala Leu Gln Met Gln Arg Lys His Leu
530 535 540
Leu Leu His His His Arg Gln His His Ile Pro Pro Ala His Met Thr
545 550 555 560
Lys Pro Glu Glu Pro Arg Asp Lys Ile Trp Asp Val Asn Gln Met Asp
565 570 575
Val Cys His Ala His Leu Leu Ser Arg Gln Ile Trp
580 585
<210>5
<211>16
<212>DNA
<213>人工序列
<220>
<223>寡核苷酸E1
<400>5
gattctccct gataag
<210>6
<211>36
<212>DNA
<213>人工序列
<220>
<223>寡核苷酸锚
<400>6
ggccaggcgt cgactagtac gggIIgggII gggIIg 36
<210>7
<211>18
<212>DNA
<213>人工序列
<220>
<223>寡核苷酸E2
<400>7
gttctccagc atctccag 18
Claims (16)
1.一种编码具有转谷氨酰胺酶活性的玉米蛋白质的核苷酸序列,其中所述核苷酸序列含有多个SEQ ID NO:1或SEQ ID NO:3中823-849位所示序列的随机重复序列并且编码SEQ ID NO:2或SEQ IDNO:4中所示的蛋白质。
2.权利要求1的核苷酸序列,其中所述序列选自SEQ ID NO:1和SEQ ID NO:3。
3.表达载体,其中所述表达载体包含权利要求1或2的核苷酸序列。
4.权利要求3的表达载体,其中所述表达载体选自含有SEQ IDNO:1的pGEMT和含有SEQ ID NO:3的pGEMT。
5.具有转谷氨酰胺酶活性的蛋白质,其中所述蛋白质由权利要求1或2的核苷酸序列编码。
6.权利要求5的具有转谷氨酰胺酶活性的蛋白质,其中所述蛋白质选自SEQ ID NO:2和SEQ ID NO:4。
7.转化细胞,其中所述转化细胞包含权利要求3或4的表达载体,并且其中所述转化细胞允许具有转谷氨酰胺酶活性的蛋白质表达。
8.权利要求7的转化细胞,其中所述转化细胞选自大肠杆菌(E.coli)菌株CECT 5705和CECT 5706。
9.权利要求7或8的转化细胞在产生权利要求5或6的具有转谷氨酰胺酶活性的蛋白质的方法中的应用,其中所述蛋白质为重组蛋白质。
10.权利要求5或6的具有转谷氨酰胺酶活性的蛋白质在食品处理、加工和转化方面的应用。
11.权利要求10的应用,用于保持或改善食品的品质、稠度、弹性、水分或粘度。
12.权利要求11的应用,其中所述食品选自奶酪、酸乳、冰淇淋、蛋黄酱和肉类。
13.权利要求12的应用,其中所述食品为鱼类。
14.权利要求10-13中任一项的应用,用于形成不同密度的明胶和制备少脂的预烹调食品。
15.权利要求3或4的表达载体在开发具有新能力的转基因植物方面的应用,所述新能力由对所述转谷氨酰胺酶的特征性功能操作所引起。
16.权利要求15的应用,其中所述功能选自植物的生长和发育、形态发生、光合作用和细胞死亡。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
ESP200201253 | 2002-05-31 | ||
ES200201253A ES2223227B1 (es) | 2002-05-31 | 2002-05-31 | Secuencia de nucleotidos de maiz codificante de una proteina con actividad transglutaminasa, y su uso. |
Publications (2)
Publication Number | Publication Date |
---|---|
CN1671846A CN1671846A (zh) | 2005-09-21 |
CN1329515C true CN1329515C (zh) | 2007-08-01 |
Family
ID=29595112
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CNB038175525A Expired - Fee Related CN1329515C (zh) | 2002-05-31 | 2003-05-23 | 编码具有转谷氨酰胺酶活性的蛋白质的玉米核苷酸序列及其应用 |
Country Status (10)
Country | Link |
---|---|
US (2) | US7262057B2 (zh) |
EP (1) | EP1535992B1 (zh) |
JP (1) | JP2005528104A (zh) |
CN (1) | CN1329515C (zh) |
AT (1) | ATE360681T1 (zh) |
AU (1) | AU2003227778A1 (zh) |
DE (1) | DE60313469T2 (zh) |
ES (2) | ES2223227B1 (zh) |
MX (1) | MXPA04011918A (zh) |
WO (1) | WO2003102128A1 (zh) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ITBO20020714A1 (it) * | 2002-11-13 | 2004-05-14 | Plantechno Srl | Farine ad uso alimentare con specifiche caratteristiche tecnologiche a bassa allergenicita'. |
EP2975130A3 (en) | 2008-06-26 | 2016-04-20 | BASF Plant Science GmbH | Plants having enhanced yield-related traits and a method for making the same |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0693556A1 (en) * | 1994-01-28 | 1996-01-24 | Ajinomoto Co., Inc. | Transglutaminase originating in japanese oyster |
CN1142536A (zh) * | 1995-02-09 | 1997-02-12 | 味之素株式会社 | 杆菌衍生的转谷氨酰胺酶 |
CN1253177A (zh) * | 1997-07-04 | 2000-05-17 | 味之素株式会社 | 生产微生物转谷氨酰胺酶的方法 |
CN1334867A (zh) * | 1998-12-28 | 2002-02-06 | 味之素株式会社 | 转谷氨酰胺酶的制备方法 |
Family Cites Families (21)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH0665280B2 (ja) | 1987-03-04 | 1994-08-24 | 味の素株式会社 | タンパクゲル化剤及びそれを用いるタンパクのゲル化方法 |
DE69333718T2 (de) * | 1992-01-14 | 2005-12-01 | Ajinomoto Co., Inc. | Gen, das für eine Fisch-Transglutaminase kodiert |
WO1994010296A1 (en) | 1992-11-03 | 1994-05-11 | Oklahoma Medical Research Foundation | Transglutaminase gene |
US5549904A (en) | 1993-06-03 | 1996-08-27 | Orthogene, Inc. | Biological adhesive composition and method of promoting adhesion between tissue surfaces |
ATE329014T1 (de) | 1994-08-26 | 2006-06-15 | Novozymes As | Mikrobielle transglutaminasen, ihre herstellung und ihre verwendung |
JP3669390B2 (ja) | 1995-02-09 | 2005-07-06 | 味の素株式会社 | バチルス属細菌由来のトランスグルタミナーゼ |
JP3637718B2 (ja) | 1996-04-10 | 2005-04-13 | 味の素株式会社 | チョコレートの製造方法 |
US5834232A (en) | 1996-05-01 | 1998-11-10 | Zymogenetics, Inc. | Cross-linked gelatin gels and methods of making them |
US5928689A (en) | 1996-06-05 | 1999-07-27 | Kraft Foods, Inc. | Method for treating PSE meat with transglutaminase |
JP3582265B2 (ja) | 1996-11-28 | 2004-10-27 | 味の素株式会社 | 改質穀粉及びこれを使用した穀粉加工食品 |
US6039901A (en) | 1997-01-31 | 2000-03-21 | Givaudan Roure Flavors Corporation | Enzymatically protein encapsulating oil particles by complex coacervation |
US6042851A (en) | 1997-12-03 | 2000-03-28 | Kikkoman Corporation | Process for producing packed tofu |
JP3867261B2 (ja) | 1998-04-08 | 2007-01-10 | 味の素株式会社 | 酵素製剤及び麺類の製造方法 |
AU757109B2 (en) | 1998-05-20 | 2003-02-06 | Novozymes North America, Inc. | A method for enzymatic treatment of wool |
JP3733748B2 (ja) | 1998-06-24 | 2006-01-11 | 味の素株式会社 | 食感が改善されたチーズホエイ蛋白、その製造方法及びその利用 |
DE69929077D1 (de) | 1998-10-13 | 2006-01-26 | Fuji Oil Co Ltd | Tofu-produkte mit exzellenter gefrierresistenz sowie verfahren zur herstellung desselben |
US20090087878A9 (en) * | 1999-05-06 | 2009-04-02 | La Rosa Thomas J | Nucleic acid molecules associated with plants |
US6270814B1 (en) | 1999-06-03 | 2001-08-07 | Kraft Foods, Inc. | Incorporation of whey into process cheese |
JP2000354498A (ja) | 1999-06-14 | 2000-12-26 | Ajinomoto Co Inc | トランスグルタミナーゼ処理による穀粉原料からの糖類の製造方法 |
WO2001062888A2 (en) | 2000-02-23 | 2001-08-30 | Hindustan Lever Limited | Improved composition of marine product |
DE60140385D1 (de) | 2000-04-14 | 2009-12-17 | Novozymes Inc | Pektinase-Behandlung von Kartoffelprodukten |
-
2002
- 2002-05-31 ES ES200201253A patent/ES2223227B1/es not_active Expired - Fee Related
-
2003
- 2003-05-23 ES ES03725225T patent/ES2286430T3/es not_active Expired - Lifetime
- 2003-05-23 MX MXPA04011918A patent/MXPA04011918A/es active IP Right Grant
- 2003-05-23 EP EP03725225A patent/EP1535992B1/en not_active Expired - Lifetime
- 2003-05-23 WO PCT/ES2003/000247 patent/WO2003102128A1/es active IP Right Grant
- 2003-05-23 JP JP2004510370A patent/JP2005528104A/ja active Pending
- 2003-05-23 CN CNB038175525A patent/CN1329515C/zh not_active Expired - Fee Related
- 2003-05-23 AT AT03725225T patent/ATE360681T1/de not_active IP Right Cessation
- 2003-05-23 DE DE60313469T patent/DE60313469T2/de not_active Expired - Fee Related
- 2003-05-23 AU AU2003227778A patent/AU2003227778A1/en not_active Abandoned
-
2004
- 2004-11-30 US US11/000,530 patent/US7262057B2/en not_active Expired - Fee Related
-
2007
- 2007-08-27 US US11/895,752 patent/US20100092606A1/en not_active Abandoned
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0693556A1 (en) * | 1994-01-28 | 1996-01-24 | Ajinomoto Co., Inc. | Transglutaminase originating in japanese oyster |
CN1142536A (zh) * | 1995-02-09 | 1997-02-12 | 味之素株式会社 | 杆菌衍生的转谷氨酰胺酶 |
CN1253177A (zh) * | 1997-07-04 | 2000-05-17 | 味之素株式会社 | 生产微生物转谷氨酰胺酶的方法 |
CN1334867A (zh) * | 1998-12-28 | 2002-02-06 | 味之素株式会社 | 转谷氨酰胺酶的制备方法 |
Non-Patent Citations (1)
Title |
---|
IMMUNOGOLD LOCALIZATION OF A TRANSGLUTAMINASE RELATED TO GRANA DEVELOPMENT IN DIFFERENT MAIZE CALL TGPAS VILLALOBOS E,PROTOPLASMA,Vol.216 2001 * |
Also Published As
Publication number | Publication date |
---|---|
ES2223227B1 (es) | 2006-04-16 |
US20060005266A1 (en) | 2006-01-05 |
WO2003102128A1 (es) | 2003-12-11 |
ES2286430T3 (es) | 2007-12-01 |
JP2005528104A (ja) | 2005-09-22 |
DE60313469D1 (de) | 2007-06-06 |
MXPA04011918A (es) | 2005-03-31 |
ATE360681T1 (de) | 2007-05-15 |
DE60313469T2 (de) | 2008-01-03 |
ES2223227A1 (es) | 2005-02-16 |
US7262057B2 (en) | 2007-08-28 |
US20100092606A1 (en) | 2010-04-15 |
CN1671846A (zh) | 2005-09-21 |
EP1535992A1 (en) | 2005-06-01 |
AU2003227778A1 (en) | 2003-12-19 |
EP1535992B1 (en) | 2007-04-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP0555649B1 (en) | Gene encoding transglutaminase derived from fish | |
JP3758187B2 (ja) | マガキ由来のトランスグルタミナーゼ | |
Raines et al. | Chloroplast fructose-1, 6-bisphosphatase: the product of a mosaic gene | |
Fisher et al. | Starch branching enzyme II from maize endosperm. | |
CZ299825B6 (cs) | Molekuly nukleové kyseliny kódující protein s enzymatickou aktivitou fruktosyltransferázy, vektor, hostitelská bunka, zpusob prípravy FFT, FFT kódovaná molekulou nukleové kyseliny, zpusob prípravy transformovaných hostitelských bunek, transformovaná | |
CA2169249A1 (en) | Bacillus-derived transglutaminase | |
JP2009195255A (ja) | スクロースホスホリラーゼ(sp)の耐熱化方法 | |
CN101687907B (zh) | 转移酶、差向异构酶、编码它们的多核苷酸及其用途 | |
CN1329515C (zh) | 编码具有转谷氨酰胺酶活性的蛋白质的玉米核苷酸序列及其应用 | |
TW201106867A (en) | Genetically purified gellan gum | |
JP3364972B2 (ja) | 魚由来トランスグルタミナーゼ遺伝子 | |
CA2351566A1 (fr) | Gene codant pour une acyltransferase de colza, et ses utilisations | |
Vinnemeier et al. | Purification and partial characterization of the Streptomyces viridochromogenes Tü494 phosphinothricin-N-acetyltransferase mediating resistance to the herbicide phosphinothricin in transgenic plants | |
CN105671074B (zh) | 一种提高植物甲硫氨酸含量的载体及其构建方法和用途 | |
JP4358006B2 (ja) | イネSSIIaの活性の制御方法、及びその変異体 | |
CN111788216A (zh) | 盐泡细胞形成控制作用剂和导入了该作用剂的植物体 | |
AU750970B2 (en) | HVD1 gene induced by salt stress | |
CA2294045C (en) | Gene encoding a protein having aurone synthesis activity | |
JP3477746B2 (ja) | 魚由来トランスグルタミナーゼ遺伝子 | |
US6881433B1 (en) | Food products containing altered starch | |
CN106459933A (zh) | 用于增加谷物产量的方法 | |
JPH08205863A (ja) | アシル−アシルキャリヤープロテイン チオエステラーゼおよびそれをコードするdna | |
Torné et al. | Biotechnological aspects of maize transglutaminases | |
JPH1175853A (ja) | 銅の利用性を制御するホメオスタシスタンパク質遺伝子 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
C17 | Cessation of patent right | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20070801 Termination date: 20100523 |