公开/公告号CN1530374A
专利类型发明专利
公开/公告日2004-09-22
原文格式PDF
申请/专利权人 中国科学院研究生院;
申请/专利号CN200410004655.2
申请日2004-03-05
分类号C07K14/415;C12N15/29;A01H1/00;
代理机构北京泛华伟业知识产权代理有限公司;
代理人王凤华
地址 100039 北京市玉泉路19号
入库时间 2023-12-17 15:30:37
法律状态公告日
法律状态信息
法律状态
2010-08-04
未缴年费专利权终止 IPC(主分类):C07K14/415 授权公告日:20060215 申请日:20040305
专利权的终止
2006-02-15
授权
授权
2004-12-01
实质审查的生效
实质审查的生效
2004-09-22
公开
公开
发明领域
本发明属于植物金属阳离子转运蛋白领域,特别是关于印度芥菜(Brassica juncea)CE(cation-efflux transporters)家族Zn、Ni、Cd、Co等重金属离子跨膜运载蛋白的基因序列和氨基酸序列。
背景技术
CE(阳离子输出转运蛋白cation-efflux transporters)家族的金属阳离子跨膜运载蛋白对生物体细胞内金属离子的胞外输出和胞内液泡的区域化隔离,提高金属离子的抗性和富集量发挥着关键作用。Nies和Silver(1995)首先在细菌中鉴定出CDF(cation diffuse facilitor proteins)家族,之后在真菌、植物和动物中均发现有这类运载蛋白。其典型的结构特征是具有6个跨膜(TM,transmembrane)结构域(TM1-6),1个N端特征氨基酸序列(SLA I/V M/L TDAA/L H L/M L T/S D)和1个C端阳离子输出域。另外,在真核生物家族成员的TM4和TM5之间含有一个可变的富含His区,位于细胞质内,推测其与金属离子的结合和专一性有关(图1)。由于缺乏CDF运载蛋白动力学的基本信息,但已知所鉴定的该家族成员具有阳离子输出功能,所以Maser等(2001)建议将其重命名为CE(cation-efflux proteins)家族更为准确。
CE家族成员具有很高的蛋白质序列差异和大小变化(300-750个残基之间),一些被认为催化重金属吸收,而另一些被认为催化重金属输出。在细胞质膜和细胞内膜上均发现有该家族的不同成员。已鉴定有特定功能的该家族成员是:哺乳动物的ZnT1、2、3和4运载蛋白,参与Zn的胞外输出(ZnT1,4)和胞内区室的输入(ZnT2,3)(Huang L,Gitschier J 1997.Nature Gen,17:292-297);酵母的COT1与Co的抗性有关,酵母的ZRC1与Zn、Cd抗性有关(Li L,kaplan J 1998.J Biol Chem,273:22181-22187);拟南芥的ZAT(AtMTP1)与Zn的抗性及Zn在根部的积累有关(Van der Zaal BJ et al.,1999.Plant Physiol,199:1047-1055)。这些蛋白的表达对于提高金属的耐性已经在植物和异源表达的酵母中得到证明。在拟南芥中过量表达Zn运载蛋白基因ZAT1,其根部比对照植物累积了高浓度的Zn,在200μM的Zn水培条件下,对照植株的根生长被抑制了85%,而转基因植株的根只被抑制了15%(Vander Zaal BJ et al.,1999.Plant Physiol,199:1047-1055);COT1和ZRC1位于酵母的液泡膜上,参与了液泡对重金属的隔离,其缺陷型酵母表现了对Co、Zn和Cd的敏感性,而过量表达则提高了酵母对Co和Zn的抗性。利用COT1和ZRC1缺陷型酵母进行功能互补实验,对于鉴定动植物CE家族成员发挥了重要作用,其中包括T.goesingense中对Ni超富集起主要作用的跨膜运载蛋白基因TgMTP1的克隆和鉴定。目前已从Zn/Cd超富集植物T.caerulescens中克隆和分离出ZTP1、ZNT1和ZNT2三种Zn运载蛋白cDNAs(Pence N S etal.,2000.Proc Natl Acad Sci USA,97:4956-4960;Assucao et al.,2001.Plant,Cell and Environment,24:217-226),正常和缺Zn条件下它们在T.caerulescens中的表达水平均远高于非超富集植物T.arvense,预示三者在植物重金属超富集中起重要作用。ZTP1的cDNA具有1340个碱基,编码的推测蛋白为393个氨基酸,与拟南芥的ZAT1 cDNA的核苷酸和氨基酸序列的一致性分别达到90%和75%,归属于CE家族,主要在叶部表达,在根部也有表达。ZTP1基因的表达与Zn供给无关,对于Zn在植物体内的螯合和液泡隔离起重要作用。ZNT1和ZNT2的cDNA分别具有1375和1520个碱基,其编码的推测蛋白分别为409和423个氨基酸,与拟南芥的ZIP4 cDNA的核苷酸序列的一致性分别达到90%和83%,氨基酸序列的一致性分别达到76%和65%。二者具有典型的ZIP家族结构特征,归属于ZIP家族,主要在根部表达,对Zn的吸收和转运起主要作用。
Persans等(2001)发现T.goesingense对Ni的超富集似乎被增强的Ni在叶片液泡内的积累所控制,并克隆了推断为液泡重金属运载蛋白的基因,其编码产物命名为金属忍耐蛋白(metal tolerant protein,TgMTPs)(Persans MW et al.,2001.Proc Natl Acad Sci USA,98:9995-10000)。这些蛋白拥有所有CE家族成员的特征,是由同一基因(TgMTP1)序列产生的未拼接(unspliced,TgMTP1t1)和拼接的(spliced,TgMTP1t2)转录产物。它们在酵母中异源表达可互补TgMTP1同源基因COT1和ZRC1的缺陷型敏感系,说明TgMTP1s运载金属离子进入酵母液泡中的方式类似于COT1和ZRC1。拼接的和未拼接的TgMTP1转录产物在推断的富含组氨酸的金属结合域内存在差异,而这些差异在其运载金属离子的专一性上得到了反映。在酵母中TgMTP1t1表达可以抗高浓度的Cd、Co和Zn,而表达TgMTP1t2可以抗高浓度的Ni。TgMTP1在T.goesingense中高水平表达,解释了该植物能在茎叶液泡中累积Ni的超富集能力。
印度芥菜(Brassica juncea,BJ)又称春菜,是一种高产、快生芥科植物,具有相对较高的多种重金属富集能力,如Cd、Cr、Ni、Zn、Cu、Au、Se、等,是目前利用植物修复技术(phytoremediation)清除污染土壤和水体中重金属和放射性物质的主要模式植物。研究表明,0.4mg/L Cd处理7天的BJ,其茎叶(干重)富集的Cd可达310mg/kg(Salt DE et al.,1995.PlantPhysiol,109(4):1427-1433),已达到Cd的超富集标准(>100mg/kg);BJ富Pb量可达其干重的3.5%(Brown K S,1995.BioScience,45(9):579-585),在富铅土壤中(2500ppmPb),每千克土壤添加22.0g的HEDTA可使BJ茎叶中Pb含量从40mg/kg提高到10,600mg/kg;BJ具有较强的硒富集和生物转化作用,其硒酸盐含量可达1575mg/kg,占总硒量的75%,有机硒含量可达630mg/kg,占总硒量的25%,而且没有其他毒性化合物生成,是补硒的理想物质;在含金土壤中BJ生长得特别茂盛,它们的根系特别善于吸收土地中的Au、Ni等贵金属,喷洒硫氰酸铵溶液,能迅速溶解土壤中的黄金,从而能提高近5倍的黄金吸收量;在切尔诺贝利核电站附近遭受核辐射污染的土壤,只有BJ生长得很旺盛,美国一些科学家认为利用印度芥菜和向日葵可以清除其周围大约90%的放射性物质Cs和Sr。因此,印度芥菜蕴藏着丰富的基因资源,利用基因工程技术挖掘和转化其金属离子的富集和耐性基因,可以培育具有产业化潜力的超富集和富集金属离子的植物品种,对于清除环境重金属污染以及提高人类食品的矿质营养、增进人类健康方面具有重要应用价值,并将产生积极的社会效益、生态效益和经济效益。目前国际上还未见印度芥菜CE家族金属阳离子跨膜运载蛋白基因的报道。
发明内容
本发明的目的在于提供印度芥菜CE家族的金属阳离子跨膜转运蛋白,以及编码该蛋白的新基因,为今后开发基因工程产品奠定物质基础。
在本发明的一个方面,提供了一种印度芥菜CE家族金属阳离子跨膜运载蛋白分子,它为如下蛋白质分子(i)或(ii):
(i)具有附图13所述的氨基酸序列;
(ii)在附图13限定的氨基酸序列经过取代,缺失或叠加一个或几个氨基酸衍生的蛋白质且与(i)的蛋白质具有相同的功能。
较佳地,所述的蛋白质分子具有附图13所述的氨基酸序列,也就是本发明的BjCET4蛋白。
在本发明的另一个方面,提供了一种DNA分子,它包括:编码所述的印度芥菜CE家族金属阳离子跨膜运载蛋白分子的核苷酸序列。
较佳地,本发明所述的DNA分子编码附图13所示的氨基酸序列;进一步优选该DNA分子为附图13所述的DNA序列,该DNA分子编码附图13所示的氨基酸序列。
图13中DNA序列的编码框(ORF)为131-1258位。由于密码子的简并性,所以与图13的131-1258位核苷酸同源性低至约70%的简并序列也能编码出图13所示的BjCET4氨基酸序列。
本发明的DNA分子是采用如下方法获得的:首先,用Cd2+胁迫处理印度芥菜,提取总RNA;再以总RNA为模板进行逆转录PCR(RT-PCR),得到印度芥菜的ss cDNA;然后根据Genebank中注册的Ni/Cd超富集植物T.goesingensed的转运蛋白基因TgMTP1和拟南芥(A.thalia na)的植物转运蛋白基因ZAT mRNA序列的N端和C端保守区设计了一对简并引物P1(5’-ggcggaattcatggagtcttcaagt-3’)和P2(5’-cccggatcccatgactg/aata/gttgtactc-3’),以胁迫处理的印度芥菜sscDNA为模板进行PCR反应,PCR产物克隆入pGEM-T Easy载体(Prome ga),由上海联众基因公司测序,序列分析采用NCBI的BLAST软件,得到了含有目标基因的片断;为获得目标基因mRNA的全长序列,依据已知序列设计引物,采用ClONTECH公司的SMARTTM RACE cDNA Amplification Kit进行5’-RACE和3’-RACE(具体操作规程参见试剂盒说明书),得到了图7所示的BjCET1全长cDNA核苷酸。
由于CE家族的多成员性,已发现的拟南芥CE家族具有5个含有6个跨膜结构域(TM)的CE成员,其编码框(ORF)的C端具有很高的核苷酸和氨基酸序列同源性。因此本发明5’-RACE依据已获得的印度芥菜CE成员核苷酸和氨基酸序列,并参照TgMTP1和ZAT的mRNA序列,在ORF的C端高保守区设计了5’-RACE的下游简并引物,以期获得印度芥菜的其它CE成员。为提高3’-RACE的效率和准确性,部分3’-RACE采用巢式PCR(Furuya Y,1993.Journal of Clinical Microbiology,31(6),1637-1640.)。PCR反应产物克隆入pGEM-T Easy载体(Promega),由上海联众基因公司测序,得到了其它三个目的基因。见图11,图12,和图13。
应用本发明的基因序列或氨基酸序列进行转基因开发基因工程产品在以下方面具有重大应用价值:
1、可提高植物或作物的重金属抗性,减少毒性重金属在植物可食部位的积累,从而提高作物产量,增加食品的安全性。
2、培育高生物量的重金属超富集植物,一方面用于重金属(如Cd,Pb,Hg等)污染土壤或水体的生态治理。另一方面,对于Zn,Ni,Se等必需矿质微量元素的富集,可以开发矿质强化食品,对于增进人类健康具有积极意义。
附图说明
图1为推测的CE家族成员的拓扑结构
其中1表示胞内部分 2表示胞外部分
3表示脂膜 4表示CE特征序列
5表示可变区
图2为以印度芥菜叶部第一链cDNA为模板的扩增产物电泳图
其中M表示分子量Maker 1,2,3和4分别表示4个电泳泳道
图3为BjCET1核苷酸和氨基酸序列片段
图4为用TMPRED软件分析得到的BjCET1跨膜结构示意图
图5为BjCET15’-RACE PCR产物电泳图
图6为BjCET1 3’-RACE PCR产物电泳图
图7为BjCET1全长cDNA核苷酸和氨基酸序列
图8为含有CE家族成员BjCET1,BjCET2,BjCET3和BjCET4 DNA片段混合物的5’-RACE PCR产物电泳图
图9为BjCET2 3’-RACE PCR产物电泳图
图10为BjCET3(左)和BjCET4(右)3’-RACE PCR产物电泳图
图11为BjCET2全长cDNA核苷酸和氨基酸序列
图12为BjCET3全长cDNA核苷酸和氨基酸序列
图13为BjCET4全长cDNA核苷酸和氨基酸序列
图14为BjCET1全长cDNA核苷酸PCR产物电泳图
图15为穿梭载体pYES2质粒图谱
图16为双价载体pBIN-mGFP5-ER序粒图谱
图17A为WT和各转基因烟草植株检测融合基因GFP的基因组DNA PCR扩增产物电泳图
其中泳道从左至右依次是阴性对照WT、阳性对照GFP、转BjCET1-GFP植株1、转BjCET1-GFP植株2、100bp DNA marker
图17B为各转基因烟草植株检测融合基因GFP的基因组DNA PCR扩增产物电泳图
其中泳道从左至右依次是转BjCET1-GFP植株2、转BjCET2-GFP植株1、转BjCET3-GFP植株1、转BjCET4-GFP植株1、100bp DNA marker
具体实施方式
实施例1印度芥菜CE家族的金属阳离子跨膜转运蛋白新基因的制备
步骤1:总RNA提取
将3周生长期的印度芥菜植株转入1/2×Hoagland液体培养基中(不含蔗糖和琼脂)预培养,每两天换一次新鲜培养液。一周之后向培养基中加入200mM CdCl2进行胁迫,每天换一次新鲜培养基,两天之后采集(收割)根和叶片。对照为在同样条件下培养的印度芥菜,但是培养基中未加CdCl2进行胁迫处理。采用QIAGEN公司的RNA提取试剂盒(Rneasy Plant Mini Kit)进行对照和CdCl2胁迫处理的样品总RNA的提取。以Rnase-free DNase I(终浓度80,000U/L)除去其中污染的DNA,经甲醛变性凝胶电泳鉴定其完整性,其中28s和18s RNA的量大约为2∶1,表明完整性很好。
步骤2:PCR引物设计和合成
根据Genebank中注册的Ni/Cd超富集植物T.goesingense和拟南芥(A.thalia na)植物转运蛋白基因TgMTP1和ZAT mRNA序列的N端和C端保守区设计了上游引物P1(5’-ggcggaattcatggagtctt caagt-3’)和下游简并引物P2(5’-cccggatcccatgact g/a at a/g ttgtac tc-3’)。术语“简并引物”是指根据某些特定的目的而选用的一组引物混合物,在寡核苷酸的某一位置(一个简并位)上可以有多个碱基存在于不同的寡核苷酸分子中,如一组简并引物中有N1,N2,N3三个简并位,在N1位上可以有三个碱基简并,N2二个,N3四个,则此简并引物中共有3*2*4=24种寡核苷酸分子。
步骤3:单链cDNA的合成、PCR和基因克隆
以印度芥菜叶片对照总RNA和CdCl2处理的印度芥菜叶片总RNA为模板,采用GIBCO BRL公司的SuperScriptTM First-Strand Synthesis System forRT-PCR(Cat.No.11904-018)试剂盒进行ss cDNA的反转录合成。操作按说明书。
以上面得到的印度芥菜叶片对照和处理印度芥菜叶片的ss cDNA为模版,p1和p2为引物进行PCR反应,反应体系(50μL)包括:20mmol/LTris-HCL(pH9.0),10mmol/L KCl,2mmol/L MgCl2,10mmol/L(NH4)2SO4,0.1%Triton X-100,0.2mmol/L dNTP mix(4种dNTP体积比为1∶1∶1∶1),P1和P2各0.4mmol/L,2 Units Taq DNA聚合酶,对照和处理样品印度芥菜叶片的sscDNA均分别取2.0μL和3μL。反应条件:94℃变性10min;94℃ 1min,退火温度50℃ 2min,72℃ 2min,10个循环,退火温度每一轮循环增加0.5℃;94℃ 1min,60℃ 1min,72℃ 2min,25个循环;72℃延伸10min。
分别取5μL PCR产物经1%琼脂糖凝胶电泳检测。电泳结果如图2所示,其中M:marker;泳道1,2:对照;泳道3,4:Cd2+处理的样品;50μL PCR反应体系所加模板量泳道1和3为2μL,泳道2和4为3μL。4个泳道均得到3条扩增带,按条带大小依次命名为B1(约1100bp)、B2(约870bp)和B3(约660bp),说明200mM CdCl2处理2d对B1,B2和B3的转录量无较大影响。
将B1、B2和B3分别切胶回收,连接到pGEM-T Easy载体上,转化感受态大肠杆菌DH5α,各挑取10个白色克隆,培养扩增,提取质粒进行限制性内切酶酶切,分别取含有插入片段的3个克隆进行测序,所测序列一致,结果显示B1为1145bp,B2为879bp,B3为666bp。各序列与GenBank/BLAST数据库进行同源性比较,结果发现B1为本研究所要克隆的金属阳离子跨膜转运蛋白基因,将该基因命名为BjCET1(图3)。B2为一未知功能的基因,与拟南芥未知蛋白基因AY096642氨基酸序列具有较高同源性,达到78%,而B3含有DnaJ-domain(结构域)(DnaJ-domain为分子伴侣系统的一部分),与Hsp70热激蛋白系统相关,为Dnaj-like(样)蛋白基因。通过GenBank/BLAST数据库进行同源性比较分析可知,BjCET1与TgMTP1核苷酸序列同源性达到85%,氨基酸序列同源性达到75%;与ZAT核苷酸序列同源性达到83%,氨基酸序列同源性达到78%。用TMPRED软件(http://www.ch.embnet.org/software/TMPRED form.html)根据基因序列分析预测基因编码的蛋白质的特性,得到的结果是BjMAP1编码的蛋白质具有CDF家族典型的蛋白结构特征:6个跨膜(TM,transmembrane)结构域(TM1-6,图4),1个N端特征氨基酸序列和1个C端阳离子输出域,另外还具有CDF真核生物家族成员在TM4和TM5之间含有一个可变的富含His特征区,存在于胞质内,推测与金属离子的结合和专一性有关。
步骤4:BjCET1全长cDNA克隆
以印度芥菜叶片CdCl2处理的总RNA为模板,采用ClONTECH公司的SMARTTM RACE cDNA Amplification Kit分别合成了5’-RACE和3’-RACE的cDNA模板(具体操作规程参见试剂盒说明书)。依据已知的BjCET1序列设计合成5’-RACE引物5p1:5’-AGTCTGCCTCGGCGTCGCTTCCCAACCA-3和3’-RACE 引物3p1:5’-gggaattcgaag tctttggtggca-3’。5’-RACE反应条件:94℃变性10min;94℃ 30s,72℃ 3min,5个循环;94℃ 30s,70℃ 30s,72℃ 3min,5个循环;94℃ 30s,68℃ 30s,72℃ 3min,25个循环;72℃延伸10min。3’-RACE反应条件:94℃变性10min;94℃ 45s,50℃ 1min,72℃ 2min 30s,25个循环;72℃延伸10min。
取5μL PCR产物经1%琼脂糖凝胶电泳检测(见图5),图5左边的泳道是分子量Marker,右边是PCR产物的电泳结果,5’-RACE得到3条扩增带,其中约600bp位置的扩增带为所预计的扩增产物,命名为5R1。3’-RACE在约1000bp位置出现所预计的扩增带(图6左边的泳道是分子量Marker,右边是PCR产物的电泳结果),命名为3R1。将5’-RACE和3’-RACE所预计的PCR扩增产物分别切胶回收,连接到pGEM-T Easy载体上,转化感受态大肠杆菌DH5α,各挑取10个白色克隆,培养扩增,提取质粒进行限制性内切酶酶切,分别取含有插入片段的3个克隆进行测序(上海联众基因公司),所测序列一致。各序列通过GenBank/BLAST软件与BjCET1已有序列进行同源性比较,重叠区同源性均达99%以上,只有2-3个碱基的差异,通过参照测序图谱,经过拼接得到BjCET1的全长cDNA序列(图7),共1481个核苷酸,ORF:216-1361,编码382个氨基酸,MW=42012。
步骤5:BjCET2,BjCET3,BjCET4全长cDNA克隆
依据已知的BjCET1序列,参照植物转运蛋白基因TgMTP1和ZAT mRNA序列,在3’端保守区设计合成5’-RACE简并引物5p3:5’-ccggatccgatagcccaa/tatgtga/gag-3’。以步骤4合成的5’-RACE cDNA产物为本次5’-RACE反应的扩增模板(具体操作规程参见试剂盒说明书)。5’-RACE反应条件:94℃变性10min;94℃ 1min,退火温度55℃ 2min,72℃ 2min,10个循环,退火温度每一轮循环增加0.5℃;94℃ 1min,60℃ 1min,72℃ 2min,25个循环;72℃延伸10min。取5μL PCR产物经1%琼脂糖凝胶电泳检测,图8左边的泳道是分子量Marker,右边是PCR产物的电泳结果,5’-RACE得到在1000-1500bp之间的一条亮带,为所预计的扩增产物,命名为5R2(下面的克隆测序实验说明了条带5R2是至少含有3条片段大小相近的DNA序列的混合物)。将5R2切胶回收,连接到pGEM-T Easy载体上,转化感受态大肠杆菌DH5α,挑取20个白色克隆,培养扩增,提取质粒进行限制性内切酶酶切,得到9个含有约1200bp大小插入片段的克隆。分别取该9个克隆进行测序(上海联众基因公司),得到4个不同序列。各序列通过GenBank/BLAST软件分析证明为4个不同的CE家族成员,其中之一为BjCET1(序列为图7中的1-1226位核苷酸),有2个重复的克隆。其它3个不同序列分别命名为BjCET2(序列为图11中的1-1089位核苷酸),BjCET3(序列为图12中的1-1114位核苷酸),BjCET4(序列为图13中的1-1123位核苷酸)。其中BjCET3有2个重复的克隆,BjCET4有3个重复的克隆。
鉴于BjCET2,BjCET3和BjCET4 ORF区域的高同源性,而3序列5’端具有较高的特异性,为了得到BjCET2,BjCET3和BjCET4片段的全长cDNA,在3’-RACE采用巢式PCR。具体实施如下:
引物设计:分别以BjCET2,BjCET3和BjCET4 5’端特异序列设计第1次PCR的上游引物。BjCET2为3p2-1:5’-GGGAACAAACAGATAGAAACTTTA TTGATC-3’;BjCET3为3p3-1:5’-GGAGCACAGGCACGACGACACGAGGC-3’;BjCET4为3p4-1:5’-GGGAGGCAAAAGTGAATCTAAAGGG-3’。然后分别在BjCET2,BjCET3和BjCET4的ORF区域靠近3’端设计第2次PCR的上游引物。BjCET2为3p2-2:5’-CTTGGGGACTCCATCCAGAGCGTTGGTGTT-3’;BjCET3为3p3-2:5’-CGTCATCT GGTGCAACCCGGAATGGAAAA-3’;BjCET4为3p4-1:5’-CCAGAGCGTTGGTGTTATGGTT GGAGGAGC-3’。两次PCR的下游引物均为UPM:5’-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T)30N-1N-3’(其中d(T)30表示30个脱氧胸酰嘧啶,N=A,G,C或T;N-1=A,G或C)。
PCR反应:以步骤4合成的5’-RACE cDNA产物为本次3’-RACE第1次PCR反应的扩增模板,50μL反应体系模板取2μL。分别将各自的第1次PCR反应产物稀释50倍,用作第2次PCR反应的扩增模板,50μL反应体系模板取3μL。PCR反应体系同步骤3。第1次PCR反应的条件为:94℃变性10min;94℃ 1min,55℃ 45s,72℃ 3min,25个循环;72℃延伸10min。第2次PCR反应的条件为:94℃变性10min;94℃ 1min,60℃ 45s,72℃ 1min30s,35个循环;72℃延伸10min。
PCR产物分析:分别取5μL PCR产物经1%琼脂糖凝胶电泳检测,图9右边的泳道是分子量Marker,左边是BjCET2 3’-RACE第2次PCR产物的电泳结果,在500-600bp位置的有一很亮的扩增带,为所预计的扩增产物,命名为3R2。图10中间的泳道是分子量Marker,左边是BjCET3 3’-RACE第2次PCR产物的电泳结果,在500bp左右的位置有一亮的扩增带,命名为3R3。右边是BjCET4 3’-RACE第2次PCR产物的电泳结果,在500-600bp的位置有一亮的扩增带,命名为3R4。将3R2,3R3和3R4 PCR扩增产物分别切胶回收,连接到pGEM-T Easy载体上,转化感受态大肠杆菌DH5α,各挑取10个白色克隆,培养扩增,提取质粒进行限制性内切酶酶切,分别取含有插入片段的3个克隆进行测序(上海联众基因公司),所测序列一致。通过GenBank/BLAST软件进行同源性比较,3R4与BjCET4重叠区同源性达100%,3R2与BjCET2,3R3与BjCET3的重叠区同源性均达99%,只有1-2个碱基的差异,差异的碱基通过参照测序图谱进行修正。
经过拼节得到BjCET2的全长cDNA序列(图11),共1336个核苷酸,ORF:64-1224,编码387个氨基酸,MW=42159。BjCET3的全长cDNA序列(图12),共1363个核苷酸,ORF:95-1249,编码385个氨基酸,MW=42359。BjCET4的全长cDNA序列(图13),共1379个核苷酸,ORF:131-1258,编码376个氨基酸,MW=41421。
步骤6:BjCET1,BjCET2,BjCET3,BjCET4全长cDNA克隆载体构建
依据已有BjCET1,BjCET2,BjCET3,BjCET4的全长cDNA序列,分别设计扩增其全长cDNA的上游引物p1和下游引物p2如下:
BjCET1:p1:5’-GGGGAAGGATAAGAGAGAGAAAAAAAG-3’,
p2:5’-GGACAAAAAGATAGAAAACTTTATTGATT-3’;
BjCET2:p1:5’-GGAGGAAGTAGTGAATCTATCAAACCGGTG-3’,
p2:5’-ggGAACAAACAGATAGAAACTTTATTGATC-3’;
BjCET3:p1:5’-GGAGCACAGGCACGACGACACGAGGC-3’,
p2:5’-gcgggGCAAAAGATTGGAAACTATTG-3’;
BjCET4:p1:5’-GGGAGGCAAAAGTGAATCTAAAGGG-3’,
p2:5’-GGGCGGGGCAAAAGGATTGAAAC-3’。
以步骤4合成的5’-RACE cDNA产物为本次PCR反应的扩增模板,50μL反应体系模板取2μL。PCR反应体系同步骤3。PCR反应的条件为:94℃变性10min;94℃ 1min,55℃ 2min,72℃ 2min,35个循环;72℃延伸10min。分别取5μL PCR产物经1%琼脂糖凝胶电泳检测,如图14均在1300-1500bp位置有一很亮的扩增带,为所预计的扩增产物。将PCR扩增产物分别切胶回收,连接到pGEM-T Easy载体上,转化感受态大肠杆菌DH5α,各挑取5-10个单克隆,培养扩增,提取质粒进行限制性内切酶酶切,分别取含有插入片段的克隆进行测序(上海联众基因公司)验证,所测序列与已知序列一致。-80℃超低温冰箱分别保存pGEM-T Easy载体上含有BjCET1,BjCET2,BjCET3,BjCET4的全长cDNA序列的DH5α菌株。
实施例2印度芥菜CE家族的金属阳离子跨膜转运蛋白新基因转化酵母的重金属抗性分析
步骤1:酵母表达载体的构建
依据穿梭载体pYES2(见图15)上的多克隆限制性酶切位点HindIII/BamHI序列,设计了BjCETs(1-4)序列的ORF区PCR引物(见表1),以pGEM-T Easy载体上的BjCETs(1-4)全长cDNA序列为模板分别扩增其ORF区域,扩增的DNA片段经HindIII/BamHI双酶切后,与HindIII/BamHI双酶切的pYES2载体进行环化连接,转化感受态大肠杆菌DH5α,在含有50μg/mL氨苄青霉素的LB培养基上进行筛选,37℃过夜培养。分别挑取5-10个单克隆,培养扩增,提取质粒进行HindIII/BamHI双酶切,取含有插入片段的克隆进行测序(上海联众基因公司)验证,所测序列与已知序列一致,得到pYES2-BjCET1、pYES2-BjCET2、pYES2-BjCET3和pYES2-BjCET4酵母表达载体。
表1:BjCETs(1-4)序列的ORF区PCR引物
基因 上游引物 下游引物
BjCET1 5’-gggAAGCTTATGGCGTCTTCAAGC-3’5’-ggcGGATCCTTAGCGCTCGATTTGTAT-3’
BjCET2 5’-gggAAGCTTATGGCGTCTTCAAGC-3 5’-ggcGGATCCTTAACGCTCGATTTGTAT-3’
BjCET3 5’-gggAAGCTTATGGCGTCCTCAAGC-3 5’-ggcGGATCCTTAGCGCTCAATTTGTAT-3’
BjCET4 5’-gggAAGCTTATGGAGCCTTCAAGC-3 5’-ggcGGATCCTTAACGCTCAATTTGTAT-3’
步骤2:转化缺陷型酵母
通过化学转化法(PEG-LiAC法)分别将pYES2-BjCET1、pYES2-BjCET2、pYES2-BjCET3和pYES2-BjCET4导入缺陷型酵母菌YK40、YK41和YK44中,其中YK40(ura3-52 his3-200,ΔCot1,交配型α)为Co/Ni缺陷型;YK41(ura3-52his3-200,ΔZRC1,交配型α)为Zn/Cd缺陷型;YK44(ura3-52 his3-200,ΔZRC,ΔCot1,交配型α)为Zn/Cd/Ni/Co缺陷型。选择培养基为SMM固体培养基:6.7g无氨基酸的酵母基本氮源(invitrogen)+20g/L葡萄糖+20mg/L尿嘧啶+20g/L琼脂粉,并通过菌斑PCR技术对单克隆酵母转化菌进行确证,挑选与已知序列大小一致的菌斑保存于-80℃超低温冰箱。
步骤3:抗重金属分析
从-80℃超低温冰箱取出YK40、YK41和YK44及其转化酵母菌划平板。YK40、YK41和YK44用YPD固体培养基(参见《精编分子生物学实验指南》,p481,科学出版社,1998),转化酵母菌用SMM固体选择培养基。在30℃培养箱培养,待长出单克隆后,分别挑取单克隆接种于SMM液体培养基(附加20mg/L尿嘧啶和20mg/L组氨酸)。30℃恒温摇床培养,转速280rpm/min,待菌液浓度摇至对数生长期,分别稀释至OD600=0.1,在含有不同重金属浓度的SMM固体选择培养基(附加20mg/L尿嘧啶和20mg/L组氨酸)上划线,30℃培养,4天后观察酵母菌的生长情况。
结果:
(1)Zn/Cd/Ni/Co缺陷型酵母yk44及其转化菌Yk44-pyes2(用p0表示),Yk44-pyes2-BjCET1(用p1表示),Yk44-pyes2-BjCET2(用p2表示),Yk44-pyes2-BjCET3(用p3表示)和Yk44-pyes2-BjCET4(用p4表示)在含有0.5mM NiCl2,0.5mM ZnCl2和0.5mM CoCl2培养基上的生长情况。只含有空载体的酵母菌Yk44-pyes2在三种培养基上均不能生长,没有单克隆菌斑出现,而转运蛋白转基因酵母却表现了不同程度的重金属抗性。在0.5mM NiCl2培养基上,四种转运蛋白转基因酵母p1-p4划线部分均长满了菌斑,表现了对Ni和Co高的抗性。在0.5mM ZnCl2培养基上,只有BjCET1和BjCET2转化酵母p1和p2划线部分有菌斑长出,说明二者对Zn有较高的抗性。在0.5mMCoCl2培养基上,BjCET1转化酵母p1出现的菌斑较少,说明BjCET1对Co的抗性要低于其它三种转运蛋白基因,但高于对照空载体转化酵母菌p0。
(2)Ni/Co缺陷型酵母yk40及其转化菌Yk40-pyes2(p0),Yk40-pyes2-BjCET1(p1),Yk40-pyes2-BjCET2(p2),Yk40-pyes2-BjCET3(p3)和Yk40-pyes2-BjCET4(p4)在含有0.5mM CoCl2,0.8mM CoCl2和1.2mMCoCl2培养基上的生长情况。转运蛋白基因转化酵母菌p1-p4均表现了对Co高的抗性,在0.5mM CoCl2和0.8mM CoCl2培养基上均有大量菌斑长出,但随Co浓度增加菌斑量也逐渐减少,在1.2mM CoCl2培养基上只有转基因酵母p2有菌斑出现,表明BjCET2比其它三个基因表现了更高的Co抗性。而只含有空载体的酵母菌p0在0.5mM CoCl2培养基上略有生长,在0.8mMCoCl2和1.2mM CoCl2培养基上均不能生长。四种基因对Co的抗性在结果(1)也得到了验证。
(3)Zn/Cd缺陷型酵母yk41及其转化菌Yk41-pyes2(p0),Yk41-pyes2-BjCET1(p1),Yk41-pyes2-BjCET2(p2),Yk41-pyes2-BjCET3(p3)和Yk41-pyes2-BjCET4(p4),在含有0.5mM CoCl2,0.8mM CoCl2和1.2mMCoCl2培养基上的生长情况。Yk41对Co不敏感,故在0.5mM CoCl2和0.8mMCoCl2培养基上p0-p4均有不少菌斑长出,但含有空载体的酵母菌p0菌斑量要少于p1-p4。在1.2mM CoCl2培养基上只含有空载体的酵母菌p0不能生长,而p1-p4转化酵母菌均有菌斑长出。再次说明转BjCET1、BjCET2、BjCET3和BjCET4基因能提高酵母菌对Co胁迫的抗性。
(4)Ni/Co缺陷型酵母yk40及其转化菌Yk40-pyes2(p0),Yk40-pyes2-BjCET1(p1),Yk40-pyes2-BjCET2(p2),Yk40-pyes2-BjCET3(p3)和Yk40-pyes2-BjCET4(p4)在含有0.5mM NiCl2和1mM NiCl2培养基上的生长情况。转运蛋白基因转化酵母菌p1-p4均表现了对Ni高的抗性,只含有空载体的酵母菌p0在0.5mM NiCl2培养基上略有生长,在1mM NiCl2培养基上已不能生长,但p1-p4均可生长,其中p2菌斑量最大,表现了对Ni更高的抗性。
(5)Ni/Co缺陷型酵母yk40及其转化菌Yk40-pyes2(p0),Yk40-pyes2-BjCET1(p1),Yk40-pyes2-BjCET2(p2),Yk40-pyes2-BjCET3(p3)和Yk40-pyes2-BjCET4(p4)在含有0.5mM ZnCl2,1mM ZnCl2和7.5mM ZnCl2培养基上的生长情况。yk40对Zn不敏感,在三个浓度梯度的培养基上p0-p4均能生长,但p1-p4酵母转化菌菌斑量明显好于转空载体的酵母菌p0,说明转BjCET1、BjCET2、BjCET3和BjCET4基因能提高酵母菌对Zn胁迫的抗性。
从以上实验还发现,转空载体的缺陷型酵母菌比其本身对重金属离子更敏感,BjCET1,BjCET2,BjCET3和BjCET4转基因酵母可不同程度挽救缺陷型酵母菌对金属离子的敏感性,从而提高了酵母菌对重金属的抗性。
实施例3印度芥菜CE家族的金属阳离子跨膜转运蛋白新基因转化烟草的重金属抗性和累积分析
步骤1:植物表达载体的构建与农杆菌转化
依据双价载体pBIN-mGFP5-ER(见图16)上的多克隆限制性酶切位点XbaI/BamHI序列,设计了BjCETs(1-4)序列的ORF区PCR引物(见表2),以pGEM-T Easy载体上的BjCETs(1-4)全长cDNA序列为模板分别扩增其ORF区域,扩增的DNA片段经XbaI/BamHI酶切后,与XbaI/BamHI双酶切的pBIN-mGFP5-ER载体进行环化连接,转化感受态大肠杆菌DH5α,在含有50μg/mL卡那霉素的LB培养基上进行筛选。37℃过夜培养。分别挑取5-10个单克隆,培养扩增,提取质粒进行XbaI/BamHI双酶切,分别取含有插入片段的克隆进行测序(上海联众基因公司)验证,所测序列与已知序列一致,得到pBIN-mGFP5-ER-BjCET1、pBIN-mGFP5-ER-BjCET2、pBIN-mGFP5-ER-BjCET3和pBIN-mGFP5-ER-BjCET4植物表达载体,其中含有CaMV 35S组成性表达强启动子和GFP绿色荧光蛋白报告基因,所表达的CE转运蛋白与GFP构成融合蛋白。应用热激法(TzVi Tzfira et al,PlantMolecular Biology Reporter,1997,15:219-235)将pBIN-mGFP5-ER-BjCET1、pBIN-mGFP5-ER-BjCET2、pBIN-mGFP5-ER-BjCET3和pBIN-mGFP5-ER-BjCET4导入农杆菌(Agrobacteria tumefaciencs)EHA105中,在含有30μg/mL利福平(Rif)和50μg/mL卡那霉素(Kan)的LB培养基上进行筛选,并通过菌斑PCR技术对单克隆转化农杆菌进行确证。
表2
基因 上游引物 下游引物
BjCET1 5’-gggTCTAGAATGGCGTCTTC 5’-ggcGGATCCgGCGCTCGATTTG
AAGC-3’ TAT-3’
BjCET2 5’-gggTCTAGAATGGCGTCTTC 5’-ggcGGATCCgACGCTCGATTTG
AAGC-3 TAT-3’
BjCET3 5’-gggTCTAGAATGGCGTCCTC 5’-ggcGGATCCgGCGCTCAATTTG
AAGC-3 TAT-3’
BjCET4 5’-gggTCTAGAATGGAGCCTTC 5’-ggcGGATCCgACGCTCAATTTG
AAGC-3 TAT-3’
步骤2:烟草转化
选用烟草品种W38(Nicotiana tabacum c.v.W38)作为转基因受体,采用叶盘法(Horsch R B et al,Science,1985,227:1229-1231)转化烟草品种W38。以MS(Murashige&Shoog)为基本培养基,其中分化再生培养基为T0:MS+1mg/L6-BA+0.1mg/L NAA+100mg/L卡那霉素+250mg/L Cef(头孢霉素)+8g/L琼脂,PH5.8;生根培养基为T1:1/2MS+0.1mg/L NAA+100mg/L卡那霉素+250mg/L Cef+8g/L琼脂,PH5.8。
挑取携带pBIN-mGFP5-ER-BjCET1、pBIN-mGFP5-ER-BjCET2、pBIN-mGFP5-ER-BjCET3和pBIN-mGFP5-ER-BjCET4改良载体的农杆菌EHA105的单菌落,接种于5ml含Rif 20mg/l和Kan 50mg/L的YEB液体培养基中,28℃振荡培养过夜。取活化过夜的农杆菌,按1∶50的比例稀释到含Rif 30mg/L和Kan 50mg/L的LB液体培养基中,继续培养至OD600值大约为0.6-0.8。5000rpm离心5min,收集菌体,用1/2 MS液体培养基洗涤菌体一次,并将其稀释至OD600值0.3-0.35。
选取约30天苗龄的烟草无菌苗,切下成熟叶片,用直径6mm的打孔器制取叶盘外植体。将新制备的外植体投入已准备好的农杆菌菌液中,振荡侵染15-20分钟后,取出用滤纸吸干附着于叶盘表面的残液,然后放在表面铺有一层滤纸的不含抗生素的T0培养基上暗处共培养两天,然后再转到T0培养基上进行筛选培养,每隔2-3周用T0培养基继代一次。待抗性芽长到1-1.5cm时,将其切下换到T1培养基上诱导生根并获得抗性植株。2周后观察发现,转基因烟草长势明显优于野生型烟草(WT),对照WT无根生出,而转基因烟草均已有根生出。
利用CTAB法提取WT及转基因烟草植株叶片基因组DNA,用GFP上游引物p1:5’-cccGGATCCAAGGAGATATAAC-3’和下游引物p2:5’-CCCGAGCTCTTATTIGTATAGTTCATCC-3’做PCR,反应体系25μL反应体系,取基因组DNA模板2μL,PCR反应体系同实施例1步骤3。PCR反应的条件为:94℃预变性10min;94℃ 1min,55℃ 2min,72℃ 2min,35个循环;72℃延伸10min。如图17A和17B,GFP片段约800bp,对照WT无扩增带,各转基因烟草植株均有800bp DNA扩增带,说明融合基因BjCET1-GFP、BjCET2-GFP、BjCET3-GFP和BjCET4-GFP已分别整合到烟草基因组中。
利用共聚焦激光扫描电镜(Confocal laser-scanning microscope),取活体植株叶片进行观察,拍照,对照WT植株只是红色叶绿素激发荧光,转基因植株叶片均有绿色激发荧光。说明所转化基因在蛋白质水平上得到表达。
步骤3:T0代烟草重金属抗性和累积分析
重金属对转基因植株组织培养分化再生的影响:分别剪取约1cm2的WT及转基因植株叶片,接种于T0分化培养基(不含抗生素,附加有200μM/L的CdCL2)上,每瓶接种10片,各接种3瓶,25℃,16h/d光照培养。约35d(天)后,计算分化苗个数(见表3)。
表3:分化苗个数
可见Cd胁迫严重抑制了野生型烟草的叶片组织分化成苗,而转CE转运蛋白基因的烟草植株,均不同程度提高了Cd胁迫条件下的组织分化成苗能力,约为WT的3倍以上,其中,转BjCET4烟草出苗数最多,约是WT的4.67倍,三者也均远高于转空载体烟草。
重金属胁迫条件下转基因植株的重金属抗性和累积分析:分别剪取约1cm2的WT及转基因植株叶片,接种于T0分化培养基(WT接种于不含抗生素的T0培养基)上,25℃,16h/d光照培养。待再生植株长至2-3cm时挑选整齐一致的再生苗,接种于不合抗生素的T1生根培养基,附加有200μM/L的CdCL2,每瓶接种3棵,各接种两瓶。12d后观察生根情况(见表4),发现,野生型与转空载体烟草苗均无根生出,而转基因烟草,均有根生出,其中转BjCET4烟草生根较多,且长,长势也优于对照。30d后,从培养瓶中收获烟草,测试计算各种指标数据见表5。其中Cd含量的测定应用原子吸收光谱法,由国家有色金属研究院测试中心完成。
由表5分析可知,Cd胁迫对烟草根的生长有明显抑制作用,转CE转运蛋白基因烟草明显提高了烟草根的抗胁迫能力,其每株根数约是WT的1.3-1.8倍,每株根长约是WT的1.4-1.6倍,而每株根鲜重约是WT的1.88-3.98倍。其中转BjCET1和BjCET4烟草植株根的抗Cd能力最具优势。通过对比转基因烟草与WT根部Cd累积量发现,根部过量表达BjCET1,比WT减少了16.92%的Cd累积量;过量表达BjCET3,比WT减少了6.15%的Cd累积量。故二者根部抗Cd能力的提高可能是通过BjCET1和BjCET3的外排作用,减少了Cd在根部的累积而获得的。参照茎叶部的Cd含量发现,二者茎叶部Cd含量最高,比WT均增加了56%。所以,过量表达BjCET1和BjCET3可以起到在烟草茎叶部富集Cd的作用,虽然Cd的富集一定程度抑制了茎叶的生长,但仍高于转空载体烟草植株。过量表达BjCET4在根部和茎叶部与WT相比均增加了Cd累积量,但主要是提高了茎叶部的Cd含量,虽然增幅低于BjCET1和BjCET3,但其茎叶部生物量却远高于二者及WT,故其对Cd的累积量在茎叶部最高。过量表达BjCET2转基因烟草,在根部的Cd累积量最高,比WT增加了41.5%,而茎叶部则比WT减少了52%,故过量表达BjCET2转运蛋白明显限制了Cd向地上部的运输累积,很适于在应用基因工程技术降低重金属在粮食和蔬菜可食物部位累积方面发挥作用。
表4:12天后的生根情况
表5:30天后的烟草生长情况
FPI04057-sequence list
SEQUENCE LISTING
<110>中国科学院研究生院
<120>印度芥菜阳离子输出转运蛋白家族的基因BjCET4及其蛋白质
<130>FPI04057
<150>CN 03119515.6
<151>2003-03-07
<160>10
<170>PatentIn version 3.1
<210>1
<211>1125
<212>DNA
<213>Brassica juncea
<220>
<221>CDS
<222>(1)..(1125)
<223>
<400>1
atg gag tct tca agt ccc caa cat tgc cac atc atc gag gtc aat cga 48
Met Glu Ser Ser Ser Pro Gln His Cys His Ile Ile Glu Val Asn Arg
1 5 10 15
ggt aaa tcc gtt gaa gaa agc aca aca att ctg gca agc aaa gcc tgc 96
Gly Lys Ser Val Glu Glu Ser Thr Thr Ile Leu Ala Ser Lys Ala Cys
20 25 30
gga gaa gcc ccc tgc ggc ttc tca gat ctc aac aac gct tcc ggt gac 144
Gly Glu Ala Pro Cys Gly Phe Ser Asp Leu Asn Asn Ala Ser Gly Asp
35 40 45
gcc caa gaa cgc aat gcc tcc atg cgc aag ctc tgc atc gcc gtg gtg 192
Ala Gln Glu Arg Asn Ala Ser Met Arg Lys Leu Cys Ile Ala Val Val
50 55 60
tta tgc ctt ctc ttc atg acc gtt gaa gtc ttt ggt ggc atc aaa gct 240
Leu Cys Leu Leu Phe Met Thr Val Glu Val Phe Gly Gly Ile Lys Ala
65 70 75 80
aac agc cta gct ata ctc acc gac gca gcg cat ctc ctc tct gac gtt 288
Asn Ser Leu Ala Ile Leu Thr Asp Ala Ala His Leu Leu Ser Asp Val
85 90 95
gct gcc ttc gcc atc tcc ctg ttc tcc ctg tgg gcc gct ggt tgg gaa 336
Ala Ala Phe Ala Ile Ser Leu Phe Ser Leu Trp Ala Ala Gly Trp Glu
100 105 110
gcg acg ccg agg cag act tat ggt ttt ttc agg att gag att ttg gga 384
Ala Thr Pro Arg Gln Thr Tyr Gly Phe Phe Arg Ile Glu Ile Leu Gly
115 120 125
gct ctt gtc tcc atc cag ctc att tgg ctc ctc act ggt att ctt gtc 432
Ala Leu Val Ser Ile Gln Leu Ile Trp Leu Leu Thr Gly Ile Leu Val
130 135 140
tat gaa gct atc atc aga ctt ctt agt gag act agt gag gtt aat ggt 480
Tyr Glu Ala Ile Ile Arg Leu Leu Ser Glu Thr Ser Glu Val Asn Gly
145 150 155 160
ttc ctt atg ttc ctt gtt gct gct ttt ggg ttg ctt gtg aat atc ata 528
Phe Leu Met Phe Leu Val Ala Ala Phe Gly Leu Leu Val Asn Ile Ile
165 170 175
atg gct gtt ctg ttg gga cat gat cat ggt cac ggt cat ggc cat ggt 576
Met Ala Val Leu Leu Gly His Asp His Gly His Gly His Gly His Gly
180 185 190
cat gat cat cac agt cat ggg gtg acg gtt acc aca cat cat cac cat 624
FPI04057-sequence list
His Asp His His Ser His Gly Val Thr Val Thr Thr His His His His
195 200 205
cat ggt cat gga gag gac aag cat cat cat cac gct cat ggg gat gaa 672
His Gly His Gly Glu Asp Lys His His His His Ala His Gly Asp Glu
210 215 220
gat gtg act gag cag ttg ctg gag aaa tca gag aag aga aag agg aac 720
Asp Val Thr Glu Gln Leu Leu Glu Lys Ser Glu Lys Arg Lys Arg Asn
225 230 235 240
atc aat gtc caa gga gct tac ctc cat gtc ctt gga gac tca atc cag 768
Ile Asn Val Gln Gly Ala Tyr Leu His Val Leu Gly Asp Ser Ile Gln
245 250 255
agc gtt ggt gtt atg att gga gga ggt atc atc tgg tac aac ccg gag 816
Ser Val Gly Val Met Ile Gly Gly Gly Ile Ile Trp Tyr Asn Pro Glu
260 265 270
tgg aag ata gtc gat ctg atc tgc acg ctt gtc ttt tcc gtg att gtc 864
Trp Lys Ile Val Asp Leu Ile Cys Thr Leu Val Phe Ser Val Ile Val
275 280 285
ttg ggg acg acc atc aac atg atc aga agc att ctt gag gtg ttg atg 912
Leu Gly Thr Thr Ile Asn Met Ile Arg Ser Ile Leu Glu Val Leu Met
290 295 300
gag agc acg ccg aga gag att gac gct acg aag ctg gag aag ggt ttg 960
Glu Ser Thr Pro Arg Glu Ile Asp Ala Thr Lys Leu Glu Lys Gly Leu
305 310 315 320
ctg gag atg gaa gaa gtg gtg gct gtt cat gag ctt cac att tgg gct 1008
Leu Glu Met Glu Glu Val Val Ala Val His Glu Leu His Ile Trp Ala
325 330 335
atc acg gtg ggg aaa gtg ttg ctt gct tgc cat gtt aat atc aca ccg 1056
Ile Thr Val Gly Lys Val Leu Leu Ala Cys His Val Asn Ile Thr Pro
340 345 350
gag gca gat gcg gac atg gtg ctt aac aag gtc att gat tac atc cgc 1104
Glu Ala Asp Ala Asp Met Val Leu Asn Lys Val Ile Asp Tyr Ile Arg
355 360 365
agg gag tac aac atc agt cat 1125
Arg Glu Tyr Asn Ile Ser His
370 375
<210>2
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<213>Brassica juncea
<400>2
Met Glu Ser Ser Ser Pro Gln His Cys His Ile Ile Glu Val Asn Arg
1 5 10 15
Gly Lys Ser Val Glu Glu Ser Thr Thr Ile Leu Ala Ser Lys Ala Cys
20 25 30
Gly Glu Ala Pro Cys Gly Phe Ser Asp Leu Asn Asn Ala Ser Gly Asp
35 40 45
Ala Gln Glu Arg Asn Ala Ser Met Arg Lys Leu Cys Ile Ala Val Val
50 55 60
Leu Cys Leu Leu Phe Met Thr Val Glu Val Phe Gly Gly Ile Lys Ala
65 70 75 80
FPI04057-sequence list
Asn Ser Leu Ala Ile Leu Thr Asp Ala Ala His Leu Leu Ser Asp Val
85 90 95
Ala Ala Phe Ala Ile Ser Leu Phe Ser Leu Trp Ala Ala Gly Trp Glu
100 105 110
Ala Thr Pro Arg Gln Thr Tyr Gly Phe Phe Arg Ile Glu Ile Leu Gly
115 120 125
Ala Leu Val Ser Ile Gln Leu Ile Trp Leu Leu Thr Gly Ile Leu Val
130 135 140
Tyr Glu Ala Ile Ile Arg Leu Leu Ser Glu Thr Ser Glu Val Asn Gly
145 150 155 160
Phe Leu Met Phe Leu Val Ala Ala Phe Gly Leu Leu Val Asn Ile Ile
165 170 175
Met Ala Val Leu Leu Gly His Asp His Gly His Gly His Gly His Gly
180 185 190
His Asp His His Ser His Gly Val Thr Val Thr Thr His His His His
195 200 205
His Gly His Gly Glu Asp Lys His His His His Ala His Gly Asp Glu
210 215 220
Asp Val Thr Glu Gln Leu Leu Glu Lys Ser Glu Lys Arg Lys Arg Asn
225 230 235 240
Ile Asn Val Gln Gly Ala Tyr Leu His Val Leu Gly Asp Ser Ile Gln
245 250 255
Ser Val Gly Val Met Ile Gly Gly Gly Ile Ile Trp Tyr Asn Pro Glu
260 265 270
Trp Lys Ile Val Asp Leu Ile Cys Thr Leu Val Phe Ser Val Ile Val
275 280 285
Leu Gly Thr Thr Ile Asn Met Ile Arg Ser Ile Leu Glu Val Leu Met
290 295 300
Glu Ser Thr Pro Arg Glu Ile Asp Ala Thr Lys Leu Glu Lys Gly Leu
305 310 315 320
Leu Glu Met Glu Glu Val Val Ala Val His Glu Leu His Ile Trp Ala
325 330 335
Ile Thr Val Gly Lys Val Leu Leu Ala Cys His Val Asn Ile Thr Pro
340 345 350
Glu Ala Asp Ala Asp Met Val Leu Asn Lys Val Ile Asp Tyr Ile Arg
355 360 365
Arg Glu Tyr Asn Ile Ser His
370 375
FPI04057-sequence list
<210>3
<211>1481
<212>DNA
<213>Brassica juncea
<220>
<221>CDS
<222>(216)..(1361)
<223>
<400>3
gaaggataag agagagaaaa aaagaaaaaa aggaccccgg aaagaaaagg aaaagagaag 60
ctgcccccat ctcatctctc gtggccgcct catacttttc tattcataaa taaaaggctt 120
cttccttttt ccttgacatt ctttaaaggg ctaggaggag tagtgaatct atcaaaccgg 180
caacttcagc ctctcgactc tcttctctct ctctg atg gcg tct tca agc ccc 233
Met Ala Ser Ser Ser Pro
1 5
caa cat tgc cac atc atc gag gtc aat cga ggt aaa tcc gtt gaa gaa 281
Gln His Cys His Ile Ile Glu Val Asn Arg gly Lys Ser Val Glu Glu
10 15 20
agc aca aca att ctg gca agc aaa gcc tgc gga gaa gcc ccc tgc ggc 329
Ser Thr Thr Ile Leu Ala Ser Lys Ala Cys Gly Glu Ala Pro Cys Gly
25 30 35
ttc tca gat ctc aac aac gct tcc ggt gac gcc caa gaa cgc aat gcc 377
Phe Ser Asp Leu Asn Asn Ala Ser Gly Asp Ala Gln Glu Arg Asn Ala
40 45 50
tcc atg cgc aag ctc tgc atc gcc gtg gtg cta tgc ctt ctc ttc atg 425
Ser Met Arg Lys Leu Cys Ile ala Val Val Leu Cys Leu Leu Phe Met
55 60 65 70
acc gtt gaa gtc ttt ggt ggc atc aaa gct aac agc cta gct ata ctc 473
Thr Val Glu Val Phe Gly gly Ile Lys Ala Asn Ser Leu Ala Ile Leu
75 80 85
acc gac gca gcg cat ctc ctc tct gac gtt gct gcc ttc gcc atc tcc 521
Thr Asp Ala Ala His Leu Leu Ser Asp Val Ala Ala Phe Ala Ile Ser
90 95 100
ctg ttc tcc ctg tgg gcc gct ggt tgg gaa gcg acg ccg agg cag act 569
Leu Phe Ser Leu Trp Ala Ala Gly Trp Glu Ala Thr Pro Arg Gln Thr
105 110 115
tat ggt ttt ttc agg att gag att ttg gga gct ctt gtc tcc atc cag 617
Tyr Gly Phe Phe Arg Ile Glu Ile Leu Gly Ala Leu Val Ser Ile Gln
120 125 130
ctc att tgg ctc ctc act ggt att ctt gtc tat gaa gct atc atc aga 665
Leu Ile Trp Leu Leu Thr Gly Ile Leu Val Tyr Glu Ala Ile Ile Arg
135 140 145 150
ctt ctt agt gag act agt gag gtt aat ggt ttc ctt atg ttc ctt gtt 713
Leu Leu Ser Glu Thr Ser Glu Val Asn Gly Phe Leu Met Phe Leu Val
155 160 165
gct gct ttt ggg ttg ctt gtg aat atc ata atg gct gtt ctg ttg gga 761
Ala Ala Phe Gly Leu Leu Val Asn Ile Ile Met Ala Val Leu Leu Gly
170 175 180
cat gat cat ggt cac ggt cat ggc cat ggt cat gat cat cac agt cat 809
His Asp His Gly His Gly His Gly His Gly His Asp His His Ser His
185 190 195
ggg gtg acg gtt acc aca cat cat cac cat cat ggt cat gga gag gac 857
Gly Val Thr Val Thr Thr His His His His His Gly His Gly Glu Asp
200 205 210
aag cat cat cat cac gct cat ggg gat gaa gat gtg act gag cag ttg 905
FPI04057-sequence list
Lys His His His His Ala His Gly Asp Glu Asp Val Thr Glu Gln Leu
215 220 225 230
ctg gag aaa tca gag aag aga aag agg aac atc aat gtc caa gga gct 953
Leu Glu Lys Ser Glu Lys Arg Lys Arg Asn Ile Asn Val Gln Gly Ala
235 240 245
tac ctc cat gtc ctt gga gac tca atc cag agc gtt ggt gtt atg att 1001
Tyr Leu His Val Leu Gly Asp Ser Ile Gln Ser Val Gly Val Met Ile
250 255 260
gga gga ggt atc atc tgg tac aac ccg gag tgg aag ata gtc gat ctg 1049
Gly Gly Gly Ile Ile Trp Tyr Asn Pro Glu Trp Lys Ile Val Asp Leu
265 270 275
atc tgc acg ctt gtc ttt tcc gtg att gtc ttg ggg acg acc atc aac 1097
Ile Cys Thr Leu Val Phe Ser Val Ile Val Leu Gly Thr Thr Ile Asn
280 285 290
atg atc aga agc att ctt gag gtg ttg atg gag agc acg ccg aga gag 1145
Met Ile Arg Ser Ile Leu Glu Val Leu Met Glu Ser Thr Pro Arg Glu
295 300 305 310
att gac gct acg aag ctg gag aag ggt ttg ctg gag atg gaa gaa gtg 1193
Ile Asp Ala Thr Lys Leu Glu Lys Gly Leu Leu Glu Met Glu Glu Val
315 320 325
gtg gct gtt cat gag ctt cac att tgg gct atc acg gtg ggg aaa gtg 1241
Val Ala Val His Glu Leu His Ile Trp Ala Ile Thr Val Gly Lys Val
330 335 340
ttg ctt gct tgc cat gtt aat atc aca ccg gag gca gat gcg gac atg 1289
Leu Leu Ala Cys His Val Asn Ile Thr Pro Glu Ala Asp Ala Asp Met
345 350 355
gtg ctt aac aag gtc att gat tac atc cgc agg gag tat aat atc agt 1337
Val Leu Asn Lys Val Ile Asp Tyr Ile Arg Arg Glu Tyr Asn Ile Ser
360 365 370
cat gtc act ata caa atc gag cgc tgaaaggcaa atcaaatatc tcaagagtat 1391
His Val Thr Ile Gln Ile Glu Arg
375 380
tttgtataag catttgcatt gacatgatga taaaatcaat aaagttttct atctttttgt 1451
ccaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1481
<210>4
<211>382
<212>PRT
<213>Brassica juncea
<400>4
Met Ala Ser Ser Ser Pro Gln His Cys His Ile Ile Glu Val Asn Arg
1 5 10 15
Gly Lys Ser Val Glu Glu Ser Thr Thr Ile Leu Ala Ser Lys Ala Cys
20 25 30
Gly Glu Ala Pro Cys Gly Phe Ser Asp Leu Asn Asn Ala Ser Gly Asp
35 40 45
Ala Gln Glu Arg Asn Ala Ser Met Arg Lys Leu Cys Ile Ala Val Val
50 55 60
Leu Cys Leu Leu Phe Met Thr Val Glu Val Phe Gly Gly Ile Lys Ala
65 70 75 80
FPI04057-sequence list
Asn Ser Leu Ala Ile Leu Thr Asp Ala Ala His Leu Leu Ser Asp Val
85 90 95
Ala Ala Phe Ala Ile Ser Leu Phe Ser Leu Trp Ala Ala Gly Trp Glu
100 105 110
Ala Thr Pro Arg Gln Thr Tyr Gly Phe Phe Arg Ile Glu Ile Leu Gly
115 120 125
Ala Leu Val Ser Ile Gln Leu Ile Trp Leu Leu Thr Gly Ile Leu Val
130 135 140
Tyr Glu Ala Ile Ile Arg Leu Leu Ser Glu Thr Ser Glu Val Asn Gly
145 150 155 160
Phe Leu Met Phe Leu Val Ala Ala Phe Gly Leu Leu Val Asn Ile Ile
165 170 175
Met Ala Val Leu Leu Gly His Asp His Gly His Gly His Gly His Gly
180 185 190
His Asp His His Ser His Gly Val Thr Val Thr Thr His His His His
195 200 205
His Gly His Gly Glu Asp Lys His His His His Ala His Gly Asp Glu
210 215 220
Asp Val Thr Glu Gln Leu Leu Glu Lys Ser Glu Lys Arg Lys Arg Asn
225 230 235 240
Ile Asn Val Gln Gly Ala Tyr Leu His Val Leu Gly Asp Ser Ile Gln
245 250 255
Ser Val Gly Val Met Ile Gly Gly Gly Ile Ile Trp Tyr Asn Pro Glu
260 265 270
Trp Lys Ile Val Asp Leu Ile Cys Thr Leu Val Phe Ser Val Ile Val
275 280 285
Leu Gly Thr Thr Ile Asn Met Ile Arg Ser Ile Leu Glu Val Leu Met
290 295 300
Glu Ser Thr Pro Arg Glu Ile Asp Ala Thr Lys Leu Glu Lys Gly Leu
305 310 315 320
Leu Glu Met Glu Glu Val Val Ala Val His Glu Leu His Ile Trp Ala
325 330 335
Ile Thr Val Gly Lys Val Leu Leu Ala Cys His Val Asn Ile Thr Pro
340 345 350
Glu Ala Asp Ala Asp Met Val Leu Asn Lys Val Ile Asp Tyr Ile Arg
355 360 365
Arg Glu Tyr Asn Ile Ser His Val Thr Ile Gln Ile Glu Arg
370 375 380
FPI04057-sequence list
<210>5
<211>1336
<212>DNA
<213>Brassica juncea
<220>
<221>CDS
<222>(64)..(1224)
<223>
<400>5
ggaggaagta gtgaatctat caaaccggtg ttaaacttcg tcctcgactc tctcctcctc 60
tga atg gcg tct tca agc ccc caa cgt agt cac atc att gag gtt aaa 108
Met Ala Ser Ser Ser Pro Gln Arg Ser His Ile Ile Glu Val Lys
1 5 10 15
gca ggc aaa gcc gtt gaa gaa agc acg aca agt ctg gca agt tta gcc 156
Ala Gly Lys Ala Val Glu Glu Ser Thr Thr Ser Leu Ala Ser Leu Ala
20 25 30
tgt gga gaa gca ccc tgt ggc ttc tca gat ctc aac aac gct tcc ggc 204
Cys Gly Glu Ala Pro Cys Gly Phe Ser Asp Leu Asn Asn Ala Ser Gly
35 40 45
gac gcc cag gaa cgc aat gct tcc atg cgc aag ctc tgt atc gct gtg 252
Asp Ala Gln Glu Arg Asn Ala Ser Met Arg Lys Leu Cys Ile Ala Val
50 55 60
gtg cta tgt ctt ctc ttc atg aca gtt gaa gtc ttt ggt ggg atc aaa 300
Val Leu Cys Leu Leu Phe Met Thr Val Glu Val Phe Gly Gly Ile Lys
65 70 75
gct aac agc ttg gct ata cta acc gat gca gcg cat ctt ctc tct gat 348
Ala Asn Ser Leu Ala Ile Leu Thr Asp Ala Ala His Leu Leu Ser Asp
80 85 90 95
gtt gct gcc ttt gct atc tcc ttg ttc tcc ttg tgg gct gct ggt tgg 396
Val Ala Ala Phe Ala Ile Ser Leu Phe Ser Leu Trp Ala Ala Gly Trp
100 105 110
gaa gct acc cca agg cag act tat ggg ttt ttc agg gtt gag att ctt 444
Glu Ala Thr Pro Arg Gln Thr Tyr Gly Phe Phe Arg Val Glu Ile Leu
115 120 125
ggt gct ctt gtc tct atc cag ctc att tgg ctt ctc act ggt att ctt 492
Gly Ala Leu Val Ser Ile Gln Leu Ile Trp Leu Leu Thr Gly Ile Leu
130 135 140
gtt tat gaa gct atc atc aga ctt ctt tca gag acc agt gag gtt aat 540
Val Tyr Glu Ala Ile Ile Arg Leu Leu Ser Glu Thr Ser Glu Val Asn
145 150 155
ggg ttc ctt atg ttt ctt gtt gct gct ttt ggg ttg ctt gtg aat atc 588
Gly Phe Leu Met Phe Leu Val Ala Ala Phe Gly Leu Leu Val Asn Ile
160 165 170 175
gta atg gct gtt ttg ctc ggg cat gat cat ggt cat ggt cat ggc cat 636
Val Met Ala Val Leu Leu Gly His Asp His Gly His Gly His Gly His
180 185 190
ggt cac ggt cat gac cat cac agt cat ggg gtg act gtt acc aca cat 684
Gly His Gly His Asp His His Ser His Gly Val Thr Val Thr Thr His
195 200 205
cat cat ggc cat gat cat ggt cat act cat gga gag gac aag cac cat 732
His His Gly His Asp His Gly His Thr His Gly Glu Asp Lys His His
210 215 220
cac gct cat ggt cat ggg gat gtg act gag cag ttg ctg gac aaa tca 780
His Ala His Gly His Gly Asp Val Thr Glu Gln Leu Leu Asp Lys Ser
225 230 235
gag aag agg aag agg aac atc aat gtc cag gga gct tat ctt cat gtc 828
FPI04057-sequence list
Glu Lys Arg Lys Arg Asn Ile Asn Val Gln Gly Ala Tyr Leu His Val
240 245 250 255
ctt ggg gac tcc atc cag agc gtt ggt gtt atg atc ggt gga gct gtc 876
Leu Gly Asp Ser Ile Gln Ser Val Gly Val Met Ile Gly Gly Ala Val
260 265 270
atc tgg tac aaa ccg gag tgg aag ata gtt gat ctg atc tgc acg ctt 924
Ile Trp Tyr Lys Pro Glu Trp Lys Ile Val Asp Leu Ile Cys Thr Leu
275 280 285
gtc ttt tcg gtt att gtc ttg ggg aca acc atc aac atg atc cga agc 972
Val Phe Ser Val Ile Val Leu Gly Thr Thr Ile Asn Met Ile Arg Ser
290 295 300
att ctt gag gtg ttg atg gag agt aca ccg aga gag att gac gct acg 1020
Ile Leu Glu Val Leu Met Glu Ser Thr Pro Arg Glu Ile Asp Ala Thr
305 310 315
aag ctg gaa aag ggt ctg ctg gaa atg gat gaa gtg gtg gct gtt cat 1068
Lys Leu Glu Lys Gly Leu Leu Glu Met Asp Glu Val Val Ala Val His
320 325 330 335
gag ctt cac att tgg gct atc acg gtg ggg aaa gtg ctg ctt gct tgc 1116
Glu Leu His Ile Trp Ala Ile Thr Val Gly Lys Val Leu Leu Ala Cys
340 345 350
cat gtt aat atc aca cca gag gca gat gcg gat atg gtg ctc aat aaa 1164
His Val Asn Ile Thr Pro Glu Ala Asp Ala Asp Met Val Leu Asn Lys
355 360 365
gtc att gat tac atc cgc ggg gag tat aat atc agt cat gtc act ata 1212
Val Ile Asp Tyr Ile Arg Gly Glu Tyr Asn Ile Ser His Val Thr Ile
370 375 380
caa atc gag cgt taaaagcaaa catctgcaga gtattttgta taagcattct 1264
Gln Ile Glu Arg
385
cattgacgat gataagatca ataaagtttc tatctgtttg ttcaaaaaaa aaaaaaaaaa 1324
aaaaaaaaaa aa 1336
<210>6
<211>387
<212>PRT
<213>Brassica juncea
<400>6
Met Ala Ser Ser Ser Pro Gln Arg Ser His Ile Ile Glu Val Lys Ala
1 5 10 15
Gly Lys Ala Val Glu Glu Ser Thr Thr Ser Leu Ala Ser Leu Ala Cys
20 25 30
Gly Glu Ala Pro Cys Gly Phe Ser Asp Leu Asn Asn Ala Ser Gly Asp
35 40 45
Ala Gln Glu Arg Asn Ala Ser Met Arg Lys Leu Cys Ile Ala Val Val
50 55 60
Leu Cys Leu Leu Phe Met Thr Val Glu Val Phe Gly Gly Ile Lys Ala
65 70 75 80
Asn Ser Leu Ala Ile Leu Thr Asp Ala Ala His Leu Leu Ser Asp Val
85 90 95
FPI04057-sequence list
Ala Ala Phe Ala Ile Ser Leu Phe Ser Leu Trp Ala Ala Gly Trp Glu
100 105 110
Ala Thr Pro Arg Gln Thr Tyr Gly Phe Phe Arg Val Glu Ile Leu Gly
115 120 125
Ala Leu Val Ser Ile Gln Leu Ile Trp Leu Leu Thr Gly Ile Leu Val
130 135 140
Tyr Glu Ala Ile Ile Arg Leu Leu Ser Glu Thr Ser Glu Val Asn Gly
145 150 155 160
Phe Leu Met Phe Leu Val Ala Ala Phe Gly Leu Leu Val Asn Ile Val
165 170 175
Met Ala Val Leu Leu Gly His Asp His Gly His Gly His Gly His Gly
180 185 190
His Gly His Asp His His Ser His Gly Val Thr Val Thr Thr His His
195 200 205
His Gly His Asp His Gly His Thr His Gly Glu Asp Lys His His His
210 215 220
Ala His Gly His Gly Asp Val Thr Glu Gln Leu Leu Asp Lys Ser Glu
225 230 235 240
Lys Arg Lys Arg Asn Ile Asn Val Gln Gly Ala Tyr Leu His Val Leu
245 250 255
Gly Asp Ser Ile Gln Ser Val Gly Val Met Ile Gly Gly Ala Val Ile
260 265 270
Trp Tyr Lys Pro Glu Trp Lys Ile Val Asp Leu Ile Cys Thr Leu Val
275 280 285
Phe Ser Val Ile Val Leu Gly Thr Thr Ile Asn Met Ile Arg Ser Ile
290 295 300
Leu Glu Val Leu Met Glu Ser Thr Pro Arg Glu Ile Asp Ala Thr Lys
305 310 315 320
Leu Glu Lys Gly Leu Leu Glu Met Asp Glu Val Val Ala Val His Glu
325 330 335
Leu His Ile Trp Ala Ile Thr Val Gly Lys Val Leu Leu Ala Cys His
340 345 350
Val Asn Ile Thr Pro Glu Ala Asp Ala Asp Met Val Leu Asn Lys Val
355 360 365
Ile Asp Tyr Ile Arg Gly Glu Tyr Asn Ile Ser His Val Thr Ile Gln
370 375 380
Ile Glu Arg
385
FPI04057-sequence list
<210>7
<211>1363
<212>DNA
<213>Brassica juncea
<220>
<221>CDS
<222>(95)..(1249)
<223>
<400>7
ggagcacagg cacgacgaca cgaggctctt cttctttttg taaataaata acccaaccac 60
cacccacctt gaatctctca gctctcttct tctg atg gcg tcc tca agc cct caa 115
Met Ala Ser Ser Ser Pro Gln
1 5
cat agt cac atc att gag gtt aat ata gca aaa cct gat gaa caa aga 163
His Ser His Ile Ile Glu Val Asn Ile Ala Lys Pro Asp Glu Gln Arg
10 15 20
aca gca ctt ggg gca agt aaa gcc tgt gga gaa gca cct tgt ggg ttc 211
Thr Ala Leu Gly Ala Ser Lys Ala Cys Gly Glu Ala Pro Cys Gly Phe
25 30 35
tca gat ctc aac aat gct tct ggt gat gca cat gaa cgc aat gct tcc 259
Ser Asp Leu Asn Asn Ala Ser Gly Asp Ala His Glu Arg Asn Ala Ser
40 45 50 55
atg cgg aag ctc tgt atc gcg gtc gtg tta tgt ctt ttg ttc atg acc 307
Met Arg Lys Leu Cys Ile Ala Val Val Leu Cys Leu Leu Phe Met Thr
60 65 70
gtt gaa gtt ttc ggc ggg atc aaa gct aat agc ttg gct ata cta acc 355
Val Glu Val Phe Gly Gly Ile Lys Ala Asn Ser Leu Ala Ile Leu Thr
75 80 85
gat gca gct cac ctt ctc tct gac gtt gct gcc ttt gcc atc tcc ttg 403
Asp Ala Ala His Leu Leu Ser Asp Val Ala Ala Phe Ala Ile Ser Leu
90 95 100
ttc tcc ttg tgg gct gct ggc tgg gaa gcc act cct agg cag acg tat 451
Phe Ser Leu Trp Ala Ala Gly Trp Glu Ala Thr Pro Arg Gln Thr Tyr
105 110 115
ggg ttt ttc agg att gag atc ctc gga gct ctt gtc tct atc cag ctc 499
Gly Phe Phe Arg Ile Glu Ile Leu Gly Ala Leu Val Ser Ile Gln Leu
120 125 130 135
att tgg ttg ctc act ggt att ctc gtc tac gaa gcc att arc aga ctt 547
Ile Trp Leu Leu Thr Gly Ile Leu Val Tyr Glu Ala Ile Ile Arg Leu
140 145 150
ctt acc gag acc act gag gtt aat gga ttc ctt atg ttc ctt gtt gct 595
Leu Thr Glu Thr Thr Glu Val Asn Gly Phe Leu Met Phe Leu Val Ala
155 160 165
gcg ttt ggg ctg ctt gtg aat atc ata atg gct gtt ctg ctc ggg cat 643
Ala Phe Gly Leu Leu Val Asn Ile Ile Met Ala Val Leu Leu Gly His
170 175 180
gat cac ggt cat gga cat ggt cat gac cat cac aac cac ggt ggg gtg 691
Asp His Gly His Gly His Gly His Asp His His Asn His Gly Gly Val
185 190 195
act gtt acc act cgt cac cat cat gat cat ggt cat act cat gga gag 739
Thr Val Thr Thr Arg His His His Asp His Gly His Thr His Gly Glu
200 205 210 215
gac aag cac cac cac gct cat ggt gat gtt act gag aag tta ctc gac 787
Asp Lys His His His Ala His Gly Asp Val Thr Glu Lys Leu Leu Asp
220 225 230
aaa tcg aag ccg gat aaa gag aag aga aag agg aac atc aat gtc caa 835
FPI04057-sequence list
Lys Ser Lys Pro Asp Lys Glu Lys Arg Lys Arg Asn Ile Asn Val Gln
235 240 245
gga gct tat ctt cat gtc ctt ggg gac tcc atc cag agc gtt ggt gtt 883
Gly Ala Tyr Leu His Val Leu Gly Asp Ser Ile Gln Ser Val Gly Val
250 255 260
atg atc gga ggg gcc gtc atc tgg tgc aac ccg gaa tgg aaa ata gta 931
Met Ile Gly Gly Ala Val Ile Trp Cys Asn Pro Glu Trp Lys Ile Val
265 270 275
gac ctg atc tgc act ttt gtc ttc tcg gtt att gtc ttg gga aca acc 979
Asp Leu Ile Cys Thr Phe Val Phe Ser Val Ile Val Leu Gly Thr Thr
280 285 290 295
atc aac atg att cga agc att ctt gaa gtg ttg atg gag agt aca ccg 1027
Ile Asn Met Ile Arg Ser Ile Leu Glu Val Leu Met Glu Ser Thr Pro
300 305 310
aga gag att gat gct aca aaa ctg gaa gag ggt ttg gtg gaa atg gaa 1075
Arg Glu Ile Asp Ala Thr Lys Leu Glu Glu Gly Leu Val Glu Met Glu
315 320 325
gaa gtg gtg gct gtt cat gag ctt cac att tgg gct atc aca gtg gga 1123
Glu Val Val Ala Val His Glu Leu His Ile Trp Ala Ile Thr Val Gly
330 335 340
aaa gtg ctg ctc gct tgc cat gtc aat atc aga cca gaa gca gat gca 1171
Lys Val Leu Leu Ala Cys His Val Asn Ile Arg Pro Glu Ala Asp Ala
345 350 355
gat atg gtg ctc aac aag gtc att gat tac atc cgc agg gag tac aat 1219
Asp Met Val Leu Asn Lys Val Ile Asp Tyr Ile Arg Arg Glu Tyr Asn
360 365 370 375
att agt cat gtc aca ata caa att gag cgc taagagcaaa ccaaacatgt 1269
Ile Ser His Val Thr Ile Gln Ile Glu Arg
380 385
gaaggaatat tctgtatcag ctttctcatt aacaaccatt aaatcaatag tttccaatct 1329
tttgcaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 1363
<210>8
<211>385
<212>PRT
<213>Brassica juncea
<400>8
Met Ala Ser Ser Ser Pro Gln His Ser His Ile Ile Glu Val Asn Ile
1 5 10 15
Ala Lys Pro Asp Glu Gln Arg Thr Ala Leu Gly Ala Ser Lys Ala Cys
20 25 30
Gly Glu Ala Pro Cys Gly Phe Ser Asp Leu Asn Asn Ala Ser Gly Asp
35 40 45
Ala His Glu Arg Asn Ala Ser Met Arg Lys Leu Cys Ile Ala Val Val
50 55 60
Leu Cys Leu Leu Phe Met Thr Val Glu Val Phe Gly Gly Ile Lys Ala
65 70 75 80
Asn Ser Leu Ala Ile Leu Thr Asp Ala Ala His Leu Leu Ser Asp Val
85 90 95
FPI04057-sequence list
Ala Ala Phe Ala Ile Ser Leu Phe Ser Leu Trp Ala Ala Gly Trp Glu
100 105 110
Ala Thr Pro Arg Gln Thr Tyr Gly Phe Phe Arg Ile Glu Ile Leu Gly
115 120 125
Ala Leu Val Ser Ile Gln Leu Ile Trp Leu Leu Thr Gly Ile Leu Val
130 135 140
Tyr Glu Ala Ile Ile Arg Leu Leu Thr Glu Thr Thr Glu Val Asn Gly
145 150 155 160
Phe Leu Met Phe Leu Val Ala Ala Phe Gly Leu Leu Val Asn Ile Ile
165 170 175
Met Ala Val Leu Leu Gly His Asp His Gly His Gly His Gly His Asp
180 185 190
His His Asn His Gly Gly Val Thr Val Thr Thr Arg His His His Asp
195 200 205
His Gly His Thr His Gly Glu Asp Lys His His His Ala His Gly Asp
210 215 220
Val Thr Glu Lys Leu Leu Asp Lys Ser Lys Pro Asp Lys Glu Lys Arg
225 230 235 240
Lys Arg Asn Ile Asn Val Gln Gly Ala Tyr Leu His Val Leu Gly Asp
245 250 255
Ser Ile Gln Ser Val Gly Val Met Ile Gly Gly Ala Val Ile Trp Cys
260 265 270
Asn Pro Glu Trp Lys Ile Val Asp Leu Ile Cys Thr Phe Val Phe Ser
275 280 285
Val Ile Val Leu Gly Thr Thr Ile Asn Met Ile Arg Ser Ile Leu Glu
290 295 300
Val Leu Met Glu Ser Thr Pro Arg Glu Ile Asp Ala Thr Lys Leu Glu
305 310 315 320
Glu Gly Leu Val Glu Met Glu Glu Val Val Ala Val His Glu Leu His
325 330 335
Ile Trp Ala Ile Thr Val Gly Lys Val Leu Leu Ala Cys His Val Asn
340 345 350
Ile Arg Pro Glu Ala Asp Ala Asp Met Val Leu Asn Lys Val Ile Asp
355 360 365
Tyr Ile Arg Arg Glu Tyr Asn Ile Ser His Val Thr Ile Gln Ile Glu
370 375 380
Arg
385
FPI04057-sequence list
<210>9
<211>1379
<212>DNA
<213>Brassica juncea
<220>
<221>CDS
<222>(131)..(1258)
<223>
<400>9
gaggcaaaag tgaatctaaa gggttttcga atcaatggtg aatattttca accaagtacc 60
ttcatcctcc tctctcttct tcttctgatt cggaaaccta ttgtgcttca ctcgcattct 120
tattacagag atg gag cct tca agc ccc caa cat agt cac atc atc gag 169
Met Glu Pro Ser Ser Pro Gln His Ser His Ile Ile Glu
1 5 10
gtc aat gca tca aaa tct gat gaa gaa cag aga aca aca acg ctt ggt 217
Val Asn Ala Ser Lys Ser Asp Glu Glu Gln Arg Thr Thr Thr Leu Gly
15 20 25
tca gtt aaa gtc tgt gga gaa aca cct tgt cat ctc aac aat gcc tct 265
Ser Val Lys Val Cys Gly Glu Thr Pro Cys His Leu Asn Asn Ala Ser
30 35 40 45
ggt gac gcc gag gaa cgc act gcc tcc atg aga aag ctc tgc atc gct 313
Gly Asp Ala Glu Glu Arg Thr Ala Ser Met Arg Lys Leu Cys Ile Ala
50 55 60
gtg gtg cta tgt ctt ctc ttc atg act gtt gaa gtc ttt ggt ggg atc 361
Val Val Leu Cys Leu Leu Phe Met Thr Val Glu Val Phe Gly Gly Ile
65 70 75
aaa gct aac agc ttg gct ata ctt acc gac gca gct cat ctc ctc tct 409
Lys Ala Asn Ser Leu Ala Ile Leu Thr Asp Ala Ala His Leu Leu Ser
80 85 90
gat gtt gcc gcc ttt gcc atc tcc ttg ttc tcc ctg tgg gct gct ggc 457
Asp Val Ala Ala Phe Ala Ile Ser Leu Phe Ser Leu Trp Ala Ala Gly
95 100 105
tgg gaa gcg act cca agg cag act tat ggg ttt ttc agg att gag att 505
Trp Glu Ala Thr Pro Arg Gln Thr Tyr Gly Phe Phe Arg Ile Glu Ile
110 115 120 125
ctc gga gct ctt gtc tct atc cag ctc att tgg ctt ctc aca ggg att 553
Leu Gly Ala Leu Val Ser Ile Gln Leu Ile Trp Leu Leu Thr Gly Ile
130 135 140
ctc gtc tat gaa gct atc att aga ctt ctt act gag acc agt gag gtt 601
Leu Val Tyr Glu Ala Ile Ile Arg Leu Leu Thr Glu Thr Ser Glu Val
145 150 155
gat ggg ttc ctt atg ttc ctt gtt gct gcg ttt ggg ctg ttg gtg aac 649
Asp Gly Phe Leu Met Phe Leu Val Ala Ala Phe Gly Leu Leu Val Asn
160 165 170
atc gta atg gct gtt ctg tta ggt cat gat cat gga cat ggt cat gga 697
Ile Val Met Ala Val Leu Leu Gly His Asp His Gly His Gly His Gly
175 180 185
cat ggt cac agt cat gat ggg gtg act gtt acc acc cat cac cat cat 745
His Gly His Ser His Asp Gly Val Thr Val Thr Thr His His His His
190 195 200 205
ggt cat act cat ggt gag gac aag cat cac tct cat ggt gat gtt acc 793
Gly His Thr His Gly Glu Asp Lys His His Ser His Gly Asp Val Thr
210 215 220
gag gag ttg cta gac aaa tca gag aag agg aag agg aac atc aat gtg 841
Glu Glu Leu Leu Asp Lys Ser Glu Lys Arg Lys Arg Asn Ile Asn Val
225 230 235
FPI04057-sequence list
caa gga gct tac ctc cac gtc ctt gga gac tta atc cag agc gtt ggt 889
Gln Gly Ala Tyr Leu His Val Leu Gly Asp Leu Ile Gln Ser Val Gly
240 245 250
gtt atg gtt gga gga gcc atc atc tgg tac aag cca gag tgg aag ata 937
Val Met Val Gly Gly Ala Ile Ile Trp Tyr Lys Pro Glu Trp Lys Ile
255 260 265
gtt gat ctg atc tgc acg ctt gtc ttc tcg gtt att gtc ttg ggg aca 985
Val Asp Leu Ile Cys Thr Leu Val Phe Ser Val Ile Val Leu Gly Thr
270 275 280 285
acc atc aac atg atc aga agc att ctc gag gtg ttg atg gag agt aca 1033
Thr Ile Asn Met Ile Arg Ser Ile Leu Glu Val Leu Met Glu Ser Thr
290 295 300
cct aga gag att gac gct acg aag ctg gaa cag ggt ttg ctg gag atg 1081
Pro Arg Glu Ile Asp Ala Thr Lys Leu Glu Gln Gly Leu Leu Glu Met
305 310 315
gag gaa gtg gtg gct gtt cat gag ctt cac att tgg gct atc acg gtg 1129
Glu Glu Val Val Ala Val His Glu Leu His Ile Trp Ala Ile Thr Val
320 325 330
ggg aaa gtg ctg ctt gct tgc cat gtt aat atc aca ccg gag gca gat 1177
Gly Lys Val Leu Leu Ala Cys His Val Asn Ile Thr Pro Glu Ala Asp
335 340 345
gct gat atg gtg ttg aac aag gtt att gat tac atc cgg aga gag tat 1225
Ala Asp Met Val Leu Asn Lys Val Ile Asp Tyr Ile Arg Arg Glu Tyr
350 355 360 365
aac atc agt cat gtc aca ata caa att gag cgt taaaaaggca tagcaaacat 1278
Asn Ile Ser His Val Thr Ile Gln Ile Glu Arg
370 375
ctcaagagtc ttttgtatca gtattctctt tattaacaat aataaaatca ataaagtttc 1338
aatccttttg caaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 1379
<210>10
<211>376
<212>PRT
<213>Brassica juncea
<400>10
Met Glu Pro Ser Ser Pro Gln His Ser His Ile Ile Glu Val Asn Ala
1 5 10 15
Ser Lys Ser Asp Glu Glu Gln Arg Thr Thr Thr Leu Gly Ser Val Lys
20 25 30
Val Cys Gly Glu Thr Pro Cys His Leu Asn Asn Ala Ser Gly Asp Ala
35 40 45
Glu Glu Arg Thr Ala Ser Met Arg Lys Leu Cys Ile Ala Val Val Leu
50 55 60
Cys Leu Leu Phe Met Thr Val Glu Val Phe Gly Gly Ile Lys Ala Asn
65 70 75 80
Ser Leu Ala Ile Leu Thr Asp Ala Ala His Leu Leu Ser Asp Val Ala
85 90 95
Ala Phe Ala Ile Ser Leu Phe Ser Leu Trp Ala Ala Gly Trp Glu Ala
100 105 110
FPI04057-sequence list
Thr Pro Arg Gln Thr Tyr Gly Phe Phe Arg Ile Glu Ile Leu Gly Ala
115 120 125
Leu Val Ser Ile Gln Leu Ile Trp Leu Leu Thr Gly Ile Leu Val Tyr
130 135 140
Glu Ala Ile Ile Arg Leu Leu Thr Glu Thr Ser Glu Val Asp Gly Phe
145 150 155 160
Leu Met Phe Leu Val Ala Ala Phe Gly Leu Leu Val Asn Ile Val Met
165 170 175
Ala Val Leu Leu Gly His Asp His Gly His Gly His Gly His Gly His
180 185 190
Ser His Asp Gly Val Thr Val Thr Thr His His His His Gly His Thr
195 200 205
His Gly Glu Asp Lys His His Ser His Gly Asp Val Thr Glu Glu Leu
210 215 220
Leu Asp Lys Ser Glu Lys Arg Lys Arg Asn Ile Asn Val Gln Gly Ala
225 230 235 240
Tyr Leu His Val Leu Gly Asp Leu Ile Gln Ser Val Gly Val Met Val
245 250 255
Gly Gly Ala Ile Ile Trp Tyr Lys Pro Glu Trp Lys Ile Val Asp Leu
260 265 270
Ile Cys Thr Leu Val Phe Ser Val Ile Val Leu Gly Thr Thr Ile Asn
275 280 285
Met Ile Arg Ser Ile Leu Glu Val Leu Met Glu Ser Thr Pro Arg Glu
290 295 300
Ile Asp Ala Thr Lys Leu Glu Gln Gly Leu Leu Glu Met Glu Glu Val
305 310 315 320
Val Ala Val His Glu Leu His Ile Trp Ala Ile Thr Val Gly Lys Val
325 330 335
Leu Leu Ala Cys His Val Asn Ile Thr Pro Glu Ala Asp Ala Asp Met
340 345 350
Val Leu Asn Lys Val Ile Asp Tyr Ile Arg Arg Glu Tyr Asn Ile Ser
355 360 365
His Val Thr Ile Gln Ile Glu Arg
370 375
机译: (nbc),一种编码蛋白质的碳酸氢盐转运蛋白家族成员的基因
机译: 抗原多肽组成,纯化基因家族,多核苷酸家族,从家族分离的恶性疟原虫基因,多核苷酸序列,蛋白质,抗原多肽,免疫原性组合物,疫苗,重组蛋白的用途,纯化的重组或合成片段抗体抗体,抗体组或抗体片段,组合物的用途,药物,诊断方法,诊断试剂盒,重组核苷酸序列,重组表达和/或克隆载体,重组表达细胞和表达载体的用途
机译: 属于Judaica ARI脂类转运蛋白转运蛋白家族的低变应性化学蛋白质,用于治疗过敏症。