CN101343316A - 一种钾离子转运相关蛋白系统及其编码基因簇与应用 - Google Patents
一种钾离子转运相关蛋白系统及其编码基因簇与应用 Download PDFInfo
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- CN101343316A CN101343316A CNA2008101193947A CN200810119394A CN101343316A CN 101343316 A CN101343316 A CN 101343316A CN A2008101193947 A CNA2008101193947 A CN A2008101193947A CN 200810119394 A CN200810119394 A CN 200810119394A CN 101343316 A CN101343316 A CN 101343316A
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Abstract
本发明公开了一种钾离子转运相关蛋白系统及其编码基因簇与应用。该系统是由如下(a)和(b)两种蛋白质组成的:(a)其氨基酸序列为序列表中的序列3,或将序列表中序列3的氨基酸残基序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与钾离子转运相关的由序列3的蛋白质衍生的蛋白质;(b)其氨基酸序列为序列表中的序列4,或将序列表中序列4的氨基酸残基序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与钾离子转运相关的由序列4的蛋白质衍生的蛋白质。实验证明,转入上述基因簇TrkAH的大肠杆菌可以在K+浓度为0.1mM、pH8.0的碱性环境中生长良好,而转入空载体的大肠杆菌在此培养条件下不能生长。
Description
技术领域
本发明涉及一种钾离子转运相关蛋白系统及其编码基因簇与应用。
背景技术
钾离子转运蛋白是细胞中负责将K+向细胞内转运的一种跨膜运输蛋白。它普遍存在于生物界,目前已经在革兰氏阳性细菌、革兰氏阴性细菌、蓝细菌、酵母、植物和古细菌等生物体内都发现了K+转运蛋白(Ballal et al.,2007;Markus&Pascal,2007;Matsuda&Uozumi,2006;Michel et al.,2006;Nakamura et al.,1994;Nakamuraet al.,1998b)。在细菌中已经发现了四种K+转运蛋白,主要是肠细菌、蓝细菌等(Barbacid et al.,1991;Bossemeyer et al.,1989;Dosch et al.,1991;Nakamura et al.,1998b;Rhoads et al.,1978)。
K+是细胞内含量最高的阳离子,对于维持细胞内各种酶的活性、抵抗外界的高渗环境和维持细胞内pH值的平衡都起到了重要作用。此外,K+还可以作为胞内的第二信使,传递信号。细菌中K+转运蛋白Trk系统受ATP激活,在质子势的驱动下,将K+转向细胞内部,同时伴随着H+的向内转运。这一功能受胞内外渗透压的调控。
K+转运蛋白在医药、发酵、环保和耐盐碱植物培育等方面具有重要的应用潜力。例如,克隆K+转运蛋白基因,将其转入植物中,可以获得耐盐碱性提高的转基因植物,这对于盐渍化土壤的利用具有重要意义(Espinosa-Ruiz et al.,1999;Su et al.,2002);又如通过K+转运蛋白基因转化微生物,使转基因微生物能够在高渗的环境中完成正常的降解污染物的功能,对于污水治理、加强环境保护具有广阔的前景。
发明内容
本发明的目的是提供一种钾离子转运相关蛋白系统及其编码基因簇。
本发明所提供的钾离子转运相关蛋白系统,来源于淀粉水解嗜碱单胞菌(Alkalimonas amylolytica)N10 CGMCC 0463,是由如下(a)和(b)中的两种蛋白质组成的:
(a)其氨基酸序列为序列表中的序列3,或将序列表中序列3的氨基酸残基序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与钾离子转运相关的由序列3的蛋白质衍生的蛋白质;
(b)其氨基酸序列为序列表中的序列4,或将序列表中序列4的氨基酸残基序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与钾离子转运相关的由序列4的蛋白质衍生的蛋白质。
其中,序列表中序列3由458个氨基酸残基组成。序列表中序列4由483个氨基酸残基组成。
为了便于所述两种蛋白的纯化,可在由序列表中序列3或4所示的氨基酸残基序列组成的蛋白质的氨基末端或羧基末端连接上如表1所示的标签。
表1标签的序列
标签 | 残基 | 序列 |
Poly-Arg | 5-6(通常为5个) | RRRRR |
Poly-His | 2-10(通常为6个) | HHHHHH |
FLAG | 8 | DYKDDDDK |
Strep-tag II | 8 | WSHPQFEK |
c-myc | 10 | EQKLISEEDL |
带有表1所示标签的蛋白可人工合成,也可先合成其编码基因,再进行生物表达得到。如带有表1所示标签的上述(a)蛋白的编码基因可通过将序列表中序列5的自5′末端第3009-4382位碱基所示的DNA序列中缺失一个或几个氨基酸残基的密码子,和/或进行一个或几个碱基对的错义突变,和/或在其5′端和/或3′端连上表1所示标签的编码序列得到。
编码上述蛋白系统的基因簇也属于本发明的保护范围。
所述基因簇具体可为如下1)-3)中任一所述的DNA分子:
1)其核苷酸序列是序列表中的序列5;
2)在严格条件下可与序列表中序列5限定的DNA序列杂交且编码上述钾离子转运相关蛋白系统的DNA分子;
3)与1)的基因具有90%以上的同源性,且编码上述钾离子转运相关蛋白系统的DNA分子。
所述步骤3)中的基因簇,与1)的基因簇最好有95%以上的同源性。
序列表中的序列5由4634个核苷酸组成,自5′末端第1558-3006位编码序列表中序列3所示的蛋白质;自5′末端第3009-4382位编码序列表中序列4所示的蛋白质。
上述严格条件可为在0.1×SSPE(或0.1×SSC),0.1%SDS的溶液中,在65℃条件下杂交并洗膜。
扩增上述基因簇全长或其任一片段的引物对也属于本发明的保护范围。
含有上述基因簇的重组载体、转基因细胞系和重组菌也属于本发明的保护范围。
所述重组载体具体可为在pUC18中插入上述基因簇得到的重组载体,如pUCAaAH。
本发明的另一个目的是提供一种培育钾离子转运能力提高的转基因生物的方法。
本发明所提供的培育钾离子转运能力提高的转基因生物的方法,是将上述基因簇导入宿主细胞中,得到钾离子转运能力提高的转基因生物。
所述基因簇是通过所述重组载体导入生物中。
本发明从淀粉水解嗜碱单胞菌(Alkalimonas amylolytica)N10 CGMCC 0463中克隆到K+转运蛋白基因簇TrkAH,通过构建含有该基因簇的重组载体,将其转入大肠杆菌感受态细胞中。实验证明,转入K+转运蛋白基因簇TrkAH的大肠杆菌可以在K+浓度为0.1mM、pH 8.0的碱性环境中生长良好,而转入空载体的大肠杆菌在此培养基中不能生长。
附图说明
图1为重组载体pUCAaAH的示意图
图2为转入重组克隆载体pUCAaAH的LB2003在不同pH条件下、在含有3mMK+的培养基中的生长曲线
图3为转入重组克隆载体pUCAaAH的LB2003在不同pH条件下、在含有0.1mM K+的培养基中的生长曲线
图4为转入重组克隆载体pUCAaAH的LB650在pH 7.4条件下的生长情况
具体实施方式
下面结合具体实施例对本发明作进一步的说明。
实施例1、与钾离子转运相关蛋白及其编码基因的获得
1、淀粉水解嗜碱单胞菌(Alkalimonas amylolytica)N10总DNA的提取
取淀粉水解嗜碱单胞菌(Alkalimonas amylolytica)N10(专性嗜碱革兰氏阴性菌,分离自中国内蒙古盐碱湖,保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏号为CGMCC 0463)的新鲜湿菌体20g,悬浮于10mL 50mm/LpH 8.0的Tris缓冲液中,加入少量溶菌酶和8ml 0.25mm/L pH 8.0的乙二胺四乙酸(EDTA),混匀后于37℃放置20min;然后向反应液中加入2mL 10%(质量百分含量)的十二烷基硫酸钠(SDS),55℃放置5分钟;用等体积的酚、氯仿组成的抽提液抽提两次,取最后一次抽提的上清液,用2倍体积的无水乙醇沉淀DNA。将回收的DNA先后用70%(体积百分含量)的乙醇和无水乙醇洗涤后,所得DNA溶于0.5mL TE缓冲液中(10mm/L Tris,1mm/L EDTA pH 8.0),加入10mg/mL RNA酶(RNase)3uL,37℃保温1h,再用等体积的酚、氯仿组成的抽提液抽提一次,取上清用2倍体积的无水乙醇沉淀DNA。将回收的DNA先后用70%(体积百分含量)的乙醇和无水乙醇洗涤后,真空干燥得到DNA。将得到的DNA溶于去离子水中,冷藏备用。
利用紫外分光光度计对上述获得的DNA进行检测,计算A260/A280和A260/A230的值,结果表明,A260/A280=1.98,A260/A230=2.18,说明获得的DNA纯度较高。
2、TrkH基因和trkA基因3’端序列的获得
取上述步骤1获得的DNA溶液10uL(含50ug DNA),用限制性内切酶BamHI部分酶切,纯化回收3-8kb的酶切产物。取5ug上述酶切产物与1ug经BamHI酶切并脱磷酸化的质粒pUC18(GIBCO-BRL公司)在20uL的连接体系中16℃反应16h,连接产物转化大肠杆菌LB650(TK1001ΔtrkH::CamrΔtrkG::Kanr)(Nakamura,T.,Yamamuro,N.,Stumpe,S.,et al.Cloning of the trkAH gene cluster and characterizationof the Trk K(+)-uptake system of Vibrio alginolyticus.Microbiology(Reading,England)(1998a).144(Pt 8),2281-2289.)的感受态细胞。将得到的重组菌在含有3Mm KCl的固体培养基平板上进行筛选。挑取单克隆菌落,提取质粒,采用Sanger双脱氧法进行测序。测序结果表明,插入的片段中含有一个长1449bp的TrkH基因的开放阅读框架(ORF)和TrkA基因3’端的部分序列。TrkH基因的核苷酸序列如序列表中序列2所示,其编码的氨基酸序列如序列表中序列4所示。
3、反向PCR构建trkA基因的5’端序列
取10μg上述步骤1获得的DNA,用限制性内切酶HindIII不完全酶切30min以后,DNA纯化试剂盒纯化回收,测定OD260值。取300ng上述回收的DNA,在300μl的体系中用T4DNA连接酶环化,4℃过夜。以上述连接产物为模板,用正向引物
5′-TCGATTTGCTGCAAGGAGGTGAAGTCGA-3′和反向引物
5′-AAGCACCACGTTTGATAAGCACGATGG-3′扩增核心区域的侧翼序列。对扩增产物进行琼脂糖凝胶电泳检测,结果发现,得到了一个8000bp左右的基因组序列,经测定发现该序列包括1100bp的trkA基因的5’端序列。
4、重组克隆载体pUCAaH、pUCAaA和pUCAaAH的构建
分别根据上述步骤2扩增得到的trkH基因序列和trkA基因的3’端序列,根据上述步骤3扩增得到的trkA基因的5’端序列设计引物,引物序列及限制性酶切位点如下:
trkH基因的正向引物trkH-H101:
5′-ACCGAATTCTGCCGAAAGTGCTGGCTGA-3′(画线部分为EcoR I酶切位点)
trkH基因的反向引物trkH-H102:
5′-AAATCTAGAAGGTGAAGTCGATATCGC-3′(画线部分为Xba I酶切位点)
trkA基因的正向引物trkA-A101:
5′-CCATCTAGAGACCGCAGCCTGCTT-3′(画线部分为Xba I酶切位点)
trkA基因的反向引物trkA-A102:
5′-CAAAAGCTTCAGCTCTGGCCTCTGCTC-3′(画线部分为HindIII酶切位点)
trkAH基因簇的正向引物trkH-H101:
5′-ACCGAATTCTGCCGAAAGTGCTGGCTGA-3′(画线部分为EcoR I酶切位点)
trkAH基因簇的反向引物trkA-A102:
5′-CAAAAGCTTCAGCTCTGGCCTCTGCTC-3′(画线部分为HindIII酶切位点)。
以上述步骤1获得的N10的总DNA为模板,利用PCR方法扩增全长trkH基因、trkA基因和trkAH基因簇序列。将trkH基因的PCR扩增产物用EcoRI和XbaI双酶切后与经同样酶切的质粒pUC18相连接,将得到的重组载体命名为pUCAaH;将trkA基因的PCR扩增产物用XbaI和HindIII双酶切后与经同样酶切的质粒pUC18相连接,将得到的重组载体命名为pUCAaA;将trkAH基因簇的PCR扩增产物用EcoRI和HindIII双酶切后与经同样酶切的质粒pUC18相连接,将得到的重组载体命名为pUCAaAH。
将重组载体pUCAaH、pUCAaA和pUCAaAH酶切后分别连接到pMD18-T载体上进行测序,测序结果表明,trkH基因已经插入到重组载体pUCAaH中,trkH基因的核苷酸序列如序列表中序列2所示,其编码的氨基酸序列如序列表中序列4所示;trkA基因已经插入到重组载体pUCAaA中,trkA基因的核苷酸序列如序列表中序列1所示,其编码的氨基酸序列如序列表中序列3所示;trkAH基因簇已经插入到重组载体pUCAaAH中,trkAH基因簇的核苷酸序列如序列表中序列5所示。
重组载体pUCAaAH的示意图如图1所示,从图中可以看出,trkA基因和trkH基因的转录方向相反。
同时,以E.coli K12的基因组DNA为模板,用下述引物PCR扩增E.coli K12中的trkH基因和trkA基因:
E.coli K12的trkH基因的正向引物:
5′-ACCGAATTCatgcattttcgcgcc-3′(画线部分为EcoR I酶切位点)
E.coli K12的trkH基因的反向引物:
5′-CAAAAGCTT TCAATGTCGCAACAAC-3′(画线部分为HindIII酶切位点)
E.coli K12的trkA基因的正向引物:
5′-ATCGgaattcGGATCTTTGTGCCGC-3′(画线部分为EcoR I酶切位点)
E.coli K12的trkA基因的反向引物:
5′-TAGCaagcttTTTAGTTGGTGCAGGTG-3′(画线部分为HindIII酶切位点)
将上述PCR扩增产物分别用HindIII和EcoR I酶切后与经同样酶切的pUC18载体连接,得到重组质粒pUCEH和pUCEA。
实施例2、钾离子转运相关蛋白系统TrkAH的碱适应性测定
将上述实施例1构建的重组克隆载体pUCAaAH转入大肠杆菌LB2003(TK1001ΔtrkA)(Nakamura,T.,Yamamuro,N.,Stumpe,S.,et al.Cloning of the trkAH genecluster and characterization of the Trk K(+)-uptake system of Vibrio alginolyticus.Microbiology(Reading,England)(1998a).144(Pt 8),2281-2289.)中,得到重组大肠杆菌KB2003AH。用二乙醇胺(DEA)缓冲液洗涤上述生长至指数生长后期的重组大肠杆菌KB2003AH 3次,分别在pH7.5(HEPES)、pH8.0(Tricine)、pH8.5(TAPS)和pH9.0(Ampso)的条件下测定细胞内k+的浓度,并计算动力学常数。结果表明,在pH 7.5时,Vmax=129nmol min-1mg-1,Km=0.37mM;在pH 8.0时,Vmax=125nmol min-1mg-1,Km=0.17mM;在pH 8.5时,Vmax=76nmol min-1mg-1,Km=0.13mM;在pH 9.0时,由于测定方法的局限性,得不到有效的结果。从以上结果可以看出,随着pH值的升高,TrkAH对K+的亲和力不断增加,根据在pH 7.5、8.0和8.5时的结果,说明在pH 8.0时,TrkAH对K+具有相对较高的亲和力和转运能力,说明TrkAH具有一定的碱适应性。
实施例3、钾离子转运相关蛋白系统TrkAH的功能实验
将上述实施例1步骤4构建的重组克隆载体pUCAaA和pUCAaAH分别转入到大肠杆菌LB2003中,得到重组菌KB2003A和KB2003AH。将得到的重组菌KB2003A和KB2003AH分别在LBK30培养基(多聚蛋白胨10g/L,酵母提取物5g/L,K2HPO4 2.74g/L,KH2PO4 0.82g/L,Na2HPO4 12.2g/L,NaH2PO4 2.65g/L)中37℃下培养12小时,挑取单克隆分别在pH 7.5、8.0、8.5和9.0的条件下,在K3培养基(KCl0.224g/L,(NH4)2SO4 1.06g/L,Na2HPO4 1.647g/L,NaH2PO4 0.359g/L,MgSO4 0.1g/L,NaCl 5.85g/L,葡萄糖4.36g/L)中37℃下培养40小时,观察重组菌的生长情况;同时以转入空载体pUC18的大肠杆菌LB650和LB2003作为对照。
重组菌KB2003A和KB2003AH在含有3mM K+培养基中的生长情况如图2所示。图2A中,KB2003-18表示转入空质粒pUC18的LB2003,KB2003EA表示转入质粒pUCEA的重组菌的生长情况,KB2003A表示转入重组克隆载体pUCAaA的重组菌的生长情况,KB2003AH表示转入重组克隆载体pUCAaAH的重组菌的生长情况。图2B、2C和2D中各条曲线代表的含义与图2A相同。
结果表明,转入TrkAH基因的重组菌可以在高pH的条件下生长,说明TrkAH起到转运钾离子的作用。
重组菌KB2003AH在含有0.1mM K+培养基中的生长情况如图3所示。图3中,LB2003AH表示转入重组克隆载体pUCAaAH的重组菌的生长情况,LB2003EA表示转入重组克隆载体pUCEA的重组菌的生长情况,LB2003-18表示转入空质粒pUC18的重组菌的生长情况。
从图3中可以看出,转入重组克隆载体pUCAaAH的大肠杆菌LB2003在pH 8.0的条件下,可以在含有0.1mM K+的K0.1液体培养基(培养基中KCl浓度为0.1mM,其余成分和K3培养基相同)中生长,而转入重组克隆载体pUCEA的大肠杆菌LB2003和转入空载体的阴性对照菌均不能生长,说明在偏碱性环境中、在极低浓度的K+条件下,TrkAH依然具有转运K+的能力。
转入重组克隆载体pUCAaAH的大肠杆菌LB650在pH 7.4的条件下,在含有100mMK+的LBK100培养基(酵母提取物5g/L,多聚蛋白胨10g/L,KCl 7g/L,HEPES 3g/L,pH7.4)中,在含有3.5%(质量百分含量)的NaCl存在的条件下仍然能保持较高的生长速率;而对照转入重组克隆载体pUCEH的大肠杆菌LB650则生长速率变慢。具体生长情况如图4所示。其中,KB650AH表示,转入重组克隆载体pUCAaAH的重组菌的生长情况,KB650EH表示转入重组克隆载体pUCAEH的重组菌的生长情况,LB650/pUC18表示转入空载体的重组菌的生长情况。结果表明,将含有trkAH基因簇的重组载体导入宿主细胞,可以增加宿主细胞对盐的耐受性。
序列表
<160>5
<210>1
<211>1374
<212>DNA
<213>嗜碱菌(Alkalimonas amylolytica)
<400>1
atgaaaatca tcattcttgg cgcaggacag gtcggcggta ccctggcgga aaatctggtc 60
ggggaaatga atgaaatcac tgtggtcgac acccatatgg agaaactgcg gttattgcag 120
gatcgattcg acttgcaggt gatccatggt tacggtgcgc acccggatat tttaaaaaaa 180
gccggcgctg aagatgccga catgctggtt gctgttacca gcagcgatga agtcaacatc 240
gttgcgtgtc aggtggccta cagtattttc cataccccgc ttaaaattgc ccgcatccgc 300
agcaatcagt acctgaaata ccgcgatcag ctgtttcgcc gagaagacat gccggtcgat 360
cactttattg cgccggaaac cctggttacc gactacatcc gccggctgat tgattaccct 420
ggagccttac aggtggtgga gtttgcgcag ggcaaagtca gcctggtcgg tttgcgcgcc 480
tattatggcg gtatgttggt tgggcatgcg ctgtcgacct taaaagtgca tatgcccaat 540
attgatgccc gggttgctgc catttatcgc cggggaacct cgatcaaacc attgggaacc 600
acggtgatcg aagccgacga tgaagtgttt tttgtcgctg ccaccaagca tatccgggta 660
gtgatgagcg agctgcaaaa gctggaaagc agttaccgcc gtattatgat tgctggcggc 720
ggcaatgtcg gctatgggct ggcaagagca ctggaaaaca actacagcgt gaagttgatt 780
gaacgcagca aagagcgggc tgagtttctg tccggcgtgc tggataatac cctggtctat 840
gtcggggata tctcggatcc ggagctgctg gaagaagaaa atatcgagca aatcgatgtc 900
tttattgccg ttaccaacga cgatgaagcc aatattatgt cggccatgct ggctaaacgg 960
atgggtgcac agaaaaccat cgtgcttatc aaacgtggtg cttatgtcga tttgctgcaa 1020
ggaggtgaag tcgatatcgc agtttcgccc cagcaggcta ctatctcggc gctgcttacc 1080
catatccgcc gtggcgatat tgtcaatgtg cactcgttgc gccgcggcgc agctgaagcc 1140
atcgaagcca tagcccatgg cgatgaaacc acctccaagg tagttggccg cgctattggc 1200
gacattaaac tgccaccggg tgctactata ggggctattg taaggggcga tgaagtgatc 1260
attgcccacg accatattcc catccaaacc gacgatcaca tcatcctgtt tttggtggat 1320
aaaaaacaca tcagcgaagt ggagaagctg ttccaggtca gtgcgatttt tatt 1374
<210>2
<211>1449
<212>DNA
<213>嗜碱菌(Alkalimonas amylolytica)
<400>2
atgcaatatc gctccattat ccgtattctg gggttactgg tcgctgtgtt cagcgtcagc 60
atgctgccgc cagccctggt gtcgctctgg tatcaggatg gtgccggtgt cccttttctg 120
ttgtcttttc tgatttgtat cgcagctggc ttgctgattt ggtaccccaa ccgcagttat 180
cagcgcgatc tgaaagttcg tgacggcttt ttgattgtgg tgatgttttg gctggtgctg 240
ggggctgtcg gtaccttgcc gctctactta tcctctgaaa cgggcatgaa tttggcggat 300
gccacttttg aggctttctc aggccttacc accaccgggg ccaccgttct gaccggcatt 360
gaaaccctgc cgaaagccat tttgttttac cgccagcaat tgcaatggct gggcggcatg 420
gggatcatcg ttttggcggt agctatcttg ccgatgctgg gcgtcggggg catgcagctg 480
tataaagcgg aaaccccggg cccggtcaaa gacaacaaag tcacaccgcg cattgccgat 540
accgccaaac acttgtggtt gatttatgtg gtgcttaccc tggcctgtgc cctggcctat 600
cgtttggcag gtatgaactg gtttgatgcc gtcggccact ccttttccac agttgccatt 660
ggcggctttt ctacctacga tgccagtatt ggccattttg acagcagcgc cataaatatg 720
atctgcgtgg tgttcctgtt gctgagcgcc atcaattacc cactgcactt tgccgccatg 780
cgtggcaaaa atattctgac ctactggcgt gaccctgaac tgcgggcttt tttgtttatt 840
cagggctccc tggtgctgct gatttttatc ggtttactgc gtagccaggt gtacgacact 900
acctgggagg cgtttgatca cggcttgttt caagcggtgt ctatttcaac gactgcgggt 960
ttcgctacgg gcggctttgc ctactggcct ttgtatctgc cgattttgct gattttttcc 1020
agtttcattg gcggctgcgc tggctctacc ggtggcggta tgaaagttgt gcgggtgttg 1080
ttgctgtttt tgcagggcaa gcgtgagctg aaccggttgg tgcatccgcg ggccatctac 1140
agcattaagt tgggccggcg gaccgtgccg gatcgggtgg tcgaagcagt ctggggcttt 1200
ttcgccgcct atgccttggt gtttgtgatt attatgctgc tattaattat gaccgggctg 1260
gataatatga ccgctttctc agcgaccgca gcctgcttaa ataaccttgg ccccgggctg 1320
ggtgacgtag catcgcattt tggtgatatc cctgatacaa gtaaatattt gctggtgatt 1380
gcgatggtgt ttggccggct ggagattttc accttgctgg tgctctttac gccggcgttt 1440
tggaaaaat 1449
<210>3
<211>458
<212>PRT
<213>嗜碱菌(Alkalimonas amylolytica)
<400>3
Met Lys Ile Ile Ile Leu Gly Ala Gly Gln Val Gly Gly Thr Leu Ala
1 5 10 15
Glu Asn Leu Val Gly Glu Met Asn Glu Ile Thr Val Val Asp Thr His
20 25 30
Met Glu Lys Leu Arg Leu Leu Gln Asp Arg Phe Asp Leu Gln Val Ile
35 40 45
His Gly Tyr Gly Ala His Pro Asp Ile Leu Lys Lys Ala Gly Ala Glu
50 55 60
Asp Ala Asp Met Leu Val Ala Val Thr Ser Ser Asp Glu Val Asn Ile
65 70 75 80
Val Ala Cys Gln Val Ala Tyr Ser Ile Phe His Thr Pro Leu Lys Ile
85 90 95
Ala Arg Ile Arg Ser Asn Gln Tyr Leu Lys Tyr Arg Asp Gln Leu Phe
100 105 110
Arg Arg Glu Asp Met Pro Val Asp His Phe Ile Ala Pro Glu Thr Leu
115 120 125
Val Thr Asp Tyr Ile Arg Arg Leu Ile Asp Tyr Pro Gly Ala Leu Gln
130 135 140
Val Val Glu Phe Ala Gln Gly Lys Val Ser Leu Val Gly Leu Arg Ala
145 150 155 160
Tyr Tyr Gly Gly Met Leu Val Gly His Ala Leu Ser Thr Leu Lys Val
165 170 175
His Met Pro Asn Ile Asp Ala Arg Val Ala Ala Ile Tyr Arg Arg Gly
180 185 190
Thr Ser Ile Lys Pro Leu Gly Thr Thr Val Ile Glu Ala Asp Asp Glu
195 200 205
Val Phe Phe Val Ala Ala Thr Lys His Ile Arg Val Val Met Ser Glu
210 215 220
Leu Gln Lys Leu Glu Ser Ser Tyr Arg Arg Ile Met Ile Ala Gly Gly
225 230 235 240
Gly Asn Val Gly Tyr Gly Leu Ala Arg Ala Leu Glu Asn Asn Tyr Ser
245 250 255
Val Lys Leu Ile Glu Arg Ser Lys Glu Arg Ala Glu Phe Leu Ser Gly
260 265 270
Val Leu Asp Asn Thr Leu Val Tyr Val Gly Asp Ile Ser Asp Pro Glu
275 280 285
Leu Leu Glu Glu Glu Asn Ile Glu Gln Ile Asp Val Phe Ile Ala Val
290 295 300
Thr Asn Asp Asp Glu Ala Asn Ile Met Ser Ala Met Leu Ala Lys Arg
305 310 315 320
Met Gly Ala Gln Lys Thr Ile Val Leu Ile Lys Arg Gly Ala Tyr Val
325 330 335
Asp Leu Leu Gln Gly Gly Glu Val Asp Ile Ala Val Ser Pro Gln Gln
340 345 350
Ala Thr Ile Ser Ala Leu Leu Thr His Ile Arg Arg Gly Asp Ile Val
355 360 365
Asn Val His Ser Leu Arg Arg Gly Ala Ala Glu Ala Ile Glu Ala Ile
370 375 380
Ala His Gly Asp Glu Thr Thr Ser Lys Val Val Gly Arg Ala Ile Gly
385 390 395 400
Asp Ile Lys Leu Pro Pro Gly Ala Thr Ile Gly Ala Ile Val Arg Gly
405 410 415
Asp Glu Val Ile Ile Ala His Asp His Ile Pro Ile Gln Thr Asp Asp
420 425 430
His Ile Ile Leu Phe Leu Val Asp Lys Lys His Ile Ser Glu Val Glu
435 440 445
Lys Leu Phe Gln Val Ser Ala Ile Phe Ile
450 455
<210>4
<211>483
<212>PRT
<213>嗜碱菌(Alkalimonas amylolytica)
<400>4
Met Gln Tyr Arg Ser Ile Ile Arg Ile Leu Gly Leu Leu Val Ala Val
1 5 10 15
Phe Ser Val Ser Met Leu Pro Pro Ala Leu Val Ser Leu Trp Tyr Gln
20 25 30
Asp Gly Ala Gly Val Pro Phe Leu Leu Ser Phe Leu Ile Cys Ile Ala
35 40 45
Ala Gly Leu Leu Ile Trp Tyr Pro Asn Arg Ser Tyr Gln Arg Asp Leu
50 55 60
Lys Val Arg Asp Gly Phe Leu Ile Val Val Met Phe Trp Leu Val Leu
65 70 75 80
Gly Ala Val Gly Thr Leu Pro Leu Tyr Leu Ser Ser Glu Thr Gly Met
85 90 95
Asn Leu Ala Asp Ala Thr Phe Glu Ala Phe Ser Gly Leu Thr Thr Thr
100 105 110
Gly Ala Thr Val Leu Thr Gly Ile Glu Thr Leu Pro Lys Ala Ile Leu
115 120 125
Phe Tyr Arg Gln Gln Leu Gln Trp Leu Gly Gly Met Gly Ile Ile Val
130 135 140
Leu Ala Val Ala Ile Leu Pro Met Leu Gly Val Gly Gly Met Gln Leu
145 150 155 160
Tyr Lys Ala Glu Thr Pro Gly Pro Val Lys Asp Asn Lys Val Thr Pro
165 170 175
Arg Ile Ala Asp Thr Ala Lys His Leu Trp Leu Ile Tyr Val Val Leu
180 185 190
Thr Leu Ala Cys Ala Leu Ala Tyr Arg Leu Ala Gly Met Asn Trp Phe
195 200 205
Asp Ala Val Gly His Ser Phe Ser Thr Val Ala Ile Gly Gly Phe Ser
210 215 220
Thr Tyr Asp Ala Ser Ile Gly His Phe Asp Ser Ser Ala Ile Asn Met
225 230 235 240
Ile Cys Val Val Phe Leu Leu Leu Ser Ala Ile Asn Tyr Pro Leu His
245 250 255
Phe Ala Ala Met Arg Gly Lys Asn Ile Leu Thr Tyr Trp Arg Asp Pro
260 265 270
Glu Leu Arg Ala Phe Leu Phe Ile Gln Gly Ser Leu Val Leu Leu Ile
275 280 285
Phe Ile Gly Leu Leu Arg Ser Gln Val Tyr Asp Thr Thr Trp Glu Ala
290 295 300
Phe Asp His Gly Leu Phe Gln Ala Val Ser Ile Ser Thr Thr Ala Gly
305 310 315 320
Phe Ala Thr Gly Gly Phe Ala Tyr Trp Pro Leu Tyr Leu Pro Ile Leu
325 330 335
Leu Ile Phe Ser Ser Phe Ile Gly Gly Cys Ala Gly Ser Thr Gly Gly
340 345 350
Gly Met Lys Val Val Arg Val Leu Leu Leu Phe Leu Gln Gly Lys Arg
355 360 365
Glu Leu Asn Arg Leu Val His Pro Arg Ala Ile Tyr Ser Ile Lys Leu
370 375 380
Gly Arg Arg Thr Val Pro Asp Arg Val Val Glu Ala Val Trp Gly Phe
385 390 395 400
Phe Ala Ala Tyr Ala Leu Val Phe Val Ile Ile Met Leu Leu Leu Ile
405 410 415
Met Thr Gly Leu Asp Asn Met Thr Ala Phe Ser Ala Thr Ala Ala Cys
420 425 430
Leu Asn Asn Leu Gly Pro Gly Leu Gly Asp Val Ala Ser His Phe Gly
435 440 445
Asp Ile Pro Asp Thr Ser Lys Tyr Leu Leu Val Ile Ala Met Val Phe
450 455 460
Gly Arg Leu Glu Ile Phe Thr Leu Leu Val Leu Phe Thr Pro Ala Phe
465 470 475 480
Trp Lys Asn
<210>5
<211>4634
<212>DNA
<213>人工序列
<400>5
tgcggccaag tcggtgccga tttcagccga gcgtcgcatg ctgcatgtgc tgccacagga 60
attttcggtg gatatgcaag aaggtattaa aagcccggtc ggcatgtccg gggtgaggat 120
ggaagcccgg gcgcacatta ttacctgcgc caatgacatg gccaaaaaca ttgaaaaatg 180
tgccgagcgc tgtggtttga aagtcgatcc tgcaagctac gccagccata ttcaggtttt 240
gcaacagcga acccgccagt tactggagcg ggaaaatctg gacgggttgg tgattcactc 300
cggccagacc aagcgcaaat tccttgatga catggattat ccctttaaag ccaacccgca 360
ctttaaggcc tggctgccgg tggtggataa cccgcactgc tggctgcaga tagatggtgt 420
taataaaccg aaactgattt tttaccggcc aaaagatttc tggcacaagg tgccggatgt 480
accgggcgat ttttgggccg agcattttga tattcaacta ttggaaaaag ccaaccaggt 540
agagcagttg ctgccctatg acaaagagcg gctggcctat cttggtgaat acctggaagt 600
tgcccaggcg ctcggtttta gcgacattaa tccggagccg gtgctgaact tcctgcatta 660
tcaccgggct tacaaaaccg agtacgagct gcagtgtctg cgccgggcca atcgcatggc 720
ggtggctggc cataaagctg ccaaagcggc attttatggc ggtggcagtg agtttgatat 780
tcagctggcc tacctggctg cagttggtca aaccgaaaac gaggtgcctt atggcaacat 840
cgtggcgctg aaccagcact gcgctatctt gcattacacg gcgctggagc ggcagaaacc 900
ggcacagcat cgcagttttc tgatcgatgc cggggctgag tttcatggct atgctgccga 960
cattacccgc agctacagtt ttgatagcgg ccacgagttt gctgagctga tcaagcgggt 1020
ggatgccatt acgctgcaga tggtcgatgg tctgaagcct ggcatcaagt acagcgaatt 1080
gcatttgcaa acgcaccaac tgattgccga agtgctggct gattttgact ttgtccgcct 1140
gggcgccgaa gccatggtgg agcaacagat cacccaggcc tttttcccgc atggccttgg 1200
gcatcacctt ggcttgcagg tgcacgatgt cggtggcttt atgctggatg agcgtggtac 1260
ccatttaccg ccgcctgagc agcatccgtt tctgcgctgc acccgcttca ttgaaccgtc 1320
catggtttat accattgaac ctggtctgta ctttatcgac tccttactgg atgaactgcc 1380
tgatgcgcag aaaaaactgc taaactggga taaaattcag gcatttaagc cttttggtgg 1440
catccggatc gaagacaacg tcatcgtgca ccgggagcgc aatgaaaata tgaccagaga 1500
gctgcagctg gattaaccag ccggcagccg tctggcacag ggccagttcg gggaactatg 1560
caatatcgct ccattatccg tattctgggg ttactggtcg ctgtgttcag cgtcagcatg 1620
ctgccgccag ccctggtgtc gctctggtat caggatggtg ccggtgtccc ttttctgttg 1680
tcttttctga tttgtatcgc agctggcttg ctgatttggt accccaaccg cagttatcag 1740
cgcgatctga aagttcgtga cggctttttg attgtggtga tgttttggct ggtgctgggg 1800
gctgtcggta ccttgccgct ctacttatcc tctgaaacgg gcatgaattt ggcggatgcc 1860
acttttgagg ctttctcagg ccttaccacc accggggcca ccgttctgac cggcattgaa 1920
accctgccga aagccatttt gttttaccgc cagcaattgc aatggctggg cggcatgggg 1980
atcatcgttt tggcggtagc tatcttgccg atgctgggcg tcgggggcat gcagctgtat 2040
aaagcggaaa ccccgggccc ggtcaaagac aacaaagtca caccgcgcat tgccgatacc 2100
gccaaacact tgtggttgat ttatgtggtg cttaccctgg cctgtgccct ggcctatcgt 2160
ttggcaggta tgaactggtt tgatgccgtc ggccactcct tttccacagt tgccattggc 2220
ggcttttcta cctacgatgc cagtattggc cattttgaca gcagcgccat aaatatgatc 2280
tgcgtggtgt tcctgttgct gagcgccatc aattacccac tgcactttgc cgccatgcgt 2340
ggcaaaaata ttctgaccta ctggcgtgac cctgaactgc gggctttttt gtttattcag 2400
ggctccctgg tgctgctgat ttttatcggt ttactgcgta gccaggtgta cgacactacc 2460
tgggaggcgt ttgatcacgg cttgtttcaa gcggtgtcta tttcaacgac tgcgggtttc 2520
gctacgggcg gctttgccta ctggcctttg tatctgccga ttttgctgat tttttccagt 2580
ttcattggcg gctgcgctgg ctctaccggt ggcggtatga aagttgtgcg ggtgttgttg 2640
ctgtttttgc agggcaagcg tgagctgaac cggttggtgc atccgcgggc catctacagc 2700
attaagttgg gccggcggac cgtgccggat cgggtggtcg aagcagtctg gggctttttc 2760
gccgcctatg ccttggtgtt tgtgattatt atgctgctat taattatgac cgggctggat 2820
aatatgaccg ctttctcagc gaccgcagcc tgcttaaata accttggccc cgggctgggt 2880
gacgtagcat cgcattttgg tgatatccct gatacaagta aatatttgct ggtgattgcg 2940
atggtgtttg gccggctgga gattttcacc ttgctggtgc tctttacgcc ggcgttttgg 3000
aaaaattaaa taaaaatcgc actgacctgg aacagcttct ccacttcgct gatgtgtttt 3060
ttatccacca aaaacaggat gatgtgatcg tcggtttgga tgggaatatg gtcgtgggca 3120
atgatcactt catcgcccct tacaatagcc cctatagtag cacccggtgg cagtttaatg 3180
tcgccaatag cgcggccaac taccttggag gtggtttcat cgccatgggc tatggcttcg 3240
atggcttcag ctgcgccgcg gcgcaacgag tgcacattga caatatcgcc acggcggata 3300
tgggtaagca gcgccgagat agtagcctgc tggggcgaaa ctgcgatatc gacttcacct 3360
ccttgcagca aatcgacata agcaccacgt ttgataagca cgatggtttt ctgtgcaccc 3420
atccgtttag ccagcatggc cgacataata ttggcttcat cgtcgttggt aacggcaata 3480
aagacatcga tttgctcgat attttcttct tccagcagct ccggatccga gatatccccg 3540
acatagacca gggtattatc cagcacgccg gacagaaact cagcccgctc tttgctgcgt 3600
tcaatcaact tcacgctgta gttgttttcc agtgctcttg ccagcccata gccgacattg 3660
ccgccgccag caatcataat acggcggtaa ctgctttcca gcttttgcag ctcgctcatc 3720
actacccgga tatgcttggt ggcagcgaca aaaaacactt catcgtcggc ttcgatcacc 3780
gtggttccca atggtttgat cgaggttccc cggcgataaa tggcagcaac ccgggcatca 3840
atattgggca tatgcacttt taaggtcgac agcgcatgcc caaccaacat accgccataa 3900
taggcgcgca aaccgaccag gctgactttg ccctgcgcaa actccaccac ctgtaaggct 3960
ccagggtaat caatcagccg gcggatgtag tcggtaacca gggtttccgg cgcaataaag 4020
tgatcgaccg gcatgtcttc tcggcgaaac agctgatcgc ggtatttcag gtactgattg 4080
ctgcggatgc gggcaatttt aagcggggta tggaaaatac tgtaggccac ctgacacgca 4140
acgatgttga cttcatcgct gctggtaaca gcaaccagca tgtcggcatc ttcagcgccg 4200
gcttttttta aaatatccgg gtgcgcaccg taaccatgga tcacctgcaa gtcgaatcga 4260
tcctgcaata accgcagttt ctccatatgg gtgtcgacca cagtgatttc attcatttcc 4320
ccgaccagat tttccgccag ggtaccgccg acctgtcctg cgccaagaat gatgattttc 4380
atgaagtttg ttctgcctca agccgtttaa tcactttagc atagaaaaag ccatccatct 4440
gctgctggcc tggcaggatt tgccagctgt tggcatgctg ctggctggac tgcagttcgg 4500
catccggggt ttgttgtaaa aaacgctgca attgttgttg attttcggcg ctgaacacgc 4560
tgcaggttgc ataaagcaag atgccgcctg gtttgagcag aggccagagc tgctggagca 4620
tacgcgcctg gatg 4634
Claims (10)
1、一种钾离子转运相关蛋白系统,是由如下(a)和(b)两种蛋白质组成的:
(a)其氨基酸序列为序列表中的序列3,或将序列表中序列3的氨基酸残基序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与钾离子转运相关的由序列3的蛋白质衍生的蛋白质;
(b)其氨基酸序列为序列表中的序列4,或将序列表中序列4的氨基酸残基序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与钾离子转运相关的由序列4的蛋白质衍生的蛋白质。
2、编码权利要求1所述蛋白系统的基因簇。
3、根据权利要求2所述的基因簇,其特征在于:所述基因簇为如下1)-3)中任一所述的DNA分子:
1)其核苷酸序列是序列表中序列5;
2)在严格条件下可与序列表中序列5限定的DNA序列杂交且编码钾离子转运相关蛋白系统的DNA分子;
3)与1)的基因具有90%以上的同源性,且编码钾离子转运相关蛋白系统的DNA分子。
4、含有权利要求2或3所述基因簇的重组载体。
5、根据权利要求4所述的重组载体,其特征在于:所述重组载体为在pUC18中插入权利要求2或3所述基因簇得到的重组载体。
6、含有权利要求2或3所述基因簇的转基因细胞系。
7、含有权利要求2或3所述基因簇的重组菌。
8、扩增权利要求2或3所述基因簇的全长或其任一片段的引物对。
9、一种培育钾离子转运能力提高的转基因生物的方法,是将权利要求2或3所述的基因簇转入生物中,得到钾离子转运能力提高的转基因生物。
10、根据权利要求9所述的方法,其特征在于:权利要求2或3所述的基因簇是通过权利要求4所述的重组载体导入生物中。
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WO2014205597A1 (zh) * | 2013-06-24 | 2014-12-31 | 创世纪转基因技术有限公司 | 一种棉花高亲和钾离子转运蛋白hkt2及其编码基因与应用 |
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CN1289523C (zh) * | 2003-11-19 | 2006-12-13 | 中国科学院上海生命科学研究院 | 水稻钾、钠离子转运基因及其应用 |
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