CN102586279A - 云南红梨PyTTG1基因及其原核表达载体和应用 - Google Patents
云南红梨PyTTG1基因及其原核表达载体和应用 Download PDFInfo
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- CN102586279A CN102586279A CN2012100649925A CN201210064992A CN102586279A CN 102586279 A CN102586279 A CN 102586279A CN 2012100649925 A CN2012100649925 A CN 2012100649925A CN 201210064992 A CN201210064992 A CN 201210064992A CN 102586279 A CN102586279 A CN 102586279A
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Abstract
本发明公开了一种云南红梨PyTTG1基因及其原核表达载体,具体涉及一种云南红梨花青素合成、根毛、叶毛和毛状体发生发育的调控蛋白PyTTG1基因及其原核表达载体,并将原核表达载体应用在制备云南红梨花青素合成及叶毛、毛状体发生发育调控蛋白PyTTG1中,具体是利用RT-PCR技术从云南红梨果皮中克隆PyTTG1基因全长cDNA,然后再利用原核表达载体将该全长基因在大肠杆菌Rosetta(DE3)中进行表达纯化,获得云南红梨PyTTG1纯化蛋白,及云南红梨PyTTG1纯化蛋白在研究TTG1-MYB、TTG1-bHLH等互作及MYB-bHLH-WD40模型构建中的应用。
Description
技术领域
本发明属于基因工程领域,具体涉及一种云南红梨花青素合成调控、叶毛和毛状体发生发育的调控基因PyTTG1基因及其原核表达载体,以及应用原核表达载体制备云南红梨花青素合成及叶毛、毛状体发生发育调控蛋白PyTTG1。
背景技术
植物花青素的合成是由转录复合物MYB-bHLH-WD40调控的。前人对MYB和bHLH研究较多,而对植物中的WD40蛋白研究较少。WD40蛋白含WD40基序(约40氨基酸的保守序列,以甘氨酸-组氨酸(GH)开始, 以色氨酸-天冬氨酸(WD)结尾)。WD40蛋白含1-10个串联的WD40基序,一般认为至少4个以上的WD40基序才能形成高级和有功能的结构。现有研究表明WD40蛋白是一个结构多样、功能各异的蛋白家族,通常它们具有两个共同特征:一是结构域折叠成β-螺旋桨结构,一是形成多蛋白可逆组装但不具任何催化活性的平台,它们主要是在真核生物的蛋白质-蛋白质复合物形成中起作用。Fritzius等人发现ProF蛋白是将激酶和底物汇集在一起的适配器蛋白。在植物中,WD40蛋白的作用主要是作为植物特异性发育事件如开花、花发育、分生组织形成、花粉发育和配子形成关键调节因子。WD40蛋白主要通过其WD结构域与其它蛋白互作,参与植物内众多代谢反应的调控。Morita等人在牵牛花中发现具有隐性基因ca(编码InWDR1蛋白)的突变体中,除CHS外,花青素合成途径的结构基因的表达都协同地减少。Walker等人首次通过定位克隆和互补试验发现TTG1蛋白调控花青素的合成和毛状体分化。拟南芥TTG1蛋白含4个WD40基序,其ttg1突变体的特点是缺乏表皮毛、种皮透明、种子不经休眠就直接萌发、植株完全缺乏花青素、具有异常的根发育模式等突变表型,这表明TTG1的功能是多效的。棉花GhTTG1和GhTTG3具有拟南芥AtTTG1的类似功能:不仅能够恢复拟南芥ttg1突变体形成毛状体和花青素,而且还能互补紫罗兰ttg1突变体在花瓣产生紫色斑点。De等人在矮牵牛中首次发现WD40蛋白PhAN11,其定位于细胞质,能够连接信号转导与转录激活,能够调控PhAN2(MYB)的功能。玉米中的PAC1与矮牵牛和拟南芥中花青素调节蛋白AN11、TTG1极为类似,且35S-PAC1能够互补ttg1突变体。Dressel等人发现WD基序中单个氨基酸的突变(W158R)就可抑制DFR基因的表达,进而导致白花表型,且MiTTG1的54.1%高GC含量是进化的信号。Matus等人在拟南芥中异位表达葡萄WDR1导致拟南芥地上部分和莲座叶中花青素的合成,并通过GFP融合蛋白技术证明WDR1定位于细胞质和细胞核。Zhang等人通过酵母双杂交和植物过表达技术发现EGL3、GL3能够与TTG1(WD40)和MYB蛋白(GL1, PAP1、PAP2、CPC、TRY)组合,形成MYB/TTG1/WD40复合物进行包括花青素生物合成和毛状体形成的多功能的调控。Brueggemann等人通过互补试验证实了苹果MdTTG1具有类似拟南芥AtTTG1的功能。Baudry等人通过遗传和分子的方法发现TT2 (MYB)、TT8 (TTG1)和TTG1(WDR) 能够形成四元复合物直接调控BAN在植物中的表达。Payne等通过酵母双杂交证实了在拟南芥中GL3/GL1/TTG1复合物调控毛状体的发育。Bouyer通过克隆分析、异位表达和微注射试验提出了毛状体形成的捕获-耗尽机制:在高浓度的毛状体促进蛋白GL3细胞中,GL3通过结合毛状体形成蛋白TTG1来耗尽临近细胞中的TTG1,从而导致毛状体形成,而周围的细胞因TTG1的减少而不能形成毛状体。而Zhao等人通过离子轰击试验证明了TTG1, GL3, GL1和GL2并不在邻近细胞间转移,而R3-MYB, CPC则可在邻近细胞间转移,提出TTG1复合物直接调节激活子和抑制子,而抑制子的运动影响拟南芥叶上毛状体的类型。TTG是多功效基因, Gonzalez等人还发现TTG1能够与MYB5和TT2(MYB)形成复合物控制外种皮的分化。
在苹果中,Brueggemann等人克隆并验证了MdTTG1基因的功能,但是并没有进行原核表达和纯化蛋白。但是,在体外验证苹果和梨中的TTG1-MYB及TTG1-bHLH的互作,需要纯化TTG1蛋白。本发明选择着色最好且稳定的‘云红梨1号’作为实验材料,克隆云南红梨‘云红梨1号’PyTTG1基因,进行原核表达和蛋白纯化的研究,将为在体外验证苹果核梨中的TTG1-MYB及TTG1-bHLH的互作,进而在苹果和梨中构建MYB-bHLH-WD40复合物模型及果树和花卉的分子育种奠定基础。
发明内容
本发明的目的在于提供一种云南红梨PyTTG1基因,该基因具有如SEQ ID NO.3所示核苷酸序列或编码如SEQ ID NO.4所示氨基酸序列的蛋白质。
本发明中PyTTG1基因来源于云南红梨。
本发明另一目的是提供南红梨PyTTG1基因的原核表达载体pET32a-PyTTG1,该载体含有PyTTG1基因和PyTTG1基因的上游有T7启动子和细菌核糖体结合位点RBS,T7启动子的下游有可被IPTG诱导的操作子序列,PyTTG1基因的N端和C端均带有6 ×His标签。
本发明另一目的是将原核表达载体应用在制备云南红梨花青素合成及叶毛、毛状体发生发育调控蛋白PyTTG1中,PyTTG1纯化蛋白应用于研究梨和苹果中TTG1与MYB和bHLH等互作中。
为了实现本发明的上述目的,本发明提供了如下的技术方案:
1、云南红梨PyTTG1基因、基因组序列的获得
(1)根据苹果MdTTG1基因(GenBank登录号为AF220203)编码框序列和原核表达载体pET-32a多克隆酶切位点,设计1对特异引物PyTTG1-F:5′- CCG CCATGGAGAACTCT ACGCAAGAATCG-3′,PyTTG1-R:5′-CCG CTCGAGAACCTTCAAAAGCTGCATCTTG-3′,在上下游引物的5′端分别加入NcoI和XhoI酶切位点及保护碱基(下划线部分为酶切位点,斜体部分为保护碱基);提取云南红梨果皮的总RNA,并以RNA为模板,用M-MLV反转录酶合成cDNA后使用PyTTG1-F和PyTTG1-R进行扩增;提取云南红梨果皮中的基因组,使用上述引物进行扩增;
(2)回收PyTTG1的cDNA和gDNA全长片段。
2、构建原核表达载体及PyTTG1 gDNA的T/A克隆
使用NcoI和XhoI对PyTTG1基因全长片段和载体pET32a进行双酶切,分别获得5′和3′末端分别带有NcoI和XhoI酶切位点的PyTTG1基因和pET32a载体,琼脂糖凝胶电泳后分别进行回收,然后在16 ℃连接16 h,获得原核表达载体pET32a-PyTTG1,进行测序比对分析;使用pMD18T载体,将回收的gDNA片段进行T/A克隆,酶切验证后,进行测序分析,将测序结果进行生物信息学分析,并上传至GenBank数据库;
3、PyTTG1的原核表达
使用热刺激法将pET32a-PyTTG1转入大肠杆菌Rosetta(DE3)中,在终浓度1 mM IPTG诱导下筛选最佳表达条件,并在最适条件下进行大量表达;
4、PyTTG1蛋白纯化
收集菌体,进行超声破碎,过镍柱进行纯化,收集纯化后的蛋白,纯化后的蛋白用于制作PyTTG1蛋白抗体和PyTTG1蛋白表达检测。
5、云南红梨PyTTG1纯化蛋白在研究MYB-TTG1-WD40复合物功能中的应用
使用Far-western技术体外验证PyTTG与PyMYB及PyTTG1的互作,为梨和苹果中MYB-bHLH-WD40复合物模型的建立奠定基础。
本发明中克隆的基因是首次在梨中扩增得到,进行原核表达和蛋白纯化,获得的PyTTG1蛋白,将解决目前因缺乏该蛋白而无法在体外研究PyTTG1所结合的蛋白的问题,为在体外验证苹果核梨中的TTG1-MYB及TTG1-bHLH的互作,进而在苹果和梨中构建MYB-bHLH-WD40复合物模型及果树花卉的分子育种中奠定了基础。
附图说明
图1为本发明云南红梨果皮的总RNA电泳示意图。
图2为本发明PyTTG1基因的扩增产物电泳示意图,其中1是DNA Marker III;2是PyTTG1基因的PCR产物。
图3为本发明PyTTG1基因PCR产物的胶回收电泳示意图,其中1是DNA Marker III;2是PyTTG1基因的PCR产物。
图4为本发明原核表达载体pET32a-PyTTG1电泳示意图,其中1是对照载体pET32a-
PyMYB;2-6是载体pET32a-PyTTG1。
图5为本发明原核表达载体pET32a-PyTTG1的酶切检测示意图,其中1是DNA Marker III;2是载体pET32a-PyTTG1 的NcoI和XhoI双酶切结果。
图6为本发明中PyTTG1蛋白与其它WD40蛋白的系统发育分析图。
图7为本发明原核表达载体pET32a-PyTTG1在16 ℃和37 ℃ IPTG终浓度为1 mM条件下,诱导0、2、4、6、8 h的表达情况示意图,其中M是蛋白质分子量标准M0431;1是pET-32a空载体转化菌未诱导菌体的总蛋白;2-5是16 ℃、IPTG终浓度为1 mM条件下,工程菌诱导2、4、6、8 h后菌体的总蛋白;6-10是37 ℃、IPTG终浓度为1 mM条件下,工程菌诱导0、2、4、6、8 h后菌体的总蛋白;11是pET-32a空载体转化菌在37 ℃ IPTG终浓度为1 mM条件下诱导8 h后菌体的总蛋白。
图8为本发明原核表达载体pET32a-PyTTG1在28 ℃、IPTG终浓度为1 mM时,诱导0、2、4、6、8 h的蛋白表达情况示意图,其中1是pET-32a空载体转化菌诱导8 h的菌体总蛋白;2-6是原核表达载体pET32a-PyTTG1诱导0、2、4、6、8 h的细菌总蛋白。
图9为本发明原核表达载体pET32a-PyTTG1在16 ℃、不同IPTG终浓度下诱导8 h的细菌总蛋白示意图,其中M是蛋白质分子量标准M0431;1-7是IPTG终浓度为分别为0、0.1、0.3、0.5、0.7、0.9、1.0 mM/L时表达载体诱导8 h的细菌总蛋白;8是pET-32a空载体转化IPTG终浓度为1 mM/L诱导8 h的细菌总蛋白。
图10为本发明PyTTG1蛋白的纯化电泳示意图,其中1是加IPTG诱导前的细菌总蛋白;2是16 ℃、IPTG终浓度0.5 mM下原核表达载体诱导8 h的细菌总蛋白;3是蛋白质分子量标准M0431;4-8是镍柱纯化PyTTG1时分别使用30、150、200、300和500 mM咪唑洗脱的结果。
图11本发明原核表达载体 pET32a-PyTTG1构建策略示意图。
具体实施方式
下面结合附图和实施例实施例对本发明作进一步详细说明,但本发明保护范围并不局限于所诉内容。
实施例中采用的试剂主要为分子生物学实验试剂,各种限制性内切酶、pfu DNA聚合酶、RNA酶抑制剂、dNTP等为大连宝生物工程有限公司产品,反转录酶为Promaga公司的,质粒提取试剂盒购自博大泰克生物技术有限公司,基因组提取试剂盒购自于上海生工生物工程技术公司,其余试剂均为国产分析纯。仪器均为分子生物学以及基因工程实验室常用仪器设备。
所有引物序列合成和基因测序均在上海捷瑞生物公司(上海,GeneRay)进行。本发明实施例中所用方法如无特别说明均为常规方法。
实施例1:云南红梨PyTTG1基因、基因组序列的获得
(1)引物设计
根据苹果MdTTG1基因(GenBank登录号为AF220203)编码框和原核表达载体pET-32a多克隆酶切位点,设计1对特异引物如下:
PyTTG1-F:5′- CCG CCATGGAGAACTCTACGCAAGAATCG-3′,
PyTTG1-R:5′-CCG CTCGAGAACCTTCAAAAGC TGCATCTTG-3′
在上下游引物的5′端分别加入NcoI和XhoI酶切位点及保护碱基(下划线部分为酶切位点,斜体部分为保护碱基)。
(2)总RNA的提取
a、使用液氮将0.1 g云南红梨红色果皮充分研磨成粉末以后,加入1 ml RNA提取缓冲液(4 M异硫氰酸胍,25 mM柠檬酸钠,0.5 % (w/v) 十二烷基肌氨酸钠,2 %(w/v) 聚乙烯吡咯烷酮,β-巯基乙醇),研磨均匀后,转入2 ml离心管中,加入1/10体积2 M醋酸钠(pH 4.2);
b、颠倒离心管数次混匀,接着加入等体积的氯仿:异戊醇(24:1),盖紧离心管盖,剧烈振荡5 min,冰上静置15 min 后,4 ℃离心15 min (12000 g/min);
c、转移上清液于新的离心管,加入等体积的氯仿,盖紧离心管盖,剧烈振荡5 min,冰上静置15 min 后,4 ℃离心15 min (12000 g/min);
d、转移上清液于新的离心管,加入等体积异丙醇,在-80 ℃沉淀RNA 30 min,再次4 ℃离心15 min (12 000 g/min),让RNA沉淀在管壁;
e、RNA沉淀经75 %乙醇清洗两次后进行真空干燥,然后加入20μl无RNase的DEPC处理水彻底溶解RNA;
f、使用1.2 %的琼脂糖凝胶电泳检测RNA(见图1)。
(3)反转录及PCR扩增(RT-PCR)
以上述总RNA为模板,用M-MLV反转录酶合成cDNA后使用PyTTG1-F和PyTTG1-R进行扩增,得到PyTTG1的cDNA序列片段。
(4)云南红梨果皮基因组的提取及PyTTG1基因组序列的扩增
使用上海生工的植物基因组提取试剂盒,提取红梨果皮的基因组;使用PyTTG1基因引物进行扩增,结果如图2。
(5)PyTTG1的cDNA和gDNA全长片段的回收
对PyTTG1 cDNA及基因组PCR产物进行1 %琼脂糖凝胶电泳后,切下目的片段;使用胶回收试剂盒,按照试剂盒说明书对目的片段进行回收,结果如图3。
实施例2:原核表达载体的构建(见图11)及PyTTG1 gDNA的T/A克隆
使用NcoI和XhoI对PCR纯化产物PyTTG1基因全长片段和载体pET32a按照说明书进行双酶切,分别获得5’端带有NcoI和3’端带有XhoI的PyTTG1片段和pET32a载体,电泳分析后分别进行胶回收,按目的基因:载体=照摩尔比3:1加样,然后加入连接液I,16 ℃连接16 h。将感受态大肠杆菌E.coli DH5α100 μl加入6 μl连接体系中混匀;混合液冰浴30 min,42 ℃热刺激45 s后,冰浴2 min;加入900 μl液体培养基SOC,37 ℃摇床200 rpm 60 min 使菌体复苏;培养结束后,常温8 000 rpm离心1 min收集菌体;在超净台上吸去上清,剩余约0.1 ml时,使用移液枪混匀,接入带有Amp抗性的LB固体平板上,用无菌三角棒涂布均匀;37 ℃过夜培养;挑取白色菌落,接种于LB液体 + Amp的培养基,37 ℃、180 rpm培养12 h后,提取质粒,结果如图4;进行酶切验证(见图5)后,再送至上海杰瑞生物工程公司测序进行测序验证插入基因的正确性,最后获得原核表达载体pET32a-PyTTG1。按照pMD18T的载体说明书进行操作,将PyTTG1基因组序列进行T/A克隆,获得载体pMD18T-gPyTTG1,使用酶切进行验证后,送上海杰瑞生物公司测序。
实施例3:PyTTG1基因及gDNA序列的生物信息学分析和GenBank登录号的获得
测序结果表明,该基因的读码框为1029 bp,该基因编码342个氨基酸,分子量为38.4485 kDa,等电点为4.87。BLASTp比对结果表明,PyTTG1与其它WD40类转录因子具有很高的同源性,如与桃中的PrTTG1的同源性为95.32%,啤酒花HuWDR的为86.69%,矮牵牛PhAN11的为83.93%,拟南芥AtTTG182的为97%,与苹果MdTTG1的同源性高达99.12%。结构域分析表明,其与苹果、棉花、拟南芥等的WD40蛋白具有相同的WD40结构域。因PyTTG1与苹果MdTTG1相似性较高(见图6),将其命名为PyTTG1,随后将其上传至GenBank数据库,获得其登录号为HQ641374。稀有密码子分析结果表明PyTTG1基因含有大约10 %的稀有密码子,其中三联体稀有密码子和二连体稀有密码子各1个,因此需要使用能够补充稀有密码子的大肠杆菌Rosetta(DE3)菌株进行原核表达。测序结果表明PyTTG1的gDNA序列与其cDNA序列完全相同,这说明该基因不含有内含子序列,在生物进化过程中高度保守,因此,TTG1(WD40蛋白)在生命代谢过程中具有重要作用。
实施例4:PyTTG1蛋白的原核表达
使用热刺激法将重组质粒pET32a-PyTTG1转化入大肠杆菌Rosetta(DE3)感受态细胞中。涂LB + Amp固体平板,挑取pET32a-PyTTG1的重组菌落于LB + Amp液体培养基中37 ℃、200 rpm振荡培养过夜,按1:100的比例接种于相同的LB培养基上培养至OD600为0.6-0.8,加IPTG至终浓度为0.5 mM,分别在37℃、28℃和16℃条件下培养0、2、4、6和8 h后收集菌液用于分析总蛋白。4 ℃、12 000 rpm离心1 min,弃上清液,沉淀用100 μl SDS凝胶加样缓冲液(Tris-HCl 50 mM,pH 6.8;SDS 2 %;DTT 100 mM;溴酚蓝0.1 %;甘油10 %)重悬,煮沸5 min后,12 000 rpm离心1 min,取20 μl上清液进行SDS-PAGE检测。用考马斯亮蓝R-250染色液(0.1 %考马斯亮蓝R-250,40 %甲醇,10 %冰乙酸)染色,脱色液(25 %甲醇,6 %冰乙酸)进行脱色后,使用凝胶成像系统进行拍照。结果显示插入有外源片段的重组质粒pET32a-PyTTG1经终浓度1 mM IPTG诱导后,在37、28和16 ℃下均诱导出了预期的蛋白分子量约59 kD(包括载体上TRX蛋白)的蛋白条带,而未诱导的转化重组质粒和pET-32a对照质粒未出现这条蛋白带(图7和图8)。表明重组质粒pET32a-PyTTG1在大肠杆菌Rosetta(DE3)中诱导表达了PyTTG1蛋白。为获得在16 ℃条件下最佳诱导的IPTG浓度,使用IPTG终浓度梯度分别为0 mM/L、0.1 mM/L、0.3 mM/L、0.5 mM/L、0.7 mM/L、0.9 mM/L、1 mM/L分别对工程菌进行诱导8 h。结果表明,当IPTG终浓度为0.5 mM/L时蛋白的表达量最高(图9)。为获得最大量的诱导目的蛋白,作者又进行了16 ℃、IPTG终浓度为0.5 mM/L条件下诱导10 h和22 h的试验,结果表明随着时间的增加,蛋白的表达量继续增加,22 h的表达量最高。
实施例5:PyTTG1蛋白的纯化,具体步骤如下:
a、菌体破碎:将在16 ℃、0.5 mM IPTG下大量诱导表达22 h的500 ml菌体,经超声破碎菌体(工作3 s,休息6 s)5 min;
b、收集上清和沉淀:将菌体破碎液4 ℃、12 000 rpm离心20 min,分别保留上清和沉淀;
c、蛋白破碎上清液使用0.22 μM滤器进行过滤,除去杂质;
d、His-Trap HP柱的预处理:使用5倍柱体积纯水洗柱;5倍柱体积结合缓冲液(磷酸钠缓冲液 20 mM(pH7.4),NaCl 0.5 M,咪唑 30 mM)平衡柱子,流速为 1 ml/min;
e、蛋白样品上柱:流速为1 ml/min,收集流出液;
f、洗柱:使用5倍柱体积结合缓冲液(20 mM 磷酸钠缓冲液 pH7.4,NaCl 0.5M,咪唑 30 mM)洗脱;
g、洗脱:分别使用5倍柱体积洗脱缓冲液(20 mM 磷酸钠缓冲液 pH7.4,NaCl 0.5 M,咪唑 30、150、500 mM)依次洗脱,收集洗脱液;
h、His-Trap HP柱的后处理:5柱体积洗脱缓冲液(磷酸钠缓冲液20 mM(pH7.4),NaCl 0.5 M,咪唑 500 mM)洗去所有蛋白;5倍柱体积纯水洗柱;5倍柱体积20 %乙醇洗柱;柱子前后封口,于4 ℃、20 %的乙醇中保存;
i、SDS-PAGE检测:分别取50 μl各咪唑梯度流出液,加入5 μl的5×蛋白上样缓冲液,混匀后,煮沸5 min,4 ℃、13 000 rpm离心5 min后,各取20 μl上样,进行SDS-PAGE分析,纯化结果如图10,最终获得PyTTG1纯化蛋白。
因为苹果中的MdTTG1和梨中的PyTTG1蛋白具有高度的相似性,所以本发明中的PyTTG1纯化蛋白可用于研究梨和苹果中TTG1-MYB和TTG1-bHLH等与其它与TTG1互作的蛋白。本发明将为苹果和梨中MYB-bHLH-WD40互作模型的建立提供物质基础。
SEQUENCE LISTING
<110> 昆明理工大学
<120> 云南红梨PyTTG1基因及其原核表达载体和应用
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 29
<212> DNA
<213> 人工序列
<400> 1
ccgccatgga gaactctacg caagaatcg 29
<210> 2
<211> 31
<212> DNA
<213> 人工序列
<400> 2
ccgctcgaga accttcaaaa gctgcatctt g 31
<210> 3
<211> 1029
<212> DNA
<213> Pyrus Pyrifolia
<400> 3
atggagaact ctacgcaaga atcgcatctc aaggcggaga actcggtgac gtacgagtcg 60
ccgtacccgc tgtacgcctt ggcgtttgtc tcgccccaaa cccgaacccg ccaccagcac 120
caccgaatcg ccgtcggcag ctttatcgag gagtactcga accgggtcga catcctttcc 180
ttcgacccgg ataccctttc aataaagccg aacccgaccc tctccttcga ccacccttat 240
ccgcccacca agctcatgtt ccacccgaat cccaacgccc tccacaaaac caacgacgtc 300
ttagcttcct ctggcgacta cctccgcctt tgggaggtcg gtgactccac cgtcgagcct 360
atccaggtgt taaataacag caagaccagc gagttttgcg ctccgctgac gtccttcgat 420
tggaacgaca ttgagccccg gcgaattggg acttccagca tcgacaccac ctgcacaatt 480
tgggacatcg aaaagggggt cgtcgagacg cagctgattg ctcacgataa agaggtgtac 540
gacatcgcgt ggggggaagc cagggttttt gcttcggttt cggctgatgg gtcggtgaga 600
attttcgatt tgagagacaa ggagcactcc actatcatct atgagagtcc tcagccagat 660
acccctttgc ttcgattggc ttggaacaag caggatttga ggtacatggc cacaattttg 720
atggacagca ataaagttgt gatcttggat atccgatcgc cgacgatgcc agtggcggag 780
ctggagaggc acagaggtag tgtgaatgct attgcttggg ccccccagag ttgtaggcac 840
atttgctctg ctggggatga cactcaggcg cttatttggg acctgcccac ggtcgctggg 900
ccgaacggaa tcgaccccat gtcgatgtac tccgcaggtg cggagattaa tcagctgcag 960
tggtctgctg cacagcctga ttggatttcc attgcatttt ctaacaagat gcagcttttg 1020
aaggtttga 1029
<210> 4
<211> 342
<212> PRT
<213> Pyrus Pyrifolia
<400> 4
Met Glu Asn Ser Thr Gln Glu Ser His Leu Lys Ala Glu Asn Ser Val
1 5 10 15
Thr Tyr Glu Ser Pro Tyr Pro Leu Tyr Ala Leu Ala Phe Val Ser Pro
20 25 30
Gln Thr Arg Thr Arg His Gln His His Arg Ile Ala Val Gly Ser Phe
35 40 45
Ile Glu Glu Tyr Ser Asn Arg Val Asp Ile Leu Ser Phe Asp Pro Asp
50 55 60
Thr Leu Ser Ile Lys Pro Asn Pro Thr Leu Ser Phe Asp His Pro Tyr
65 70 75 80
Pro Pro Thr Lys Leu Met Phe His Pro Asn Pro Asn Ala Leu His Lys
85 90 95
Thr Asn Asp Val Leu Ala Ser Ser Gly Asp Tyr Leu Arg Leu Trp Glu
100 105 110
Val Gly Asp Ser Thr Val Glu Pro Ile Gln Val Leu Asn Asn Ser Lys
115 120 125
Thr Ser Glu Phe Cys Ala Pro Leu Thr Ser Phe Asp Trp Asn Asp Ile
130 135 140
Glu Pro Arg Arg Ile Gly Thr Ser Ser Ile Asp Thr Thr Cys Thr Ile
145 150 155 160
Trp Asp Ile Glu Lys Gly Val Val Glu Thr Gln Leu Ile Ala His Asp
165 170 175
Lys Glu Val Tyr Asp Ile Ala Trp Gly Glu Ala Arg Val Phe Ala Ser
180 185 190
Val Ser Ala Asp Gly Ser Val Arg Ile Phe Asp Leu Arg Asp Lys Glu
195 200 205
His Ser Thr Ile Ile Tyr Glu Ser Pro Gln Pro Asp Thr Pro Leu Leu
210 215 220
Arg Leu Ala Trp Asn Lys Gln Asp Leu Arg Tyr Met Ala Thr Ile Leu
225 230 235 240
Met Asp Ser Asn Lys Val Val Ile Leu Asp Ile Arg Ser Pro Thr Met
245 250 255
Pro Val Ala Glu Leu Glu Arg His Arg Gly Ser Val Asn Ala Ile Ala
260 265 270
Trp Ala Pro Gln Ser Cys Arg His Ile Cys Ser Ala Gly Asp Asp Thr
275 280 285
Gln Ala Leu Ile Trp Asp Leu Pro Thr Val Ala Gly Pro Asn Gly Ile
290 295 300
Asp Pro Met Ser Met Tyr Ser Ala Gly Ala Glu Ile Asn Gln Leu Gln
305 310 315 320
Trp Ser Ala Ala Gln Pro Asp Trp Ile Ser Ile Ala Phe Ser Asn Lys
325 330 335
Met Gln Leu Leu Lys Val
340
Claims (4)
1.云南红梨PyTTG1基因,其特征在于:PyTTG1基因具有如SEQ ID NO.3所示核苷酸序列或编码如SEQ ID NO.4所示氨基酸序列的蛋白质。
2.根据权利要求1所述的云南红梨PyTTG1基因,其特征在于:基因来源于云南红梨。
3.权利要求1所述云南红梨PyTTG1基因的原核表达载体pET32a-PyTTG1,其特征在于:该载体含有PyTTG1基因,PyTTG1基因的上游有T7启动子和细菌核糖体结合位点RBS,T7启动子的下游有可被IPTG诱导的操作子序列,PyTTG1基因的N端和C端均带有6×His标签。
4.权利要求3的所述的原核表达载体在制备云南红梨花青素合成及叶毛、毛状体发生发育调控蛋白PyTTG1中的应用。
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