CN109337884B9 - 一种丙酮酸激酶基因及其应用 - Google Patents
一种丙酮酸激酶基因及其应用 Download PDFInfo
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- CN109337884B9 CN109337884B9 CN201811573141.7A CN201811573141A CN109337884B9 CN 109337884 B9 CN109337884 B9 CN 109337884B9 CN 201811573141 A CN201811573141 A CN 201811573141A CN 109337884 B9 CN109337884 B9 CN 109337884B9
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Abstract
本发明公开了一种丙酮酸激酶基因，该丙酮酸激酶的基因序列如SEQ ID NO.1所示。该丙酮酸激酶基因的编码氨基酸序列如SEQ ID NO.2所示。该丙酮酸激酶的基因序列来源于蒺藜苜蓿。本发明从蒺藜苜蓿克隆出丙酮酸激酶基因，并对其功能进行了系统鉴定，发现了该基因不仅可以在体外催化磷酸烯醇式丙酮酸形成丙酮酸，还可以参与类黄酮的生物合成。
Description
技术领域
本发明属于基因工程的技术领域，具体涉及一种丙酮酸激酶基因及其用途。
背景技术
丙酮酸激酶(PK)，是糖酵解过程中的关键调节酶之一。PK通过转磷酸化作用将磷酸烯醇式丙酮酸(PEP)的磷酸基团转移到ADP，形成一分子的ATP和一分子的丙酮酸。丙酮酸激酶通过糖酵解途径对代谢流的控制至关重要，更重要的是它的底物磷酸烯醇式丙酮酸和产物丙酮酸都参与了多种代谢过程。如：丙酮酸进入线粒体的三羧酸循环，而磷酸烯醇式丙酮酸作为糖酵解产物向蛋白质和脂肪酸合成转化的中间物质，其含量影响植物体内的多个代谢过程。磷酸烯醇式丙酮酸两次参与生成莽草酸的过程，第一次是磷酸烯醇式丙酮酸作为底物在DAHP(3-Deoxy-D-arabinoheptulosonate7-phosphate)合成酶作用下合成DAHP，另一个是磷酸烯醇式丙酮酸与莽草酸-3-磷酸(Erythrose-4-phosphate)在EPSP(5-Enolpyruvylshikimate3-phosphate)合成酶作用下生成EPSP的过程。综上，磷酸烯醇式丙酮酸是连接两个途径的重要桥梁式中间产物，而丙酮酸激酶正是影响磷酸烯醇式丙酮酸含量的一个关键酶。
丙酮酸激酶存在于所有的生物中，非植物来源的丙酮酸激酶已经被大量研究，但是植物中丙酮酸激酶的研究还非常欠缺，在植物中丙酮酸激酶多是以多拷贝的形式存在，这种进化上的扩张在很大程度上增加了丙酮酸激酶基因表达和蛋白活性的多样性，从而使得植物更灵活地适应各种复杂多变的环境条件。丙酮酸激酶在植物发育及代谢过程中发挥多重功能，对丙酮酸激酶功能的研究对了解植物体内复杂代谢和发育过程都具有重要意义。但是，目前植物，特别是豆科模式植物蒺藜苜蓿中的丙酮酸激酶基因功能还是未知的，鉴于丙酮酸激酶的多重功能，以及其在植物代谢中的作用和潜在的应用价值，克隆和鉴定具有功能、特异性和生物活性的植物丙酮酸激酶基因具有重要的理论和应用价值。
发明内容
为了解决现有技术中的问题，本发明提供了一种丙酮酸激酶基因及其应用，实现的目的为，本发明从豆科植物蒺藜苜蓿中克隆出具有催化活性的丙酮酸激酶基因，并对其功能进行了鉴定，产生了具有活性的丙酮酸，同时丙酮酸激酶基因也参与了类黄酮的代谢，所以也为在其它生物中利用生物技术调节类黄酮的生物合成提供了重要的基因资源和技术方法。
为了实现上述目的，本发明提供的技术方案为，本发明提供了一种丙酮酸激酶基因，其特征在于，该丙酮酸激酶的基因序列如SEQ ID NO.1所示。
进一步的，该丙酮酸激酶基因的编码氨基酸序列如SEQ ID NO.2所示。
进一步的，该丙酮酸激酶的基因序列来源于蒺藜苜蓿。
本发明还提供了一种包含所述的丙酮酸激酶基因的质粒。
本发明还提供了所述的丙酮酸激酶基因在提高类黄酮含量中的应用。
本发明还提供了所述的质粒在提高类黄酮含量中的应用。
上述技术方案中，本发明从蒺藜苜蓿克隆出丙酮酸激酶基因，并对其功能进行了系统鉴定，发现了该基因可以在细菌中产生具有活性的重组蛋白，参与类黄酮的生物合成。
附图说明
图1为MtPK1与其它植物PK氨基酸序列的多重比对分析；
图2为植物丙酮酸激酶的进化树分析；
图3为MtPK1的亚细胞定位；
图4为重组蛋白MtPK1的的SDS-PAGE胶图；
图5为pH对重组MtPK1蛋白反应速率的影响；
图6为PEP浓度对重组MtPK1蛋白反应速率的影响；
图7为ADP浓度对重组MtPK1蛋白反应速率的影响；
图8为MtPK1的组织表达谱；
图9为PCR鉴定MtPK1在蒺藜苜蓿中过表达的转基因株系；
图10为R108、NF0791和过表达株系中花青素的相对含量。
具体实施方式
以下结合附图对本发明做出进一步的说明。
实施例1
本发明发现了一种丙酮酸激酶基因MtPK1，其基因序列如SEQ ID NO.1所示，SEQ ID NO.1所示的序列中ATG为起始密码子，TGA为终止密码子。该基因编码的蛋白质，氨基酸序列如SEQ ID NO.2所示。其用途为在其它生物中异源表达丙酮酸，在蒺藜苜蓿中的过量表达可以产生类黄酮。
本发明丙酮酸激酶基因的鉴定方法包括以下步骤：
(1)MtPK1基因全长片段的克隆
根据赛默飞世尔科技公司提供的Phusion高保真DNA聚合酶说明书进行PCR反应(表1，表2)。
表1Phusion酶反应体系
其中的Forward primer的序列为：ATGATGGCAGAGAAGAAACC；Reverse primer的序列为：TCATTTCACAGTCAAGATTTTG
表2.PCR程序
(2)目的条带的胶回收
根据Takara生物医学技术(北京)公司提供的说明书进行目的片段加A反应。按照下表配置反应体系(表3)，72℃反应15min。加A反应完成后，回收目的条带。
根据北京康为世纪生物科技有限公司提供的琼脂糖凝胶DNA回收试剂盒说明书对目的条带进行胶回收。称取离心管的重量，从琼脂糖凝胶中切取单一目的条带的DNA，放入离心管中，称量并计算凝胶净重。
1)加入100μL BufferPG/100mg凝胶。
2)50℃水浴温育，其间温和地上下颠倒离心管至胶块完全溶解。
3)向吸附柱中加入200μl BufferPS，以活化柱子。室温13000g离心1min，倒掉收集管中的废液。
4)待胶溶液降至室温，将其加入吸附柱，静置2min。室温13000g离心1min，倒掉收集管中的废液。
5)向吸附柱中加入450μl BufferPW，静置5min。室温13000g离心1min。倒掉收集管中的废液。
6)室温13000g离心1min。将吸附柱放到新的离心管中，向吸附膜中间位置悬空滴加50μl Buffer EB，室温放置2min。室温13000g离心1min。琼脂糖凝胶电泳确定所收集到DNA的条带大小，并用NanoDrop 2000测定浓度，-20℃保存。
表3回收片段加A体系
(3)pCXSN与目的DNA片断的连接
根据Takara生物医学技术(北京)公司提供的说明书进行目的回收片段与Xcm I酶切后回收的pCXSN载体连接。按照下表配置反应体系(表4)，轻弹混匀，16℃金属浴过夜。
表4T4DNA连接酶体系
(4)质粒的小量提取
1)取50μL菌液接种于5mL含Carb的LB液体培养基中，37℃过夜振荡培养。
2)将过夜培养的菌液加入2mL无菌离心管中，12000g离心1min，倒掉上清。
3)吸取250μL P1(确认添加RNAseΙ)加入上述菌体沉淀中，用枪头吹打使菌体充分混匀。
4)吸取250μL P2加入离心管中，温和地上下颠倒7次，使细菌充分裂解。
5)吸取350μL N3加入离心管中，立马轻柔地颠倒10次，使充分混合。
6)室温12000g，离心5min。
7)吸取上清至吸附柱中，室温12000g离心1min。
8)倒掉收集管中的废液，向吸附柱中加入150μL PB。
9)室温12000g离心1min。
10)向吸附柱中加入400μL PW，室温12000g离心1min。
11)将吸附柱放入一个新的1.5mL离心管中，吸取50μL EB悬空加入吸附膜的中间部位，室温放置5min。室温12000g离心1min，-20℃保存质粒。
所得转化子抽提质粒进行双酶切鉴定。鉴定为阳性的转化子经克隆扩培后，送样测序。
(5)大肠杆菌(DH5α和M15)感受态细胞的转化
1)取大肠杆菌感受态细胞置于冰上融化。
2)加5μL质粒，混匀后置于冰上30min。
3)42℃热击1min，迅速取出，并立即轻柔地置于冰上2min。
4)加入500μL LB液体培养基，混匀。37℃，振荡培养1h。
5)用灭菌的涂布棒将菌液涂布于含Carb的LB固体培养基，超净台晾干后密封。
6)37℃，过夜静置培养。
7)用无菌枪头挑取单克隆进行菌液PCR鉴定。
通过对MtPK1基因进行克隆及测序分析发现，MtPK1全长为2629bp，含有3个外显子和2个内含子，编码一条含有497个氨基酸的蛋白序列，该蛋白质预测的等电点(pI)为8.14，相对分子质量约为55.68kD。
实施例2
根据NCBIgenbank和KEGG SSDB数据库中的注释，分别从拟南芥、水稻和蒺藜苜蓿获得了13、10和12个丙酮酸激酶的全长序列，用于序列比对和进化分析。
使用DNAMAN进行氨基酸序列同源比对分析，并根据Pfam 31.0数据库对其所含的保守结构域进行分析；使用Mega7软件按照Neighbor-joining法构建系统进化树。
本发明对MtPK1编码的氨基酸序列进行了生物信息学分析。将MtPK1的氨基酸序列和其同源蛋白的氨基酸序列进行多重序列比对分析，比对分析结果见图1，图1中丙酮酸激酶的β桶状结构域和α/β结构域分别在8-350和367-493氨基酸位置处；实线代表丙酮酸激酶保守的结构域活性位点。Consensus表示序列完全一致的氨基酸，用黑色强调保守的氨基。由图1的结果可知，所有的丙酮酸激酶都含有一个保守的丙酮酸激酶活性位点：[LIVAC]-x-[LIVM]-[LIVM]-[SAPCV]-K-[LIV]-E-[NKRST]-x-[DEQHS]-[GSTA]-[LIVM]。该位点富含赖氨酸，可能起主要的催化作用。更重要的是，在Pfam 31.0数据库对这些序列所含的结构域进行分析发现，除了含有一个保守的丙酮酸激酶活性位点外，在这些丙酮酸激酶的N端都有一个β桶状结构域(PF00224.14)，在它们的C端有一个α/β结构域(PF02887.9)，进一步说明了它们都属于丙酮酸激酶家族。
根据图2展示的进化树，这些丙酮酸激酶明显聚成两个主要的簇：胞质丙酮酸激酶(Cytosolic PK)和质体丙酮酸激酶(Plastidial PK)，编码类丙酮酸激酶的基因At3g49160独成一簇。PKc同工酶一致地聚成两个小簇(Cytosolic-1和Cytosolic-2)，PKp同工酶也聚成两个小簇(Plastidial-α和Plastidial-β)。胞质丙酮酸激酶MtPK1与MtPK2、MtPK4聚在一簇，它们的亲缘关系最相近，氨基酸序列同源性高达99％。分布在Cytosolic-2的4个胞质丙酮酸激酶(MtPK5、MtPK6、MtPK9和MtPK10)与其序列同源性高达68％。同属于胞质丙酮酸激酶的OsPK1(Os11g0148500)与MtPK1的同源性也为68％。
实施例3
用带限制性酶切位点的引物MtPK1SalF和MtPK1BamR克隆出MtPK1的编码序列，PCR产物经SalI和BamHI消化后连入同样的限制性内切酶消化后的载体pJIT163-hGFP以构建融合基因MtPK1-hGFP。将测序正确的结构导入拟南芥叶原生质体。25℃孵育16h，莱卡激光扫描共聚焦显微镜检测荧光以pJIT163-hGFP为正对照。
为了进一步确定MtPK1属于胞质丙酮酸激酶，本发明构建了CAMV 35S启动子驱动的MtPK1-hGFP融合表达的瞬时表达载体，并转化到拟南芥叶肉细胞原生质体里，利用Leica激光共聚焦显微镜观察荧光蛋白的定位情况。细胞质区域中GFP的绿色荧光信号清晰可见，而叶绿体的自发荧光区域几乎没有绿色荧光。图3中顶层为在拟南芥原生质体细胞中瞬时表达GFP，底层为瞬时表达MtPK1-GFP融合蛋白，通过Leica激光共聚焦显微镜观察绿色荧光蛋白的定位情况。GFP和Bright field分别表示绿色荧光和明视场。图3的结果表明：MtPK1是一个胞质定位的丙酮酸激酶，这与前面利用拟南芥、水稻和蒺藜苜蓿丙酮酸激酶构建的进化树分析而推测的细胞质定位结果一致。
实施例4
本发明丙酮酸激酶基因编码蛋白的功能鉴定方法包括以下步骤：
步骤(1)、(2)同实施例1的步骤(1)、(2)
(1)pQE30载体和目的DNA片断的连接。
酶切回收后的目的条带和载体按照下表反应体系进行连接(表5)。
表5T4DNA连接酶体系
(2)重组蛋白的诱导、纯化和酶活力的测定
1)取20μL菌液于2mL添加了Carb的LB液体培养基中，37℃过夜振荡培养。
2)将过夜培养的菌液加入300mL含100mg/L Carb和高温高压灭菌的50％葡萄糖的LB液体培养基中，37℃摇床培养至OD600值为0.5-0.6，取1mL菌液，收集菌体作为对照。
3)在300mL菌液中加入300μL的1M IPTG，终浓度为0.3mM，于16℃培养24h。
4)4℃，9000g离心5min，收集菌体。
5)加入10倍体积的lysis buffer和1mg/mL的溶菌酶，冰上静置30min。
6)用超声破碎仪对细胞破碎10min，释放蛋白。
7)4℃，9000g离心30min，收集上清。
8)将样品加入8倍柱体积washbuffer活化过的镍柱，待样品都流过亲和柱填料后，用20倍柱体积的washbuffer冲洗杂蛋白，最后利用5倍柱体积elutebuffer洗脱目标蛋白。蛋白收集在Millpore(30kDa)的管中，4℃，4000g离心15min浓缩蛋白。
9)加入1mL 100mM Tris-HCl，4℃，4000g离心15min，以洗去盐和咪唑。
10)以500：20的比例加入考马斯亮蓝进行染色，λ595nm测定重组蛋白的浓度；同时，SDS-PAGE电泳，确认重组蛋白的大小和纯度。
11)丙酮酸激酶活性的测定：500μL酶活反应体系：50mM HEPES–KOH(pH 6.4)，25mM KCl，12mM MgCl2，2mM PEP，1mMADP，1mM DTT，5％(w/v)PEG8000，0.15mM NADH和2units/ml乳酸脱氢酶。在室温28℃条件下，反应5min，于λ340nm测定吸光值。
12)NADH标准曲线的制作：配制不同浓度0、12.5、25、50、75、100和150nmol的NADH溶液，在λ340nm测定吸光值的变化。根据吸光值和NADH的浓度制作标准曲线。根据NADH的标准曲线计算丙酮酸激酶的活性。
为了对MtPK1蛋白的丙酮酸激酶活性进行检测，将MtPK1基因构建在pQE30载体上，利用原核表达系统进行融合蛋白的表达。利用镍离子亲和树脂对融合蛋白进行分离纯化，经SDS-PAGE电泳确认目的蛋白的纯度(图4)。在ADP存在的条件下，以K+和Mg2+作为辅因子，丙酮酸激酶能催化PEP转化成丙酮酸，在还原型辅酶Ⅰ(NADH)存在情况下，丙酮酸被乳酸脱氢酶(LDH)转化为乳酸。NADH在340nm处有强烈的光吸收，根据吸光值的改变来计算丙酮酸激酶的活性。结果表明，在底物和辅因子都处于饱和状态时，被纯化的带His标签的重组蛋白表现出很广泛的pH谱，且在pH 6.5左右时达到最适pH(图5)。此外，在最适pH时，MtPK1对PEP(图6)和ADP(图7)的动力学常数Km值分别是149μM和106μM。综上表明，MtPK1是一个保守的且有活跃的催化活性的胞质丙酮酸激酶。
实施例5
(1)农杆菌感受态细胞的转化
1)取-80℃冰箱保存的农杆菌感受态细胞于冰上冻融。
2)向感受态中加入2μL质粒，轻弹混匀，置于冰上15min。
3)放于液氮中，速冻1min；37℃，热激5min。
4)向上述液体中加入500μL不加抗生素的LB液体培养基，28℃振荡培养3h。
5)用灭菌的涂布棒将菌液涂布于加相应抗生素的LB固体培养基上，密封后28℃静置培养。
6)挑取单克隆进行菌落PCR，确认培养菌落阳性后，-80℃保存菌种。
(2)转基因植株的转化
1)菌株的活化：-80℃保存的pCXSN-MtPK1/EHA105菌株冻融后吸取20μL接种于2mL含有抗生素的LB培养基中，28℃过夜振荡培养。
1)扩大培养：取1mL活化的菌接种于100mL含抗生素的LB培养基，28℃振荡培养至OD600＝0.6。
2)取5-8周的蒺藜苜蓿嫩叶加1滴吐温-20(表面活性剂)放自来水下冲洗至无泡沫。
3)加入30mL 10％次氯酸钠消毒15min；同时，6000g离心5min收集摇好的菌株，用无菌的SH3a液体重悬，备用。
4)用无菌水冲洗消毒的叶片3-4次，至无次氯酸钠残留。
5)用手术刀将叶片切出伤口，将切好的叶片与菌液混匀，侵染1h。
6)平铺于SH3a固体培养基，暗处共培养2d。
7)移于加抗生素的SH3a培养基，诱导愈伤。
8)4-5周后将愈伤移至SH9固体培养基上，诱导转基因植株。
10)转基因植株地上部分长出后移于1/2SH9培养基诱导生根。
11)待转基因植株的根系发育良好，将其移栽于装有灭菌土壤的培养瓶中，置于组培室炼苗。
12)炼苗成功的植株移栽于人工气候室(25±2℃；18h光照+6h黑暗)。
13)提取转基因植株的DNA筛选转基因阳性苗。
14)提取阳性苗的RNA，对其表达水平进行分析。筛选出高表达株系
15)观察阳性苗表型，并对高表达株系的代谢物含量进行分析。
(3)花青素含量的测定
1)取同一生长环境下的蒺藜苜蓿新鲜组织样品。
2)加入液氮研磨后准确称取50mg，加入5倍体积的甲醇(含体积比为0.1％的盐酸)，超声30min，4℃过夜。
3)超声30min，10000rmp离心5min，取上清。
4)加入750μL水和750μL氯仿，振荡离心，取上清。
5)用紫外分光光度计在530nm处测定。以矢车菊素-3-O-葡萄糖的摩尔光吸收作为标准品估算花青素的含量。
为了解析蒺藜苜蓿中丙酮酸激酶基因MtPK1的作用机制，通过荧光定量RT-PCR的方法对MtPK1在蒺藜苜蓿根、茎、叶、花和开花后10d、12d、16d、20d、24d、36d收获的种子中的表达情况进行了研究。研究结果见图8，MtPK1基因在不同组织的表达情况；MtPK1为采集不同部位包括根、茎、叶、花、豆荚和授粉后10天、12天、16天、20天、24天、36天种子材料后提取RNA并反转为cDNA后，以Mtactin2为内参，进行qRT-PCR。图8的结果表明，在蒺藜苜蓿的各个部位以及种子发育的各个时期都能检测到MtPK1的表达。其中，在茎、豆荚及10d、12d、16d、20d和36d种子中的表达水平较高，在根和24d种子中的表达水平最高。
类黄酮中的花青素主要存在于蒺藜苜蓿的茎和叶中，类黄酮中的原花青素只在种子中积累，这与MtPK1的表达情况相似，说明MtPK1与花青素和原花青素的生物合成相关。
为了进一步证明MtPK1在花青素和原花青素合成中的作用，本发明将烟草花叶病毒CaMV 35S启动子驱动的丙酮酸激酶基因MtPK1连接到真核表达载体pCXSN上，将融合基因转入到农杆菌EHA105中。根据农杆菌介导的蒺藜苜蓿叶片转化的方法，在黑暗条件下先诱导出愈伤后置于光下开始再生苗的诱导。首先，使用35S:pCXSN-MtPK1/GV3101菌株转化进蒺藜苜蓿突变体NF0791中，共得到两棵抗性苗。同时还将MtPK1基因转入蒺藜苜蓿野生型R108中，经PCR鉴定获得3棵转基因阳性苗OE-10、OE-26和OE-30(图9)。过量表达植株的茎叶花青素积累水平不一，表现为大多数转基因株系花青素含量增加，茎及叶基部呈紫色。对过表达株系中花青素的含量进行测定，发现OE-10花青素的含量显著高于野生型和突变体(图10)。结果表明，花青素含量较高的材料MtPK1表达量高，这些数据表明了茎部花青素的积累与MtPK1的表达水平呈正相关。
综上可知，蒺藜苜蓿MtPK1基因编码一个丙酮酸激酶，MtPK1蛋白催化特异的底物磷酸烯醇式丙酮酸，形成丙酮酸，pH值会影响MtPK1的催化效率，与已知的其它植物的丙酮酸激酶不同，MtPK1是一个全新的植物丙酮酸激酶。蒺藜苜蓿MtPK1蛋白是一个定位于细胞质的蛋白，可以在细菌和植物中表达，表达的重组蛋白具有活性，鉴于MtPK1的过量表达可以提高类黄酮的含量，特别是其中的花色素的含量，因此可以用于微生物或者植物代谢工程生产提高类黄酮化合物的产量。
本实施例中无特殊说明的操作手法均为现有技术，故不在此过多解释。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
序列表
<110> 中国农业科学院北京畜牧兽医研究所
<120> 一种丙酮酸激酶基因及其应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1494
<212> DNA
<213> MtPK1基因(Medicago
truncatula)
<400> 1
atgatggcag agaagaaacc caaaacaaag atcgtgtgca cgctgggacc tgcatcgagg 60
tctgttccaa tggtggagaa gcttctacaa gcaggcatga atgtcgctcg tttcaacttc 120
tctcatggtt cctatgaata tcatcaggaa acgcttgata atcttagaac cgctatgcaa 180
aataccggta ttctctgcgc cgtcatgctt gacactaagg ggccggagat tcgaaccgga 240
tttctcaagg atggaaagcc tgtccaactg aaacaaggtc aggaaataac catttcaacc 300
gactatgaca taaaaggaga tgagaatatg atctgtatga gctacaaaaa gttggcttat 360
gacgtgaagc ccggaagcat tgtattatgc gcagatggca ccatatcatt taaagtttta 420
tcatgtgaca aaaaagctgg tttggttcga tgctgctgtg aaaactctgc catgcttggc 480
gaaagaaaga atgttaatct tcctggagtc atagtggatc tcccaacatt gactgagaaa 540
gacaaggaag atatcatggt atggggagtt cctaataata ttgacatgat tgcactttct 600
tttgttcgaa aaggttctga tctggtggaa gttcgcaagt tgttgggaaa acatgctaag 660
aacatacttc tcatgtcaaa ggttgaaaac caagaaggag ttgcaaattt tgatgaaatc 720
cttacaaatt cagatgcatt tatggtggca cgtggcgacc ttggaatgga aattccaata 780
gagaagatat ttctagcaca aaaagtgatg atttataagt gtaatatcca aggaaagccg 840
gttgtcactg caacgcagat gttggagtca atgatcaaat cacctaggcc aaccagagct 900
gaagctactg atgtcgcgaa tgcagttctg gatggcacag attgtgtcat gcttagtggt 960
gaaactgctg ctggagctta tccagaactt gctgttcgaa ctatggctaa aatttgtgtt 1020
gaagctgaga gtaccatcaa ctatggagat gtatttaaaa ggataatgga gcactcacca 1080
gtaccgatgg gcccattgga gagtctagct tcttctgcgg ttaaaatggc aaactcagct 1140
aaagcagcac ttatattggt tttaactaga ggagggagta ctgcaaaatt agtggctaaa 1200
tatagggcag gcatgccaat tctttctgtt gtcgttcctg agattaagac cgataccttt 1260
gattggtcct gcagtgatga ggtccctgcc agacatagct tgatattccg aggattgatt 1320
ccagtactga gtgcgggttc tgctagagct tctcatgcag aaacaacaga agaggcacta 1380
gacttcgcca ttcagtatgc caaaacaaaa ggtctttgca ataacgggga ttctgtggtg 1440
gctctgcatc gtgtaggtgt cgcatcaatc atcaaaatct tgactgtgaa atga 1494
<210> 2
<211> 497
<212> PRT
<213> MtPK1蛋白(Medicago
truncatula)
<400> 2
Met Met Ala Glu Lys Lys Pro Lys Thr Lys Ile Val Cys Thr Leu Gly
1 5 10 15
Pro Ala Ser Arg Ser Val Pro Met Val Glu Lys Leu Leu Gln Ala Gly
20 25 30
Met Asn Val Ala Arg Phe Asn Phe Ser His Gly Ser Tyr Glu Tyr His
35 40 45
Gln Glu Thr Leu Asp Asn Leu Arg Thr Ala Met Gln Asn Thr Gly Ile
50 55 60
Leu Cys Ala Val Met Leu Asp Thr Lys Gly Pro Glu Ile Arg Thr Gly
65 70 75 80
Phe Leu Lys Asp Gly Lys Pro Val Gln Leu Lys Gln Gly Gln Glu Ile
85 90 95
Thr Ile Ser Thr Asp Tyr Asp Ile Lys Gly Asp Glu Asn Met Ile Cys
100 105 110
Met Ser Tyr Lys Lys Leu Ala Tyr Asp Val Lys Pro Gly Ser Ile Val
115 120 125
Leu Cys Ala Asp Gly Thr Ile Ser Phe Lys Val Leu Ser Cys Asp Lys
130 135 140
Lys Ala Gly Leu Val Arg Cys Cys Cys Glu Asn Ser Ala Met Leu Gly
145 150 155 160
Glu Arg Lys Asn Val Asn Leu Pro Gly Val Ile Val Asp Leu Pro Thr
165 170 175
Leu Thr Glu Lys Asp Lys Glu Asp Ile Met Val Trp Gly Val Pro Asn
180 185 190
Asn Ile Asp Met Ile Ala Leu Ser Phe Val Arg Lys Gly Ser Asp Leu
195 200 205
Val Glu Val Arg Lys Leu Leu Gly Lys His Ala Lys Asn Ile Leu Leu
210 215 220
Met Ser Lys Val Glu Asn Gln Glu Gly Val Ala Asn Phe Asp Glu Ile
225 230 235 240
Leu Thr Asn Ser Asp Ala Phe Met Val Ala Arg Gly Asp Leu Gly Met
245 250 255
Glu Ile Pro Ile Glu Lys Ile Phe Leu Ala Gln Lys Val Met Ile Tyr
260 265 270
Lys Cys Asn Ile Gln Gly Lys Pro Val Val Thr Ala Thr Gln Met Leu
275 280 285
Glu Ser Met Ile Lys Ser Pro Arg Pro Thr Arg Ala Glu Ala Thr Asp
290 295 300
Val Ala Asn Ala Val Leu Asp Gly Thr Asp Cys Val Met Leu Ser Gly
305 310 315 320
Glu Thr Ala Ala Gly Ala Tyr Pro Glu Leu Ala Val Arg Thr Met Ala
325 330 335
Lys Ile Cys Val Glu Ala Glu Ser Thr Ile Asn Tyr Gly Asp Val Phe
340 345 350
Lys Arg Ile Met Glu His Ser Pro Val Pro Met Gly Pro Leu Glu Ser
355 360 365
Leu Ala Ser Ser Ala Val Lys Met Ala Asn Ser Ala Lys Ala Ala Leu
370 375 380
Ile Leu Val Leu Thr Arg Gly Gly Ser Thr Ala Lys Leu Val Ala Lys
385 390 395 400
Tyr Arg Ala Gly Met Pro Ile Leu Ser Val Val Val Pro Glu Ile Lys
405 410 415
Thr Asp Thr Phe Asp Trp Ser Cys Ser Asp Glu Val Pro Ala Arg His
420 425 430
Ser Leu Ile Phe Arg Gly Leu Ile Pro Val Leu Ser Ala Gly Ser Ala
435 440 445
Arg Ala Ser His Ala Glu Thr Thr Glu Glu Ala Leu Asp Phe Ala Ile
450 455 460
Gln Tyr Ala Lys Thr Lys Gly Leu Cys Asn Asn Gly Asp Ser Val Val
465 470 475 480
Ala Leu His Arg Val Gly Val Ala Ser Ile Ile Lys Ile Leu Thr Val
485 490 495
Lys
Claims (3)
1.过表达丙酮酸激酶MtPK1在提高蒺藜苜蓿花青素含量中的应用，其特征在于，所述丙酮酸激酶MtPK1的氨基酸序列如SEQ ID NO.2 所示。
2.如权利要求1所述的应用，其特征在于，所述的丙酮酸激酶MtPK1基因的核苷酸序列如SEQ ID NO.1 所示。
3.包含如权利要求2中所述的丙酮酸激酶基因MtPK1的质粒在提高蒺藜苜蓿花青素含量中的应用。
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