CN1807608B - γ-生育酚甲基转移酶基因、其表达载体及应用 - Google Patents
γ-生育酚甲基转移酶基因、其表达载体及应用 Download PDFInfo
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- CN1807608B CN1807608B CN 200610001855 CN200610001855A CN1807608B CN 1807608 B CN1807608 B CN 1807608B CN 200610001855 CN200610001855 CN 200610001855 CN 200610001855 A CN200610001855 A CN 200610001855A CN 1807608 B CN1807608 B CN 1807608B
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Abstract
本发明分别公开了源于玉米、大豆的γ-生育酚甲基转移酶、其编码基因及它们在植物基因工程领域中的用途。本发明采用RACE及RT-PCR技术，分别从油料作物高油玉米115、大豆(郑9525)中获得SEQ ID NO：1和SEQ IDNO：3所示的γ-生育酚甲基转移酶的全长cDNA编码序列，并通过原核表达获得了有生物活性的γ-生育酚甲基转移酶蛋白，同时还比较了不同来源的γ-TMT酶比活的差异。本发明分别将从玉米、大豆中分离的γ-生育酚甲基转移酶cDNA编码序列转化模式植物，经HPLC分析转基因植物叶子中的α-生育酚的含量比野生对照约提高2～5倍。本发明为培育高维生素E含量的油料作物新品种开辟了一条新途径。
Description
技术领域
本发明涉及编码γ-生育酚甲基转移酶的基因，尤其涉及油料作物玉米、大豆分离、克隆的γ-生育酚甲基转移酶基因、其表达载体以及它们的应用，属于植物基因工程领域。
背景技术
维生素E是一类人体所必需的脂溶性维生素，具有重要的生理功能。它能够清除脂质过氧化所产生的自由基而稳定保护生物膜的脂双层，使细胞免受过氧化物的伤害(Brigeliusb-flohE R，Traber MG，Vitamin E：function andmetablism，The FASEB J，1999，13，1145-1155)。因而被作为一种有效的抗氧化剂广泛地应用在医药、食品、饲料等行业中。大量动物实验证明，在每公斤饲料中添加40～100IU(国际单位)的维生素E，具有提高动物免疫力，改善肉质，提高动物的繁殖性能，缓解其应激反应等显著效果。另外，在饲料中添加维生素E能增加肉品的稳定性，防止肉类腐败、变味、变色，并延长上架时间。近20年来的临床研究表明，维生素E对于人体健康更具有重要作用。每天吸收10～13.4IU的维生素E才能维持肌肉、中枢神经系统和血管系统的正常生理功能。每天吸收100～1000IU的维生素E则可提高机体免疫力，延缓人体衰老，降低或防治心血管疾病和癌症的发生。
维生素E的生产主要通过化学合成和天然提取获得。化学方法合成的产品主要以醋酸酯的形式存在，副产物多且生物活性低。天然维生素E主要从植物油或其精炼副产物中提取获得。在生理活性和安全性上均明显优于合成维生素E，活性效力约为合成维生素E的1.3～1.4倍，因而正逐渐代替合成产品成为主流。随着生活水平的提高，人们对健康越来越重视，世界范围内对天然维生素E的需求量正以10％～15％的年增长率不断增长，使之成为世界上发展速度最快的维生素产品之一。
天然维生素E由8种生育酚异构体组成。根据侧链饱和度可分为生育酚(tocopherol)和三烯生育酚(tocotrienol)两大类。每类依芳香环上甲基数目和位置的不同又分别分为α、β、γ、δ生育酚和α、β、γ、δ三烯生育酚。
生育酚和三烯生育酚的基本结构
这8种同系物在植物中的含量及相对活性均不尽相同，α-生育酚的生物活性最高(α、β、γ、δ相对活性分别为100％、50％、10％和3％，α-三烯生育酚约为30％，其他三烯生育酚为10％左右.Kamal-Eldin A，Appelqvist LA，The chemistry and antioxidant properties of tocopherols and tocotrienols，Lipids，1996，31，671-701)，并可被人体优先吸收和利用，这是由于在肝脏中的运送α-生育酚的转移蛋白优先结合α-生育酚的特性决定的(Torbert R.Rocheford，Jeffrey C.Wong，Cem O.Egesel，Robert J.Lambert，Enhancement of Vitamin ELevels in Corn，Journal of the American college of nutrition，2002，Vol.21，No.3,191-198)。国际上对于维生素E产品的质量标准也主要以α-生育酚的含量和活性来判定。
但是α-生育酚在植物种子中的含量很低，即使在用于提取维生素E的各油料作物种子中的含量也很低，仅为7％～20％，而其生物合成前体γ-生育酚的含量可占总生育酚含量的50～70％。因此大幅度提高植物特别是油料作物中高活性α-生育酚的含量，改变其所占比例将会为改良作物品质、提高其农业附加值带来启示，具有良好的社会和经济效益。
研究植物维生素E合成途径发现，通过甲基转移作用将其前体γ-生育酚转化为α-生育酚的γ-生育酚甲基转移酶(γ-TMT)可提高α-生育酚的含量和比例。目前，该酶的基因已从拟南芥(Arabidopsis thaliana)、蓝藻(David Shintaniand Dean DellaPenna，Elevating the Vitamin E Contentof Plants ThroughMetabolic Engineering，Science，1998，Vol.282，2098-2100)、甘蓝(Brassicaoleracea)等中克隆得到(欧阳青，樊春涛，孙卉，张玉满，白双义，蔡文启，结球甘蓝γ-生育酚甲基转移酶cDNA的克隆、分析及其异源表达酶蛋白的功能研究，自然科学进展，2003，Vol.13，No.7，709-71)。拟南芥中功能验证表明，该基因的过量表达并不能增加转基因植物中生育酚的总含量，而是可以改变生育酚的组成，使植物中的α-生育酚的含量由原来的10％转化为90％以上，由此可使植物中维生素E的生理活性大大提高。对来源于甘蓝的γ-TMT基因的克隆及在植物中表达的结果也同样获得了相同的效果。虽然这一设想已得到了证实，但至今未见从主要油料作物中分离克隆γ-TMT新基因，并将其转化到油料作物中来提高α-生育酚的含量和改变α-生育酚分布的报道。
发明内容
本发明首先所要解决的技术问题是克服现有技术的不足，分别从油料作物玉米和大豆中分离、克隆得到新的γ-生育酚甲基转移酶cDNA。
本发明首先所要解决的技术问题是通过以下两个技术途径来实现的：
1、一种从玉米中分离的编码γ-生育酚甲基转移酶的cDNA序列，该cDNA序列具有以下(a)、(b)、(c)或(d)的核苷酸序列：
(a)具有SEQ ID NO：1所示的核苷酸序列；或
(b)在严谨条件下能与(a)所示的核苷酸序列的互补序列杂交的核苷酸序列，且该核苷酸序列编码具有γ-生育酚甲基转移酶活性的蛋白；或
(c)编码SEQ ID NO：2所示氨基酸序列的核苷酸序列；或
(d)编码具有γ-生育酚甲基转移酶功能的蛋白衍生物的核苷酸序列，该蛋白衍生物通过将SEQ ID NO：2所示的氨基酸序列的一个或多个氨基酸残基替换、缺失或插入而获得。
优选的，本发明从玉米中分离的编码γ-生育酚甲基转移酶的cDNA序列具有SEQ ID NO：1所示的核苷酸序列。
2、一种从大豆中分离的编码γ-生育酚甲基转移酶的cDNA序列，该cDNA序列具有以下(a)、(b)、(c)或(d)的核苷酸序列：
(a)具有SEQ ID NO：3所示的核苷酸序列；或
(b)在严谨条件下能与(a)所示的核苷酸序列的互补序列杂交的核苷酸序列，该核苷酸序列编码具有γ-生育酚甲基转移酶活性的蛋白；或
(c)编码SEQ ID NO：4所示氨基酸序列的核苷酸序列；或
(d)编码具有γ-生育酚甲基转移酶功能的蛋白衍生物的核苷酸序列，该蛋白衍生物通过将SEQ ID NO：4所示的氨基酸序列的一个或多个氨基酸残基替换、缺失或插入而获得。
优选的，本发明从玉米中分离的编码γ-生育酚甲基转移酶的cDNA序列具有SEQ ID NO：3所示的核苷酸序列。
本发明所要解决的再一个技术问题是提供一类γ-生育酚甲基转移酶。
本发明所要解决的再一个技术问题是通过以下两个技术途径来实现的：
1、一种玉米γ-生育酚甲基转移酶，其含有以下(a)或(b)的氨基酸序列：
(a)具有SEQ ID NO：2所示的氨基酸序列；或
(b)将SEQ ID NO：2所示的氨基酸序列通过一个或多个氨基酸残基的替换、缺失或插入而获得的仍具有γ-生育酚甲基转移酶功能的蛋白衍生物。
优选的，本发明玉米γ-生育酚甲基转移酶具有SEQ ID NO：2所示的氨基酸序列。
2、一种大豆γ-生育酚甲基转移酶，含有以下(a)或(b)的氨基酸序列：
(a)具有SEQ ID NO：4所示的氨基酸序列；或
(b)将SEQ ID NO：4所示的氨基酸序列通过一个或多个氨基酸残基的替换、缺失或插入而获得的仍具有γ-生育酚甲基转移酶功能的蛋白衍生物。
优选的，本发明大豆γ-生育酚甲基转移酶具有SEQ ID NO：4所示的氨基酸序列。
本发明所要解决的另一个技术问题是将本发明从玉米、大豆中分离到的γ-生育酚甲基转移酶cDNA应用于培育转基因植物。
本发明所要解决的另一个技术问题是通过以下技术途径来实现的：
一种应用本发明γ-生育酚甲基转移酶cDNA的方法，包括：
构建含有本发明γ-生育酚甲基转移酶cDNA的植物表达载体，将该植物表达载体转化到受体植物中，筛选、培育阳性转基因植物。
所述的受体植物优选为油料作物，例如可以是花生、玉米、油菜、大豆、向日葵、芝麻等。
本发明所要解决的又一个技术问题是提供一种制备γ-生育酚甲基转移酶的方法。
本发明所要解决的又一个技术问题是通过以下技术途径来实现的：
一种制备γ-生育酚甲基转移酶的方法，包括以下步骤：
构建含有权利要求3、4、7或8任意一项所述的cDNA序列的重组原核表达载体，用所述的原核表达载体转化宿主细胞，培养所转化的宿主细胞，诱导重组γ-生育酚甲基转移酶表达，收集、纯化所表达的重组γ-生育酚甲基转移酶。
上述方法中，所述的重组原核表达载体优选为pET-sigMTMT或pET-sigSTMT；所述的宿主细胞优选为大肠杆菌BL21。
本文中，所述的“多个”通常意味着2～60个，优选为2～15个，这些取决于γ-生育酚甲基转移酶的三维结构中氨基酸残基的位置或氨基酸的种类；所述的“替换”是指分别用不同的氨基酸残基取代一个或多个氨基酸残基；所述的“缺失”是指氨基酸序列的改变，其中分别缺少一个或多个氨基酸残基；所述的“插入”是指氨基酸序列的改变，相对天然分子而言，所述改变导致添加一个或多个氨基酸残基；所述的“严谨条件”是指杂交液为5～6×SSC，42～75℃杂交过夜，室温至37℃用2×SSC洗涤一至两次，优选的，所述的“严谨条件”为杂交液为6×SSC，68℃杂交过夜，37℃用2×SSC洗涤两次。
本发明的详细描述：
本发明采用3’RACE(Rapid Amplification of cDNA End)和5’RACE及RT-PCR技术，分别从油料作物高油玉米115、大豆(郑9525)中获得如SEQ IDNO：1和SEQ ID NO：3所示的γ-TMT的全长cDNA编码序列。与发表的来源于拟南芥、结球甘蓝、高粱(Sorghum bicolor)的γ-TMT基因的cDNA编码序列和推导的氨基酸序列进行了同源性比较(图2、3)。比较发现，本发明克隆获得的玉米γ-TMT与高梁γ-TMT的同源性较高，核苷酸同源性为78.8％，氨基酸同源性为76.9％。本发明获得的大豆γ-TMT与结球甘蓝γ-TMT同源性较高，核苷酸同源性为58.5％，氨基酸同源性为63.2％，而玉米、大豆γ-TMT二者的核苷酸同源性为55.4％，氨基酸同源性为62.9％(利用DNASTAR软件中的clustal V方法分析)。为比较不同来源的γ-TMT蛋白的酶活性，分别将来源于玉米、大豆、拟南芥和结球甘蓝的γ-TMT基因的cDNA编码序列(去除自身信号肽)构建到原核表达载体pET-30a(+)上，在相同的条件下进行诱导表达，并利用亲和层析纯化目的蛋白。以γ-生育酚和S-腺苷甲硫氨酸(SAM)为底物，在体外使四种来源的酶进行催化反应，通过测定生成的产物α-生育酚的含量来确定酶活，同时测定纯化蛋白的浓度，从而计算比较出四种来源不同的γ-TMT基因所表达的蛋白比活之间存在较大差异，玉米来源的γ-TMT的比活最高，甘蓝的比活最低。
此外，本发明分别构建了含有玉米、大豆γ-TMT基因的组成型植物表达载体，通过农杆菌介导的方法分别转化模式植物拟南芥和烟草。获得PCR、ELISA均为阳性的转基因植株。经HPLC分析转基因拟南芥和烟草叶子中的α-生育酚的含量提高2～5倍。
本发明首次克隆油料作物玉米、大豆的γ-TMT基因的cDNA全序列。并通过原核表达获得了有生物活性的酶蛋白。比较了不同来源的γ-TMT酶比活的差异。并通过转化模式植物进一步验证了基因功能并为通过分子育种提高油料作物创造新品种提供了一条新思路。
附图说明
图1为不同来源的γ-TMT基因cDNA同源比较进化图。
图2为不同来源的γ-TMT基因推导的氨基酸序列同源比较进化图。
图3为原核表达载体的构建图。
图4为γ-TMT蛋白的诱导表达SDS-PAGE电泳图。
1：蛋白marker；2：纯化的拟南芥γ-TMT蛋白；3：纯化的甘蓝γ-TMT蛋白；4：纯化的玉米γ-TMT蛋白；5：纯化的大豆γ-TMT蛋白。
图5为亲和层析γ-TMT蛋白的SDS-PAGE电泳图。
图6为γ-TMT酶活性分析薄层层析图。
1：γ-生育酚标准品；2、甘蓝来源的pET-TMT反应产物；3、大豆来源的pET-TMT反应产物；4、玉米来源的pET-TMT反应产物；5、拟南芥来源的pET-TMT反应产物；6、α-生育酚标准品。
图7：植物表达载体构建图。
以下通过实施例来进一步描述本发明的有益效果，应该理解的是，这些实施例仅用于例证的目的，决不限制本发明的保护范围。
具体实施方式
实施例1玉米γ-生育酚甲基转移酶基因的克隆
1.1总RNA的提取
总RNA的提取按Promega公司的RNAgents Total RNA Isolation System kit和进行。取生长20天的高油玉米115号叶片1g在液氮中研磨成粉末，加入到冰预冷的600L变性液(26mmol/L醋酸钠，0.5％十二烷基肌氨酸pH 4.0，0.125mol/L β-巯基乙醇，4mol/L硫氰酸胍)中，然后依次加入60L 2mol/L醋酸钠(pH4.0)，混匀，加入600L酚∶氯仿∶异戊醇(25∶24∶1，pH4.7)，剧烈振荡，在冰上放置15min，4℃下10000g离心20min，将上清吸出，加入等体积的异丙醇，-20℃放置30min，4℃下10000g离心10min，弃去上清，沉淀加入1mL冰预冷的75％乙醇洗涤后，沉淀RNA自然干燥，用Nuclease-free水溶解后备用。紫外测定总RNA的含量及纯度，取5g的总RNA做反转录获得cDNA的一链。42℃水浴1min，加入1μL(200U)SuperScriptTM II RT，轻轻混合，42℃保温50min。70℃水浴15min，终止该反应，离心10～20s，将其放置在37℃下，加入1μL RNAase mix，轻轻混匀，37℃反应30min，反应结束后，将样品短暂离心，放在冰上。
Oligo(dT)(10μmol/L) 1.5μL
样品总RNA 10μL(5μg)
DEPC ddH2O 4μL
70℃保温10min，冰上放置1min，短暂离心
10×PCR buffer 2.5μL
MgCl2(25mmol/L) 2.5μL
dNTP mix(2.5mmol/L) 2μL
DTT(0.1mol/L) 2.5μL
总体积 25μL
1.2cDNA3’末端的快速扩增
在获得cDNA的第一链后，根据来源于高粱(Sorghum bicolor)的γ-TMT基因中的保守序列设计5’端引物Vep 1：5’TGTGGCATTGGAGGCAGCTCAAGGTA，Oligo(dT)为3’端引物，进行cDNA3’末端的扩增。PCR扩增条件为：94℃4min，94℃1min，52℃45s，72℃45s(30个循环)，72℃10min。扩增出约600bp的片段，回收后连到pGM-T Easy(天为时代公司)载体上，构建重组质粒pT-3’MTMT，并转化E.coli DH5α，酶切鉴定，获得阳性克隆后测序(上海博亚生物技术有限公司)。
1.3cDNA5’末端的快速扩增
按GibcoBRL公司5′RACE System for Rapid Amplification of cDNA Ends，Version2.0kit操作。取5ug总RNA进行反转录(如1.1所述)，获得cDNA第一链。经RNaseH处理，用末端转移酶在合成的第一链cDNA3’端加poly(dC)。以锚定引物AnchorP：5’GGCCACGCGTCGACTAGTACGGGGGGGGGG为5’端特异引物，根据获得的γ-TMT基因3’端部分序列设计3’端特异引物VeP2：5’TGGCAGCATGTCACGATGATTAT，进行cDNA5’末端的扩增。PCR扩增条件为：94℃4min，94℃45s，55℃45s，72℃45s(30个循环)，72℃10min。扩增出约600bp的DNA片段，并将此片段按上述方法克隆于pGM-T Easy载体上，构建重组质粒pT-5’MTMT，鉴定出阳性克隆并测序。
通过3’RACE和5’RACE获得的部分片段测序结果可得到如序列表SEQID NO：1所示的玉米γ-TMT基因的全长cDNA序列，其所推导的氨基酸序列如SEQ ID NO：2所示。
1.4RT-PCR扩增cDNA的编码区序列
根据上述步骤获得的γ-TMT基因的全长cDNA序列设计引物，进行RT-PCR，扩增该基因的cDNA编码区序列。
上游引物MaiP 1：5’tctagaATGGCTCACGCGGCGCTGCTC(5’端含一个XbaI酶切位点)；下游引物MaiP2：5’gagctcCTACGCGGCTCCAGGCTTGCGAC(5’端含一个SacI酶切位点)。反应条件为：94℃4min，94℃1min，58℃45s，72℃1min20s(30个循环)，72℃10min。扩增出约1000bp的DNA片段，并将此片段按上述方法克隆于pGM-T Easy载体上，构建重组质粒pT-MTMTcDNA，并转化大肠杆菌DH5α，鉴定出阳性克隆并测序。
实施例2大豆γ-生育酚甲基转移酶基因的克隆
2.1总RNA的提取
选取生长20天的大豆品种(郑9525)提取总RNA，采用上述1.1的方法获得反转录的cDNA第一链。
2.2cDNA3’末端的快速扩增
在获得cDNA的第一链后，根据来源于拟南芥的γ-TMT基因中的保守序列设计5’端引物S-TMT1：5’TAGTGGATGTTGGGTGTGG，Oligo(dT)为3′端引物，进行cDNA3’末端的扩增。PCR扩增条件为：94℃4min，94℃1min，55℃45s，72℃45s(30个循环)，72℃10min。扩增出约600bp的片段，回收后连到pGM-T Easy载体上，构建重组质粒pT-3’SoyTMT，并转化E.coli DH5α，酶切鉴定，获得阳性克隆后测序(上海博亚生物技术有限公司)。
1.3cDNA5’末端的快速扩增
按GibcoBRL公司5′RACE System for Rapid Amplification of cDNA Ends，Version2.0kit操作。取5ug总RNA进行反转录(如上所述)，获得cDNA第一链。经RNaseH处理，用末端转移酶在合成的第一链3’端加poly(dC)。以锚定引物AnchorP：5’GGCCACGCGTCGACTAGTACGGGGGGGGGG为5’端特异引物，根据获得的γ-TMT基因3’端部分序列设计3’端特异引物S-TMT2：5’AGGCATATGCTCACCACTCT，进行cDNA5’末端的扩增。PCR扩增条件为：94℃4min，94℃45s，52℃45s，72℃45s(30个循环)，72℃10min。扩增出约600bp的DNA片段，并将此片段按上述方法克隆于pGM-T Easy载体上，构建重组质粒pT-5’soyTMT，鉴定出阳性克隆并测序。
通过3’RACE和5’RACE获得的部分片段测序结果可得到如序列表SEQID NO：.3所示的大豆γ-TMT基因的全长cDNA序列，所推导的氨基酸序列如SEQ ID NO：4所示。
1.5RT-PCR扩增cDNA的编码区序列
根据上述步骤获得的γ-TMT基因的全长cDNA序列设计引物，进行RT-PCR，扩增该基因的cDNA编码区序列。上游引物S-rTMTF0：5’CCCGGGACCACCATGGCCACCGTGGTGAGGAT(5’端含一个SmaI酶切位点)；下游引物S-rTMTR0：5’GATATCCTTTTTTTTTCGAACAACCCCTTTC(5’端含一个EcoRV酶切位点)，反应条件为：94℃4min，94℃1min，56℃45s，72℃1min20s(30个循环)，72℃10min。扩增出约1000bp的DNA片段，并将此片段按上述方法克隆于pGM-T Easy载体上，构建重组质粒pT-STMTcDNA，并转化大肠杆菌DH5α，鉴定出阳性克隆并测序。
实施例3含γ-TMT基因原核表达载体的构建及诱导表达
3.1内源信号肽的去除
通过软件分析推导的氨基酸序列发现，玉米γ-TMT在N-末端存在一个将蛋白定位到叶绿体膜上的导肽序列，为使γ-TMT基因在原核中正确表达并获得有活性的蛋白，我们根据其基因序列在5’端设计引物MTMTsig：5’CCCGGGGCCTCGTCGACGGCTCAGG(5’端有一个SmaI酶切位点)，并与MaiP2：5’gagctcCTACGCGGCTCCAGGCTTGCGAC(5’端含一个SacI酶切位点)进行PCR反应(以质粒pT-MTMTcDNA为模板)以去除叶绿体导肽序列，反应条件为：94℃4min，94℃1min，58℃45s，72℃1min(30个循环)，72℃10min。扩增产物为916bp，回收后克隆于pGM-T Easy载体上构建重组质粒pT-sigMTMT，转化大肠杆菌DH5α，筛选到阳性转化子后进行测序。
大豆的γ-TMT基因同样存在叶绿体导肽序列，同上述原理与操作，设计引物S-rTMTF2：5’CCCGGGGTAGCGAGCTCGGAGAGAGGG(5’端有一个SmaI酶切位点)，并与S-rTMTR0：5’gatatcCTTTTTTTTTCGAACAACCCCTTTC(5’端含一个EcoRV酶切位点)进行PCR(以质粒pT-STMTcDNA为模板)，去除叶绿体导肽序列，构建重组质粒pT-sigSTMT，转化大肠杆菌，筛选到阳性转化子后进行测序。
3.2含γ-TMT基因的原核表达载体的构建
3.2.1含玉米来源的γ-TMT基因的原核表达载体的构建
利用SmaI、SacI酶切处理重组质粒pT-sigMTMT，切下916bp的片段，同时用EcoRV、SacI酶切处理表达载体pET-30a(+)(Novagen公司)，将此916bp片段连于pET-30a(+)载体上构建重组质粒pET-sigMTMT(图3)。转化大肠杆菌BL21，经酶切鉴定获得阳性克隆。
3.2.2含大豆来源的γ-TMT基因的原核表达载体的构建
利用SmaI、NotI酶切处理重组质粒pT-sigSTMT，切下916bp的片段，同时用EcoRV、NotI酶切处理载体pET-30a(+)，将此916bp片段连于pET-30a(+)载体上构建重组质粒pET-sigSTMT(图3)。转化大肠杆菌BL21，经酶切鉴定获得阳性克隆。
3.3γ-TMT蛋白的诱导表达
挑取含重组质粒pET-sigMTMT的BL21单菌落于10mL LB液体培养基(含50mg/L的Kan)中，37℃振摇培养过夜。次日按1∶20的比例转接于50mL LB培养基(含50mg/L Kan)中，37℃振荡培养至OD＝0.6～0.8，加入IPTG至终浓度0.4mmol/L，于28℃诱导表达3h。离心收集菌体，弃上清液。同时以含有pET-30a(+)空载体的菌株和含有重组质粒pET-sigMTMT但未经IPTG诱导的菌株做对照。SDS-PAGE分析表明，经IPTG诱导的含有重组质粒pET-sigMTMT的大肠杆菌BL21在约38kD的位置有一条明显的蛋白带(图5，LaneIII)，而作为对照的两菌株在此处无蛋白带(图4，Lane I、II)。说明来源于玉米的γ-TMT基因获得了表达。大豆来源的γ-TMT基因也用上述方法和操作进行诱导，SDS-PAGE分析表明也获得了表达(图4，LaneIV)。
实施例4表达γ-TMT的酶活性分析及比活对比
为比较不同来源的γ-TMT基因所表达的酶蛋白的活性，我们根据GenBank中搜寻到的拟南芥和结球甘蓝的序列设计引物克隆获得了拟南芥和结球甘蓝的γ-TMT基因的cDNA编码序列，并依据上述方法和操作，去掉其信号肽序列克隆到原核表达载体pET-30a(+)上，进行诱导并获得表达。将四个来源不同的γ-TMT基因在相同条件下进行诱导表达。诱导条件为：IPTG终浓度0.4mmol/L，于28℃诱导表达3h。离心收集600mL培养液的菌体，无菌水洗涤1次，重悬于6mL 20mM pH7.5Na2HPO4-NaH2PO4磷酸缓冲液中(含0.5MNaCl，10mM咪唑)，超声波破碎至破碎后的菌液与未破碎的菌液相比明显澄清为止。4℃，10000rpm，20min离心。取10-20μL上清作SDS-PAGE，其余上清用于蛋白纯化。
由于所采用的pET30a(+)表达系统所表达的蛋白质为融合蛋白，其N末端带有6个连续的组氨酸，可以通过金属离子层析柱(Ni2N TA Agarose)进行亲和纯化.。采用AKTA FPLC蛋白纯化系统(Amersham pharmacia biotech公司)；HiTrap Chelating HP(柱体积1mL)对诱导获得的四个蛋白进行纯化。纯化条件是：
平衡缓冲液：20mM pH7.5Na2HPO4-NaH2PO4磷酸缓冲液(含0.5M NaCl，10mM咪唑)，洗脱缓冲液：20mM pH7.5Na2HPO4-NaH2PO4磷酸缓冲液(含0.5M NaCl，500mM咪唑)。采用线性梯度洗脱，流速为：1mL/min。SDS-PAGE分析表明，纯化获得单一条带的蛋白(图5)。
获得纯化的γ-TMT蛋白后，对其酶活进行定性与定量研究，比较四者之间的比活。酶反应体系：取300μL纯化酶液，加入5μL S-腺苷甲硫氨酸SAM(0.35mg)，2.5μL 1M的二硫苏糖醇(DTT)，0.3mgγ-生育酚，补缓冲液(pH7.5)至总反应体系为500μL，30℃反应3h后，加入500μL氯仿∶甲醇(2∶1)终止反应，各样品剧烈涡旋1min后，10000rpm，10min，离心分相，将下层氯仿相转入一新的离心管中，用高压氮气吹干。溶于20μL无水乙醇中。取3μL样品点样于TLC板(硅胶GF254板)上，在二氯甲烷中室温展层。并以标准品γ-生于酚和α-生于酚(Sigma公司)作为对照。室温干燥TLC板，在紫外光(253nm)下观察色谱斑点(图6)。同时取样品进行HPLC定量测定。
分析条件：
高效液相色谱仪(日本岛津)，
色谱分析条件：
流动相：正己烷∶异丙醚(90∶10 V/V)
色谱柱：硅胶柱：粒度5μL，4.6×25cm；
检测波长：激发波长：298nm；发射波长：325nm
柱温：40℃流速：1..5ml/min 分析时间：20min。
得到经酶活反应生成的α-生育酚的含量，从而获得四个酶的活性。γ-TMT的酶活定义为：在pH7.5、30℃条件下每小时生成的1μg α-生育酚所需要的酶量为1个酶活单位(U)。用BioRad蛋白分析法测定纯化的蛋白浓度，并由此计算出四个酶蛋白的比活性(定义为：每毫克蛋白所含的酶活力单位)。见表1
表1不同来源的γ-TMT酶蛋白比活性比较
以上数值为三次平均值结果
表1结果显示，来源不同的γ-TMT基因所表达的蛋白比活之间存在较大差异，玉米来源的γ-TMT的比活最高，甘蓝的比活最低。
实施例5含γ-TMT基因的组成型植物表达载体的构建及其重组农杆菌的制备
分别用XbaI、SacI对载体pBI121和重组质粒pT-MTMTcDNA进行双酶切，回收约1kb的玉米γ-TMT片段及13kb的载体大片断，酶连，转化，构建重组质粒pBI121-MTMT，并经酶切鉴定出阳性克隆。分别用SmaI、SacI对载体pBI121和重组质粒pT-STMTcDNA进行双酶切，回收约1kb的大豆γ-TMT片段及13kb的载体大片段，酶连，转化，构建重组质粒pBI121-STMT，并经酶切鉴定出阳性克隆(图7)。
将重组质粒pBI121-MTMT、pBI121-STMT分别电击转化感受态农杆菌LBA4404，加入1mL YEB液体培养基28℃培养3小时，取200μL涂YEB抗性平板(Kan 50mg/L，Rf 50mg/L)，28℃培养。挑取单菌落提取农杆菌质粒，并通过PCR、酶切进行鉴定分别获得含质粒pBI121-MTMT和pBI121-STMT的农杆菌。
实施例6含γ-TMT基因的植物遗传转化
6.1烟草的转化和再生
分别挑取鉴定正确的农杆菌的单菌落，接种于5mLYEB(Kan 50mg/L，Rif50mg/L)液体培养基中，28℃，250rpm培养36小时。按照1∶200转接于50mLYEB(Kan 50mg/L，Rif 50mg/L)液体培养基中，28℃，培养至OD600＝0.8～1.0。5,000rpm离心收集菌体，用等体积的无菌的MS重悬即成浸染液。利用叶盘转化法转化烟草。选取长出8到10片叶子的烟草的叶片，顺着叶脉将叶片剪成1cm2的叶盘，浸泡入菌液中10分钟，取出，在无菌滤纸上吸干菌液，放入MS平板(6BA 2mg/mL，IAA 0.2mg/mL)中暗培养2天后将转化处理的叶盘浸于MS液体中10分钟，在无菌滤纸上吸干，转入含有抗性的MS平板(6BA 2mg/mL，IAA 0.2mg/mL，kan 100mg/mL，CB 500mg/mL)上，光照培养(24℃，光照16小时)，20天更换平板，直至分化出芽，切下抗性芽转到生根培养基上(MS+kan 100mg/mL，CB 500mg/mL)，生根至长成植株。
6.2转化拟南芥
挑取鉴定正确的农杆菌的单菌落，接种于5mL YEB(Kan 50mg/L，Rif50mg/L)液体培养基中，28℃，250rpm培养36小时。按照1∶300转接至200mLYEB(Kan 50mg/L，Rif 50mg/L)液体培养基中，28℃，培养至OD600≈2.0。5,000rpm离心收集菌体重悬于约2倍体积的10％的蔗糖溶液(现用现配)，至OD600≈1.0左右，加入0.02％(v/v)的Silwet 70(Sigma公司)。选用具大量未开放的花蕾的拟南芥进行转化。将拟南芥倒置，使花蕾朝下浸入渗透液中，停留20～40秒，取出花蕾，轻轻抖落植株上的多余渗透液，将植株正置。转化后的植株用硬塑料板保护后盖好保鲜膜。在22℃培养(光照16小时)，直到开花结荚收集种子。将收集到的T0代的种子消毒处理后撒在1/2MS培养基上(含有Kan 50mg/L，1％蔗糖的)。用封口膜封好平皿，4℃春化48～72小时。22℃培养(16小时光照)。具有卡那抗性的苗在抗性平板上能够正常生长呈绿色，而非抗性苗则黄化死去。待平板上的绿苗长出四片真叶时可将其移至土中，盖上保鲜膜保水两天后揭去，培养到幼苗长出8～10片真叶且较粗壮时可剪取叶片，提基因组DNA，进行PCR检测。
实施例7阳性转基因植物的筛选及其生育酚含量的测定
7.1转基因阳性植株的鉴定
分别取T0代转基因烟草及经抗性筛选的T1代拟南芥植株叶片约30～50mg，提取植物基因组DNA，以此为模板进行PCR扩增，筛选阳性植株。筛选转玉米γ-TMT基因阳性植株所用的引物为：基因内部引物VeP1：5’TGTGGCATTGGAGGCAGCTCAAGGTA和载体终止子引物35terP：5’TATGATAATCATCGCAAGACC。扩增条件为：94℃4min，94℃1min，60℃45s，72℃1min(30个循环)，72℃10min。扩增出约800bp片段即说明此植株为PCR阳性植株。筛选转大豆γ-TMT基因阳性植株所用的引物为载体启动子引物35Pro：5’AAAGGAAAGGCCATCGTTG和基因内部引物S-rTMTR05’GATATCTTTTTTTTTCGAACAACCCCTTTC。扩增条件为：94℃4min，94℃1min，58℃45s，72℃1min 30s(30个循环)，72℃10min。扩增出约1.2kb片段即说明此植株为PCR阳性植株。
7.2高表达阳性植株的鉴定
取上述PCR阳性的转基因植株和未转基因的野生型植株叶片各150mg用液氮研磨后转入含700μL抽体液(150mM Tris-HCL pH8.0，25％甘油，2％(w/v)聚乙烯吡洛烷酮，1mmol/L PMSF)的1.5mL离心管中。混匀，在冰上静置3～4小时，12000rpm 4℃离心20min，吸取上清液备用。通过ELISA方法鉴定高表达阳性植株。首先，以实施例4所述纯化的玉米、大豆γ-TMT蛋白为抗原，用包被液稀释成2μg/mL，取100μL点于酶标板上，4℃包被过夜。将过夜包被板取出于37℃放置1小时。用磷酸缓冲液PBST洗板3～5次。每孔加入200μL 3％BSA(牛血清白蛋白组分V)溶液，37℃温育1小时。PBST洗板，加入100μL稀释的一抗溶液(以亲和层析纯化的蛋白免疫的兔抗γ-TMT血清，稀释比例1∶10000倍)，37℃放置1小时，同时做好对照。PBST洗板，加入二抗稀释液(蜡根过氧化物酶标记的羊抗兔IgG，1∶5000倍稀释)37℃放置1小时，PBST洗板，加底物OPD显色至颜色明显后，加入50μL 2M硫酸终止反应。在波长492nm下测定。ELISA反应明显的样品说明玉米、大豆来源的γ-TMT基因在烟草和拟南芥中有较高的表达。
7.3阳性转基因植株生育酚含量的测定
挑选ELISA反应明显的阳性植株和未转基因的野生型植株叶片各100mg用液氮研磨后转入含600μL甲醇-氯仿(2∶1)抽体液的1.5mL离心管中，充分震荡混匀，然后加入200μL氯仿和200μL无菌水，充分混匀。12000rpm离心15min，取下层有机相转入新的离心管中，氮气吹干，溶于400μL无水乙醇中。通过HPLC分析发现，转玉米和大豆γ-TMT基因的阳性转基因烟草和拟南芥叶片中α-生育酚的含量普遍有所提高，其中三分之一的阳性转基因植株叶片中α-生育酚的含量提高2～5倍(表2)。
表2野生植株与阳性转基因植株α-生育酚含量的对比
序列表.txt
SEQUENCE LISTING
<110>中国农业科学院生物技术研究所
<120>γ-生育酚甲基转移酶基因、其表达载体及应用
<130>p00345
<160>4
<170>PatentIn version 3.3
<210>1
<211>1059
<212>DNA
<213>玉米(Zea mays)
<400>1
atggctcacg cggcgctgct ccattgctcc cagtcctcca ggagcctcgc agcctgccgc 60
cgcggcagcc actaccgcgc cccttcgcac gtcccgcgcc actcccgccg tctccgacgc 120
gccgtcgtca gcctgcgtcc gatggcctcg tcgacggctc aggcccccgc gacggcgccg 180
ccgggtctga aggagggcat cgcggggctg tacgacgagt cgtcggggct gtgggagaac 240
atctggggcg accacatgca ccacggcttc tacgactcga gcgaggccgc ctccatggcc 300
gatcaccgcc gcgcccagat ccgcatgatc gaggaggcgc tcgccttcgc cggtgtccca 360
gcctcagatg atccagagaa gacaccaaaa acaatagtcg atgtcggatg tggcattggt 420
ggtagctcaa ggtacttggc gaagaaatac ggagcgcagt gcactgggat cacgttgagc 480
cctgttcaag ccgagagagg aaatgctctc gctgcagcgc aggggttgtc ggatcaggtt 540
actctgcaag ttgctgatgc tctggagcaa ccgtttcctg acgggcagtt cgatctggtg 600
tggtccatgg agagtggcga gcacatgccg gacaagagaa agtttgttag tgagctagca 660
cgcgtggcgg ctcctggagg gacaataatc atcgtgacat ggtgccatag gaacctggat 720
ccatccgaaa cctcgctaaa gcccgatgaa ctgagcctcc tgaggaggat atgcgacgcg 780
tactacctcc cggactggtg ctcaccttca gactatgtgg acattgccaa gtcactgtct 840
ctcgaggata tcaagacagc tgactggtcg gagaacgtgg ccccgttttg gcccgccgtg 900
ataaaatcag cgctaacatg gaagggcttc acctctctgc tgacgaccgg atggaagacg 960
atcagaggcg cgatggtgat gccgctaatg atccagggct acaagaaggg cctcatcaaa 1020
ttcaccatca tcacctgtcg caagcctgga gccgcgtag 1059
<210>2
<211>352
<212>PRT
<213>玉米(Zea mays)
<400>2
Met Ala His Ala Ala Leu Leu His Cys Ser Gln Ser Ser Arg Ser Leu
1 5 10 15
Ala Ala Cys Arg Arg Gly Ser His Tyr Arg Ala Pro Ser His Val Pro
20 25 30
Arg His Ser Arg Arg Leu Arg Arg Ala Val Val Ser Leu Arg Pro Met
35 40 45
Ala Ser Ser Thr Ala Gln Ala Pro Ala Thr Ala Pro Pro Gly Leu Lys
50 55 60
Glu Gly Ile Ala Gly Leu Tyr Asp Glu Ser Ser Gly Leu Trp Glu Asn
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Ile Trp Gly Asp His Met His His Gly Phe Tyr Asp Ser Ser Glu Ala
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Ala Ser Met Ala Asp His Arg Arg Ala Gln Ile Arg Met Ile Glu Glu
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Ala Leu Ala Phe Ala Gly Val Pro Ala Ser Asp Asp Pro Glu Lys Thr
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Pro Lys Thr Ile Val Asp Val Gly Cys Gly Ile Gly Gly Ser Ser Arg
130 135 140
Tyr Leu Ala Lys Lys Tyr Gly Ala Gln Cys Thr Gly Ile Thr Leu Ser
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Pro Val Gln Ala Glu Arg Gly Asn Ala Leu Ala Ala Ala Gln Gly Leu
165 170 175
Ser Asp Gln Val Thr Leu Gln Val Ala Asp Ala Leu Glu Gln Pro Phe
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Pro Asp Gly Gln Phe Asp Leu Val Trp Ser Met Glu Ser Gly Glu His
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Met Pro Asp Lys Arg Lys Phe Val Ser Glu Leu Ala Arg Val Ala Ala
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Pro Gly Gly Thr Ile Ile Ile Val Thr Trp Cys His Arg Asn Leu Asp
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Pro Ser Glu Thr Ser Leu Lys Pro Asp Glu Leu Ser Leu Leu Arg Arg
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Ile Cys Asp Ala Tyr Tyr Leu Pro Asp Trp Cys Ser Pro Ser Asp Tyr
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Val Asp Ile Ala Lys Ser Leu Ser Leu Glu Asp Ile Lys Thr Ala Asp
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Trp Ser Glu Asn Val Ala Pro Phe Trp Pro Ala Val Ile Lys Ser Ala
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Leu Thr Trp Lys Gly Phe Thr Ser Leu Leu Thr Thr Gly Trp Lys Thr
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Ile Arg Gly Ala Met Val Met Pro Leu Met Ile Gln Gly Tyr Lys Lys
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Gly Leu Ile Lys Phe Thr Ile Ile Thr Cys Arg Lys Pro Gly Ala Ala
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<210>3
<211>1053
<212>DNA
<213>大豆属大豆(Glycine max(L.)Merill)
<400>3
atggccaccg tggtgaggat cccaacaatc tcatgcatcc acatccacac gttccgttcc 60
caatcccctc gcactttcgc cagaatccgg gtcggaccca ggtcgtgggc tcctattcgg 120
gcatcggcag cgagctcgga gagaggggag atagtattgg agcagaagcc gaagaaggat 180
gacaagaaga agctgcagaa gggaatcgca gagttttacg acgagtcttc tggcttatgg 240
gagaacattt ggggcgacca catgcaccat ggcttttatg actcggattc cactgtttcg 300
ctttcggatc atcgtgctgc tcagatccga atgatccaag agtctcttcg ctttgcctct 360
gtttctgagg agcgtagtaa atggcccaag agtatagttg atgttgggtg tggcataggt 420
ggcagctcta gatacctggc caagaaattt ggagcaacca gtgtaggcat cactctgagt 480
cctgttcaag ctcaaagagc aaatgctctt gctgctgctc aaggattggc tgataaggtt 540
tcctttcagg ttgctgacgc tctacagcaa ccattctctg acggccagtt tgatctggtg 600
tggtccatgg agagtggaga gcatatgcct gacaaagcta agtttgttgg agagttagct 660
cgggtagcag caccaggtgc cactataata atagtaacat ggtgccacag ggatcttggc 720
cctgacgaac aatccttaca tccatgggag caagatctct taaagaagat ttgcgatgca 780
tattacctcc ctgcctggtg ctcaacttct gattatgtta agttgctcca atccctgtca 840
cttcaggaca tcaagtcaga agattggtct cgctttgttg ctccattttg gccagcagtg 900
atacgctcag ccttcacatg gaagggtcta acttcactct tgagcagtgg acaaaaaacg 960
ataaaaggag ctttggctat gccattgatg atagagggat acaagaaaga tctaattaag 1020
tttgccatca ttacatgtcg aaaacctgaa taa 1053
<210>4
<211>350
<212>PRT
<213>大豆属大豆(Glycine max(L.)Merill)
<400>4
Met Ala Thr Val Val Arg Ile Pro Thr Ile Ser Cys Ile His Ile His
1 5 10 15
Thr Phe Arg Ser Gln Ser Pro Arg Thr Phe Ala Arg Ile Arg Val Gly
20 25 30
Pro Arg Ser Trp Ala Pro Ile Arg Ala Ser Ala Ala Ser Ser Glu Arg
35 40 45
Gly Glu Ile Val Leu Glu Gln Lys Pro Lys Lys Asp Asp Lys Lys Lys
50 55 60
Leu Gln Lys Gly Ile Ala Glu Phe Tyr Asp Glu Ser Ser Gly Leu Trp
65 70 75 80
Glu Asn Ile Trp Gly Asp His Met His His Gly Phe Tyr Asp Ser Asp
85 90 95
Ser Thr Val Ser Leu Ser Asp His Arg Ala Ala Gln Ile Arg Met Ile
100 105 110
Gln Glu Ser Leu Arg Phe Ala Ser Val Ser Glu Glu Arg Ser Lys Trp
115 120 125
Pro Lys Ser lle Val Asp Val Gly Cys Gly Ile Gly Gly Ser Ser Arg
130 135 140
Tyr Leu Ala Lys Lys Phe Gly Ala Thr Ser Val Gly Ile Thr Leu Ser
145 150 155 160
Pro Val Gln Ala Gln Arg Ala Asn Ala Leu Ala Ala Ala Gln Gly Leu
165 170 175
Ala Asp Lys Val Ser Phe Gln Val Ala Asp Ala Leu Gln Gln Pro Phe
180 185 190
Ser Asp Gly Gln Phe Asp Leu Val Trp Ser Met Glu Ser Gly Glu His
195 200 205
Met Pro Asp Lys Ala Lys Phe Val Gly Glu Leu Ala Arg Val Ala Ala
210 215 220
Pro Gly Ala Thr Ile Ile Ile Val Thr Trp Cys His Arg Asp Leu Gly
225 230 235 240
Pro Asp Glu Gln Ser Leu His Pro Trp Glu Gln Asp Leu Leu Lys Lys
245 250 255
Ile Cys Asp Ala Tyr Tyr Leu Pro Ala Trp Cys Ser Thr Ser Asp Tyr
260 265 270
Val Lys Leu Leu Gln Ser Leu Ser Leu Gln Asp Ile Lys Ser Glu Asp
275 280 285
Trp Ser Arg Phe Val Ala Pro Phe Trp Pro Ala Val Ile Arg Ser Ala
290 295 300
Phe Thr Trp Lys Gly Leu Thr Ser Leu Leu Ser Ser Gly Gln Lys Thr
305 310 315 320
Ile Lys Gly Ala Leu Ala Met Pro Leu Met Ile Glu Gly Tyr Lys Lys
325 330 335
Asp Leu Ile Lys Phe Ala Ile Ile Thr Cys Arg Lys Pro Glu
340 345 350
Claims (3)
1.SEQ ID NO：3所示的核苷酸序列在提高植物α-生育酚含量中的用途。
2.按照权利要求1所述的用途，其特征在于，包括：构建含有SEQ ID NO：3所示核苷酸序列的植物重组表达载体；将所述植物重组表达载体转化到植物中。
3.SEQ ID NO：4所示的氨基酸序列在提高植物α-生育酚含量中的用途。
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| CN102010874A (zh) * | 2010-09-10 | 2011-04-13 | 兰州大学 | 白沙蒿γ-生育酚甲基转移酶(As-γ-TMT)基因及用途 |
| CN103087999B (zh) * | 2011-11-03 | 2014-09-03 | 中国农业科学院北京畜牧兽医研究所 | 紫花苜蓿γ-生育酚甲基转移酶基因及编码的蛋白和应用 |
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| CN106939308B (zh) * | 2016-01-04 | 2020-06-30 | 深圳市农科集团有限公司 | 转基因玉米事件gt1外源插入片段旁侧序列及其应用 |
| CN107022554B (zh) * | 2017-06-09 | 2020-03-13 | 华中农业大学 | 调控玉米生育酚合成的核苷酸序列及其应用 |
| CN108239654A (zh) * | 2017-12-31 | 2018-07-03 | 青岛袁策生物科技有限公司 | 一种提高水稻籽粒维生素含量的方法 |
| CN113817705B (zh) * | 2021-10-27 | 2023-03-24 | 陕西海斯夫生物工程有限公司 | 一种γ-生育酚甲基转移酶突变体及其在提高α-生育酚及α-生育三烯酚产量的应用 |
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