CN109536518A - 一种八氢番茄红素脱氢酶基因RKcrtI及其应用 - Google Patents
一种八氢番茄红素脱氢酶基因RKcrtI及其应用 Download PDFInfo
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Abstract
本发明公开了一种八氢番茄红素脱氢酶基因RKcrtI，其核苷酸序列如SEQ ID NO：1所示，该基因编码的氨基酸序列如SEQ ID NO：2所示；该基因来源于红冬胞酵母YM25235，将该基因与载体连接，转入酵母细胞；其能够促进红冬孢酵母YM25235产β‑类胡萝卜素，为大规模商业化生产β‑类胡萝卜素奠定基础。
Description
技术领域
本发明属于生物技术领域和遗传工程领域，涉及一种八氢番茄红素脱氢酶基因RKcrtI，其是从红冬孢酵母（Rhodosporidium kratochvilovae）YM25235中克隆的基因，将该基因直接与载体连接，转入酵母细胞，来提高这个基因的表达水平并最终促进类胡萝卜素的合成。
背景技术
类胡萝卜素作为一类具有重要功能的脂溶性色素，广泛分布于自然界的动物、植物、真菌、藻类和细菌中，其营养价值高，具有抗癌、抗氧化、抗心脑血管疾病等功效。因此，已被广泛应用到食品、饲料、保健品及化妆品等行业。尽管许多研究者已经对如何提高类胡萝卜素的产量进行了大量的研究，但类胡萝卜素的产量和质量仍远远不能满足人们的需求。目前，类胡萝卜素的生产方法包括植物提取法、化学合成法和生物转化法。化学合成法生产类胡萝卜素虽然成本小，但是生物活性低，另因其毒副作用，已被限制使用；植物提取法因植物中类胡萝卜素含量很低，提取成本比较高，也是很少使用；而生物合成法提取的类胡萝卜素是纯天然的，对人体无毒无害，因此用生物合成法提取类胡萝卜素是目前最具发展潜力，最受欢迎的一种提取方法。生物合成法提取类胡萝卜素的途径很多，其中最常用的有两种：一是用培养盐藻来提取类胡萝卜素，这种方法需要在高盐份的海域环境，所以只能在少数地区养殖，生产的局限性很非常大，不宜大范围推广；二是用微生物发酵法生产类胡萝卜素，目前能够发酵产生类胡萝卜素的微生物主要有真菌、细菌和酵母菌等。
具有合成类胡萝卜素功能的微生物中，存在四个关键基因，它们分别编码一种类胡萝卜素合成关键酶。这四种关键酶分别是牻牛儿基牻牛儿基焦磷酸合成酶（GGPPs）、八氢番茄红素合成酶、八氢番茄红素脱氢酶、番茄红素环化酶。牻牛儿基牻牛儿基焦磷酸合成酶(GGPPS)是萜类物质合成的一个重要分支点酶，是合成番茄红素及其他类胡萝卜素最直接的前体，人们已从多种植物中分离出GGPPS基因；这一类基因最初是在绿薄荷中发现的；八氢番茄红素合成酶(PSY)是催化类胡萝卜素生物合成的一个特异性酶，它将2分子的牻牛儿基牻牛儿基焦磷酸 (GGPP)催化合成八氢番茄红素，因此它也被认为是类胡萝卜素代谢通路中最重要的酶。PSYl和PSY2分别催化番茄果实和叶片中八氢番茄红素的合成。人们利用分子生物学方法及转基因技术，将八氢番茄红素合成酶的cDNA转入到番茄中，组成性地过量表达八氢番茄红素合成酶，结果如预料般地提高了番茄中的类胡萝卜素的含量；八氢番茄红素脱氢酶(phytoene dehydrogenase)由PDS和ZDS两种酶活性构成，能催化八氢番茄红素向番茄红素转化。
目前，未见与本发明有关的基因序列的公开。
发明内容
本发明目的是提供一种八氢番茄红素脱氢酶基因RKcrtI的，其是从红冬孢酵母（Rhodosporidium kratochvilovae）YM25235中分离得到，该基因核苷酸序列如SEQ ID NO:1所示或该核苷酸序列的片段，或与SEQ ID NO:1互补的核苷酸序列，该基因序列长为1656bp（碱基），该基因编码的氨基酸序列如SEQ ID NO:2所示。
本发明另一目的是提供一种含有八氢番茄红素脱氢酶基因RKcrtI的重组表达载体，是将SEQ ID NO:1所示基因直接与不同表达载体（质粒、病毒或运载体）连接所构建的重组载体。可用本领域技术人员熟知的方法来构建含编码红冬孢酵母YM25235 类胡萝卜素合成关键酶RKcrtI的核苷酸序列和合适的转录/翻译调控元件的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的编码红冬孢酵母YM25235 类胡萝卜素合成关键酶RKcrtI的核苷酸序列可有效连接到表达载体的恰当启动子上，以指导mRNA合成。这些启动子的代表性例子有：大肠杆菌的lac或trp启动子；λ噬菌体的PL启动子；真核启动子包括CMV早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、反转录病毒的LTRs和其它一些已知的可控制基因在原核细胞或真核细胞或其病毒中表达的启动子；表达载体还包括翻译起始用的核糖体结合位点和转录终止子等；在载体中插入增强子序列将会使其在高等真核细胞中的转录得到增强；增强子是DNA表达的顺式作用因子，通常大约有10-300bp，作用于启动子以增强基因的转录，如腺病毒增强子。
本发明另一目的是将上述八氢番茄红素脱氢酶基因RKcrtI应用在生产β-类胡萝卜素中。
本发明从红冬孢酵母（Rhodosporidiumkratochvilovae）YM25235的总RNA基因中分离得到潜在的与红冬孢酵母产生类胡萝卜素合成相关的八氢番茄红素脱氢酶基因序列RKcrtI，该基因全长1656bp；红冬孢酵母YM25235中RKcrtI基因的过表达会引起细胞内这个基因的转录水平提高，继而翻译成更多的相应酶蛋白，引起菌株中β-类胡萝卜素产量的提高。本研究结果有助于阐明红冬孢酵母YM25235中产β-类胡萝卜素机制，为揭示微生物提高β-类胡萝卜素产量机制提供参考，将有助于通过基因工程手段对其进行改造来提高β-类胡萝卜素含量，对β-类胡萝卜素的工业化生产提供有良好的应用前景和经济效益，为大规模商业化生产β-类胡萝卜素奠定基础。
附图说明
图1为本发明的红冬孢酵母YM25235的RKcrtI基因PCR扩增图；
图2为重组质粒pRHRKcrtI的质粒图谱；
图3为重组质粒pRHRKcrtI限制性酶切分析；其中：1、DNA 分子量标记DL10000；2、空质粒pRH2304的BamHⅠ、EcoRⅤ双酶切；3、重组质粒pRHRKcrtI的BamHⅠ、EcoRⅤ双酶切；4、RKcrtI基因的PCR 产物；5、DNA 分子量标记DL2000；
图4 重组质粒pRHRKcrtI转化红冬孢酵母YM25235阳性克隆验证；1、DNA分子量标记DL5000；2、野生型菌株特异性基因条带；3、重组菌株特异性基因条带；4、特异性基因的cDNA条带；5、DNA分子量标记DL2000；
图5为RKcrtI基因过表达的红冬孢酵母YM25235产类胡萝卜素的高效液相色谱分析A:YM25235/pRH2304；B: YM25235/pRHRKcrtI。
图6为两株转基因菌株中β-类胡萝卜素含量结果对比图。
具体实施方式
下面结合附图和实施例对本发明作进一步详细说明，但本发明保护范围不局限于所述内容，实施例中使用的试剂和方法，如无特殊说明，均采用常规试剂和使用常规方法。
实施例1：从红冬孢酵母（Rhodosporidiumkratochvilovae）YM25235中分离类胡萝卜素合成相关酶-八氢番茄红素脱氢酶RKcrtI的核苷酸序列
采用生工生物工程（上海）股份有限公司的UNlQ-10 柱式Trizol总RNA抽提试剂盒（产品编号: SK1321）提红冬孢酵母YM25235的总RNA ，然后按照TaKaRa公司试剂盒PrimeScript ®RT reagent Kit With gDNA Eraser(Perfect Real Time)进行反转录合成cDNA ，取0.5μL为模板进行聚合酶链式反应，根据转录组测序中发现的RKcrtI序列，设计特异性引物RKcrtI-F和RKcrtI-R（引物1和引物2），以上述得到的cDNA模板，在PCR仪（BIOER公司）上进行PCR扩增，反应所用引物、扩增体系和扩增条件如下：
引物1：RKcrtI-F：5`-CGGGATCCATGAACGGCCACGCCAAG-3`（SEQ ID NO:3）
引物2：RKcrtI-R：5`-CTCGATATCCTACTGCTTGAGGTACAC-3`（SEQ ID NO:4）
（GGATCC为BamH I酶切位点，GATATC为EcoRV酶切位点）；
PCR扩增体系如下（50 μL）：
扩增条件：95℃预变性5 min，再用95℃变性30 s，60℃退火30 s，72℃延伸90 s进行30个循环，最后72℃彻底延伸5 min，反应完后取产物2μL，然后在浓度为1%的琼脂糖凝胶中,进行电泳分析，结果如图1所示，扩增得到大小约1600bp的片段，用琼脂糖凝胶DNA回收试剂盒（北京索莱宝科技有限公司）回收，把回收片段连接至到pMD-18T（TaKaRa公司产品）中，连接产物转化到用CaCl2法处理的大肠杆菌DH5α，在含氨苄青霉素（100 µg/mL）的LB固体平板上过夜培养，挑取平板上生长的白色菌落，通过菌落PCR验证阳性克隆。将验证阳性的克隆子接入LB液体培养基（含100 µg/mL氨苄青霉素）中过夜培养，用高纯质粒小量制备试剂盒（离心柱型）（北京百泰克生物技术有限公司）提取质粒，经测序（昆明硕擎生物科技有限公司），测序结果显示所扩增得到的片段大小为1656bp，命名为RKcrtI，序列组成如SEQ IDNO:1所示的核苷酸序列。
实施例2：RKcrtI基因过表达载体pRHRKcrtI的构建
以反转录的YM25235 cDNA 为模板，用RKcrtI-F 和RKcrtI-R 作为引物扩增RKcrtI的编码序列，获得的RKcrtI片段大小约1600bp，将扩增获得的RKcrtI片段经BamHⅠ、EcoRⅤ两个限制性内切酶进行酶切后，连接到表达载体 pRH2304上获得重组质粒pRHRKcrtI（图2）；将获得的重组质粒转入大肠杆菌 DH5α中扩增，再经菌落 PCR 验证后提取重组质粒，并用BamHⅠ、EcoRⅤ对pRHRKcrtI进行双酶切验证；结果表明，重组质粒pRHRKcrtI经双酶切后产生了1.6 kb 和10.7 kb左右的两个条带（图 3 第3泳道），这两个条带分别与RKcrtI片段和pRH2304载体经双酶切后的片段大小一致，初步表明重组质粒pRHRKcrtI构建成功；用测序引物进行测序，将酶切验证正确的质粒送出测序进一步进行验证；测序结果表明，测序所获得的序列与目的序列完全一致，没有出现任何碱基突变和缺失等。
实施例3：RKcrtI基因过表达对红冬孢酵母YM25235中β-胡萝卜素合成的影响
1、农杆菌介导转化红冬孢酵母YM25235
利用农杆菌介导法将重组质粒pRHRKcrtI转化至红冬孢酵母YM25235中，以含潮霉素B（HygromycinB）终浓度为150 µg/mL的 YPD 培养基筛选转化子，然后按照上海生工生物工程股份有限公司DNA 提取试剂盒说明书中步骤提取酵母转化子的基因组 DNA，后进行PCR验证如图4所示。从图4可知RKcrtI序列已经整合到YM25235基因组中，泳道3可以看出PCR扩增可以获得两条带，一条是菌株自身包含内含子序列的基因组片段，一条带是转化整合的cDNA编码序列片段，未转化RKcrtI基因的对照菌株YM25235中只扩增得到包含内含子序列的基因组片段（泳道2）。
2、RKcrtI基因过表达与红冬孢酵母类胡萝卜素含量变化的分析
将含pRHRKcrtI的过表达菌株在28℃条件下培养，提取类胡萝卜素，并测定其中β-胡萝卜素的含量，以转入空质粒pRH2304的红冬孢酵母菌株为对照，进行高压液相分析，液相图谱如图5、6所示，图5A图是含空质粒pRH2304菌株总类胡萝卜素样品液相图谱；图5B是过表达菌株pRHRKcrtI总类胡萝卜素样品液相图谱；该菌株YM25235能合成β-胡萝卜素、红酵母素、圆酵母素等类胡萝卜素，由图可看出，其中过表达菌株YM25235/pRHRKcrtI的β-胡萝卜素（如图5中箭头所示）合成量比含空质粒pRH2304的对照菌株有明显提高，过表达菌株YM25235/pRHRKcrtI中β-类胡萝卜素合成量是对照菌的2.57倍；结果显示RKcrtI基因过表达能够促进红冬孢酵母YM25235中终产物β-胡萝卜素的合成，即RKcrtI核酸序列确实与红冬孢酵母中β-类胡萝卜素合成有关。
序列表
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Claims (3)
1.一种八氢番茄红素脱氢酶基因RKcrtI，其核苷酸序列如SEQ ID NO:1所示，该基因编码的氨基酸序列如SEQ ID NO:2所示。
2.一种含有权利要求1所述八氢番茄红素脱氢酶基因RKcrtI的重组表达载体。
3.权利要求1所述的八氢番茄红素脱氢酶基因RKcrtI在生产β-类胡萝卜素中的应用。
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