CN116286880B - 一种过氧化物酶体增值因子基因RkPEX11及其用途 - Google Patents
一种过氧化物酶体增值因子基因RkPEX11及其用途 Download PDFInfo
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Abstract
本发明公开了一种过氧化物酶体增值因子基因RkPEX11，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，该基因是红冬孢酵母（Rhodosporidium kratochvilovae）YM25235中获得的，将该基因通过转化转入到红冬孢酵母YM25235中，实验结果显示RkPEX11基因的过表达会促进YM25235菌株中类胡萝卜素合成水平的提高；本发明通过基因工程手段对微生物进行改造，来提高微生物体内类胡萝卜素的产量，为类胡萝卜素大规模商业化生产奠定基础。
Description
技术领域
本发明属于生物技术领域和遗传工程领域，涉及一种过氧化物酶体增值因子基因RkPEX11，具体涉及从红冬孢酵母（Rhodosporidium kratochvilovae）YM25235中克隆的过氧化物酶体增值因子基因RkPEX11以及将该基因直接与不同载体连接，转入酵母细胞，提高该基因在酵母里的表达水平并最终促进类胡萝卜素的合成。
背景技术
过氧化物酶体增值因子PEX11是一种过氧化物酶体扩增蛋白（即参与过氧化物酶体生物发生或组织的蛋白质），其活性是过氧化物酶体增殖期间单个过氧化物酶体膜的管状和裂变所必需的。它是过氧化物酶体增殖非诱导条件下表达最高的过氧化物酶，也是最丰富的过氧化物酶体膜蛋白。PEX11被证明它招募了线粒体裂变机制，该机制也负责新形成的过氧化物酶体的裂变。酿酒酵母的PEX11已被证明可以募集线粒体裂变机制到过氧化物酶体中，从而使过氧化物酶体的增殖成为可能，这个过程对于有效的脂肪酸β氧化至关重要。
类胡萝卜素（carotenoid）是所有光合生物及一些非光合原核生物和真菌合成的一类重要的天然色素的总称，属于类萜化合物，普遍存在于动物、高等植物、真菌、藻类中。典型的类胡萝卜素是由8个异戊二烯单元首尾相连而成的C40萜类化合物及其衍生物，其结构中的共轭双键的作用导致其各种类胡萝卜素的颜色差异。天然类胡萝卜素对于生物体具有十分广泛的作用。类胡萝卜素是人体内维生素A的主要来源，同时还具有抗氧化、免疫调节、抗癌、延缓衰老等功效，对视觉系统有保健作用，可预防夜盲症、干眼症、角膜溃疡症等病症；对皮肤组织的保健，维生素A是维持一切上皮组织完整所必需的，而β-胡萝卜能在人体内转化成维生素A，维持正常的体表、消化道、呼吸道、生殖泌尿道有重要意义，可避免皮肤多屑、角质化、表皮细胞硬鳞状、多角质血疹性皮肤干燥症等皮肤疾病。β-胡萝卜素对细胞膜的稳定性也具有良好的作用，抵抗不良环境等。在工业上类胡萝卜素被用作食品着色剂、营养强化剂和抗氧化剂等。叶黄素可作为食品着色剂用于面条、色拉调味品等食品中；在一些调味品中，利用番茄红素具有超强的抗氧化活性，代替亚硝酸盐防止食物腐败变质，有效的延长保质期。
随着人们对天然类胡萝卜素制品的需求量的增加，天然类胡萝卜素的产量与质量已无法满足市场需求。目前，类胡萝卜素的生产方法主要包括植物提取法、化学合成法和生物合成法。在食品工业中，合成化合物带来了许多负面影响，合成食品安全问题越来越受到社会的关注。而且由于存在生物活性低且生成色素有一定的毒副作用，已被许多国家和地区限制使用；植物提取法由于植物中类胡萝卜素含量较低，提取成本比较高、工艺又复杂等原因，使用也较少；而生物合成法提取的类胡萝卜素是纯天然的，对人体安全无毒副作用，因此用生物合成法提取类胡萝卜素是目前最具发展潜力的一种提取方法。目前来说，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，其中利用酵母发酵生产类胡萝卜素的研究最为常见，且酵母属于我国饲料行业中确认的饲用微生物。因此，以酵母为生产菌种用微生物发酵法来生产天然类胡萝卜素从而满足人们的需求是一条十分可行的道路。
在发酵工艺中，提高类胡萝卜素合成底物-乙酰辅酶A是促进类胡萝卜素合成的有效方法。因为红酵母具有营养要求简单、生产周期短等许多优点，被大量用于研究生产类胡萝卜素，可用于生产类胡萝卜素的红酵母主要集中于胶红酵母、粘红酵母、深红酵母、法夫红酵母、小红酵母五种。通过诱变改良菌种、发酵工艺优化等手段使得红酵母的类胡萝卜素产量进行提升一直受到研究者的广泛关注。
目前尚未见过氧化物酶体增值因子基因在促进酵母菌生产类胡萝卜素的相关报道。
发明内容
本发明提供了一种过氧化物酶体增值因子基因RkPEX11，该基因是从红冬孢酵母（Rhodosporidium kratochvilovae）YM25235中分离得到，该基因核苷酸序列如SEQ ID NO:1所示或为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735bp（碱基），该基因编码的氨基酸序列如SEQ ID NO:2所示的多肽或其片段。
本发明目的通过以下技术方案实现:
1、从红冬孢酵母（Rhodosporidium kratochvilovae）YM25235中扩增获得过氧化物酶体增值因子基因RkPEX11；
2、过氧化物酶体增值因子基因RkPEX11连接到表达载体 pRH2034上获得重组过表达质粒；
3、将重组过表达质粒pRHRkPEX11转化至红冬孢酵母YM25235中用于生产类胡萝卜素。
本发明的优点和技术效果：
本发明从红冬孢酵母（Rhodosporidium kratochvilovae）YM25235的总RNA基因中获得的过氧化物酶体增值因子基因RkPEX11在红冬孢酵母YM25235中过表达，会引起细胞内这个基因的转录水平的提高，说明外源基因在菌体内发生转录，继而翻译成相应蛋白，使得酵母内的过氧化物酶体增值，进而促进了脂肪酸的β氧化，产生大量的乙酰辅酶A，引起细胞内与类胡箩卜素合成相关的酶的表达量的提高。本研究结果有助于阐明红冬孢酵母YM25235中产类胡萝卜素机制，有助于通过基因工程手段对其进行改造来提高类胡萝卜素含量，对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，为生物合成法生产类胡萝卜素提供理论支持。
附图说明
图1为本发明的红冬孢酵母YM25235的RkPEX11基因PCR扩增图；
图2为重组质粒pRHRkPEX112的质粒图谱；
图3为重组质粒pRHRkPEX11转化红冬孢酵母YM25235的PCR验证；其中1、DNA 分子标量DL2000；2、空白对照组（未添加模板）； 3、以YM25235基因组为模板的PCR产物(含有内含子)；4、以质粒pRHRkPEX11为模板的PCR产物，5、以YM25235/pRHRkPEX11-11菌株基因组为模板的PCR产物；
图4为过表达菌株YM25235/pRHRkPEX11与对照菌株YM25235/pRH2034的总类胡萝卜素含量。
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（Rhodosporidium kratochvilovae）YM25235中分离获取过氧化物酶体增值因子基因RkPEX11
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 ，然后采用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 cDNA为模板进行聚合酶链式反应，根据转录组测序中发现的RkPEX11序列，设计特异性引物RkPEX11-F和RkPEX11-R，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：
RkPEX11-F：5’-ATCACTCACCATGGCGGATCCGATGACCGTCGTTCACCAGC-3’
RkPEX11-R：5’-CCGGTCGGCATCTACGATATCCTACTTGCTGGCGCCGAG-3’ ；
（GATCCG为BamH I酶切位点，GATATC为EcoR V酶切位点）；
PCR扩增体系如下（50μL）：
扩增条件为：94℃预变性5min，再用94℃变性30s，58℃退火30s，72℃延伸1min，进行30个循环，最后72℃彻底延伸10min，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750bp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，连接产物转化到感受态大肠杆菌DH5α，在含氨苄青霉素（100µg/mL）的LB固体平板上过夜培养，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100µg/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，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735bp，将获得的片段命名为RkPEX11，核苷酸序列如SEQ ID NO:1所示。
实施例2：过表达载体pRHRkPEX11的构建
以反转录的YM25235 cDNA 为模板，用RkPEX11-F 和RkPEX11-R 作为引物扩增RkPEX11的编码序列，获得的RkPEX11片段大小约735bp，将扩增获得的RkPEX11片段与被BamH I、EcoRⅤ两个限制性内切酶进行酶切后的载体连接，连接到表达载体 pRH2034上获得重组质粒pRHRkPEX11（图2）。将获得的重组质粒转入感受态大肠杆菌 DH5α中扩增，经菌落 PCR 验证后提取重组质粒；用测序引物进行测序。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：RkPEX11基因与红冬孢酵母中类胡萝卜素合成关系的分析
1、转化红冬孢酵母YM25235
利用农杆菌介导法将重组质粒pRHRKPEX11转化至红冬孢酵母YM25235中，以含潮霉素B（终浓度为150µg/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，结果如图3所示。
2、红冬孢酵母中类胡萝卜素含量检测
将含pRHRkPEX11的过表达菌株在28℃条件下培养168 h后，提取类胡萝卜素，并以转入空质粒pRH2034的红冬孢酵母菌株为对照，紫外-可见分光光度计在445nm下测定总类胡萝卜素的含量（mg/g干菌体），结果如图4所示；结果表明，过表达菌株YM25235/pRHRkPEX11的总类胡萝卜素合成量相较于含空质粒pRH2034的对照菌株有着明显提高，含空质粒pRH2034的对照菌株的类胡萝卜素合成量为4.99mg/g，而过表达菌株YM25235/pRHRkPEX11类胡萝卜素合成量为9.88mg/g，即过表达菌株YM25235/pRHRkPEX11类胡萝卜素合成量是对照菌的1.97倍，证明了RkPEX11基因能够促进总类胡萝卜素的合成。
Claims (2)
1.一种过氧化物酶体增值因子基因RkPEX11，其核苷酸序列如SEQ ID NO:1所示。
2.权利要求1所述的过氧化物酶体增值因子基因RkPEX11在促进红冬孢酵母（Rhodosporidium kratochvilovae）生产类胡萝卜素中的应用。
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