CN116286900B - 一种乙酸渗透酶A基因RkAcpa及其应用 - Google Patents
一种乙酸渗透酶A基因RkAcpa及其应用 Download PDFInfo
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Abstract
本发明公开了一种乙酸渗透酶A基因RkAcpa，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；该基因分离自红冬孢酵母（Rhodosporidium kratochvilovae）YM25235，将该基因与载体连接，转入红冬孢酵母细胞中，实验结果显示过表达RkAcpa基因会使得该菌株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，为大规模商业化生产类胡萝卜素奠定基础。
Description
技术领域
本发明属于基因工程技术领域，涉及一种乙酸渗透酶A基因RkAcpa及其在提高红冬孢酵母(Rhodosporidium kratochvilovae)类胡萝卜素产量中的应用。
背景技术
乙酸渗透酶A基因在丝状真菌球曲霉---构巢曲霉(Aspergillus nidulans)利用乙酸盐途径中被发现，该基因位于V染色体上，但它与其他五个参与乙酸酯代谢的基因座(fac(AN5626)、acuD(AN5634)、acuG(AN5604)、acuH(AN5336)、acuN(AN5746)并没有密切的联系。Jen1和Ady2是两个负责离子形式的乙酸转运子，Ady2是酿酒酵母中发现的乙酸摄入转运(AceTr)家族的成员之一，该家族蛋白拥有相似的大小以及5～6个预测的跨膜结构，是一类在古细菌、真细菌和一些简单或复杂真核生物中发现的具有保守功能域的一类转运蛋白。Acpa基因与Ady2基因具有高度同源性，因此，Acpa、Jen1、Ady2是同一个家族蛋白。
研究发现，Acpa突变菌株较于野生菌株不可以利用乙酸盐，这与哺乳动物MTC(单羧酸盐转运蛋白)相似。在酿酒酵母中，五种蛋白质显示出与人类MCT转运蛋白的实质相似，但是没有一种蛋白质对羧酸的跨质膜运输起到作用，大多数主要与细胞内结构有关。因此，Acpa是第一个实验支持的丝状真菌MCT的候选蛋白。在酿酒酵母中，已经描述了两种功能性MCTs：一种是由乳酸、乙酸酯、丙酸和丙酮酸共享的高亲和力转运蛋白，另一种是亲和力较低且底物特异性更有限的转运蛋白，因为它只运输乙酸酯、丙酸和甲酸；前者被发现为Jen1(TC-2.A.1.12.2)，是一种膜蛋白，属于主要辅助超家族(TC-2.A.1)，后者取决于酵母Acpa同源物Ady2的完整性。
类胡萝卜素是一类橙黄色、橙红色或红色的多烯类化合物，具有重要的营养价值，β-胡萝卜素、α-胡萝卜素和β-隐黄素是动物体内合成维生素A的前体物质，缺乏维生素A可导致夜盲症、干眼症、角膜溃疡等。
天然色素主要来源于植物和微生物，其中，植物源色素主要采用植物原料直接提取法制备，但植物的生长周期较长使得植物在大规模应用中受限；相对于植物源色素，采用微生物源色素发酵合成法制备，具有工艺绿色、稳定、发酵原料易得、发酵周期短等优势。因此，采用基因工程技术改造发酵菌株制备微生物源类胡萝卜素成为了发酵类胡萝卜的新方法。
目前，关于乙酸渗透酶A基因Acpa在促进微生物生产类胡萝卜素中鲜有报道。
发明内容
本发明提供了一种乙酸渗透酶基因RkAcpa，该基因是从红冬孢酵母(Rhodosporidium kratochvilovae)YM25235中分离得到，其核苷酸序列如SEQ ID NO:1所示，该基因序列长为831bp，编码氨基酸序列如SEQ ID NO:2所示的多肽，将该基因与载体连接，转入红冬孢酵母细胞中，这个基因表达水平的提高促进了红冬孢酵母中类胡萝卜素的合成。
本发明目的通过以下技术方案实现：
1、从红冬孢酵母YM25235中提取总RNA，然后反转录成cDNA，以合成cDNA为模板，采用扩增RkAcpa的特异引物，通过聚合酶链式反应扩增获得目的序列，将载体pRH2034进行双酶切、回收，用一步克隆法将目的片段和载体连接，获得连接产物重组质粒pRHRkAcpa，将重组质粒pRHRkAcpa转入大肠杆菌中，通过PCR筛选出阳性单克隆，重组质粒pRHRkAcpa用BamHⅠ、EcoRⅤ两个限制性内切酶进行酶切验证，验证阳性的克隆子培养后提取质粒，测序，获得片段大小为831bp的乙酸渗透酶基因RkAcpa；
2、利用PEG介导的原生质体法将重组载体pRHRkAcpa转化至红冬孢酵母YM25235中，筛选转化子，获得含有pRHRkAcpa的过表达菌株，含有pRHRkAcpa的过表达菌株经YPA培养基(酵母浸粉1％、蛋白胨2％、乙酸钠2％)培养，提取色素，利用紫外-可见分光光度计测量总类胡萝卜素的含量。
本发明提供了一种生产类胡萝卜的新方法，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，从红冬孢酵母YM25235的提取的总RNA反转录的cDNA中分离得到乙酸渗透酶基因RkAcpa，红冬孢酵母YM25235中RkAcpa基因的过表达引起细胞内这个基因的转录水平的提高，继而翻译成相应蛋白，引起细胞内与类胡萝卜素合成相关的酶的表达量的提高；红冬孢酵母YM25235具有生产周期短、遗传稳定、生产安全等优点，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，为揭示微生物提高类胡萝卜素产量机制提供参考，对类胡萝卜素的工业化生产提供良好的应用前景和经济效益，为大规模商业化生产类胡萝卜素奠定基础；本发明方法简单，易操作，适于工业化生产和市场推广应用。
附图说明
图1为本发明的红冬孢酵母YM25235的RkAcpa基因的PCR扩增图；1.DNA分子量标记DL2000；2.阴性对照；3.基因RkAcpa的cDNA片段；
图2为重组质粒pRHRkAcpa的质粒图谱；
图3为菌落PCR验证电泳图；1.DNA分子量标记DL2000；2.基因RkAcpa的cDNA片段；3-7为转化子；
图4为重组质粒pRHRkAcpa限制性酶切分析；其中：1.DNA分子量标记DL10000；2.阴性对照3.质粒pRH2034的BamHⅠ和EcoR V双酶切；4.重组质粒pRHRkAcpa的BamHⅠ、EcoR V双酶切；5.基因RkAcpa的cDNA片段；6.DNA分子量标记DL2000；
图5为重组质粒pRHRkAcpa转化红冬孢酵母YM25235阳性克隆验证；1.DNA分子标量DL2000；2.阴性对照；3.以YM25235基因组扩增的PCR产物；4.以质粒pRHRkAcpa扩增的PCR产物；5.以YM25235/pRHRkAcpa菌株基因组扩增的PCR产物；
图6为过表达菌株YM25235/pRHRkAcpa与对照菌株YM25235的类胡萝卜素含量比较结果。
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，使用常规方法；
实施例1：从红冬孢酵母YM25235中分离乙酸渗透酶基因RkAcpa及过表达载体pRHRkAcpa的构建
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)提取红冬孢酵母YM25235的总RNA，然后按照Vazyme公司试剂盒(产品编号：R212-02)HiScriptⅡ1st Strand cDNA Synthesis Kit(+gDNAwiper)中的操作说明进行反转录合成cDNA，取1μLcDNA为模板进行聚合酶链式反应，根据转录组测序中发现的RkAcpa序列，设计特异性引物RkAcpa-F和RkAcpa-R，以上述得到的cDNA模板，采用引物RkAcpa-F和RkAcpa-R，在PCR仪上进行PCR扩增，反应所用引物、扩增体系和扩增条件如下：
RkAcpa-F：
(SEQ ID NO:3)(双下划线为上游载体末端同源序列，单下划线为BamHⅠ酶切位点)
RkAcpa-R：(SEQ ID NO:4)(双下划线为下游游载体末端同源序列，单下划线为EcoRV酶切位点)；
PCR扩增体系如下(50μL)：
扩增条件：95℃预变性3min，再用95℃变性15s，62℃退火15s，72℃延伸55s，共32个循环，最后72℃彻底延伸10min；反应后取产物2μL，在浓度为1.5％的琼脂糖凝胶中进行电泳分析，结果如图1所示；扩增得到大小约为800bp的片段，命名为RkAcpa；将pRH2034经BamHⅠ、EcoRⅤ两个限制性内切酶进行双酶切；将以上两个片段用多功能DNA回收试剂盒(北京百泰克生物技术有限公司，产品编号：DP1502)回收，将回收后的两个片段用无缝克隆试剂盒(ClonExpress II One Step Cloning Kit C112，南京诺唯赞生物科技有限公司)进行连接，获得重组质粒pRHRkACPA，连接体系如下(20μL)：
使用移液器轻轻吹打混匀，短暂离心将反应液收集至管底，然后在PCR仪(北京六一生物科技有限公司)37℃反应30min；降至4℃或立即置于冰上冷却。
取10μL获得的连接产物加到100μL DH5α感受态细胞中，轻弹管壁混匀，冰浴30min，42℃水浴热激90s后立即放置在冰上冷却90s，向连接体系中加入900μL LB液体培养基，于37℃、100rpm振荡孵育1h，在5000rpm下离心10min后弃900μL上清，剩余约100μL左右LB培养基轻柔吹打悬浮菌体并涂布LB固体平板(含有100μg/mL壮观霉素)，于37℃倒置培养12～16h，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100μg/mL壮观霉素)中过夜培养，随机挑取平板上生长的5个白色菌落并编号为1-5号，通过菌落PCR进行阳性克隆验证，结果见图3，从图中可以看出挑取的五株单克隆菌株通过菌落PCR均扩增出与目的片段大小相同的特异性条带，说明挑取的五株DH5α菌株中均成功转入重组质粒；提取质粒(Star Prep快速质粒小提试剂盒，北京康润诚业生物科技有限公司)，用BamHⅠ、EcoRⅤ对pRHRkAcpa进行双酶切验证；结果见图4，结果表明，重组质粒pRHRkAcpa经双酶切后产生了850bp和10kb左右的两个条带(图4第4泳道)，这两个条带分别与RkAcpa片段和pRH2034载体经双酶切后的片段大小一致，初步表明重组质粒pRHRkAcpa构建成功；将酶切验证正确的质粒送出测序进一步进行验证，经测序(上海生工生物工程股份有限公司)，测序结果显示所扩增得到的片段大小为831bp，序列组成如SEQID NO:1所示的核苷酸序列，命名为RkAcpa，与RkAcpa基因的cDNA片段大小一致，说明成功构建表达载体pRHRkAcpa，重组载体pRHRkAcpa的质粒图谱见图2。实施例2：RkAcpa基因过表达的红冬孢酵母YM25235中类胡萝卜素含量的分析
1、转化红冬孢酵母YM25235
挑取成功转入正确重组载体pRHRkAcpa的DH5α菌株单克隆接入LB液体培养基(含100μg/mL壮观霉素)中过夜培养，用StarPrep快速质粒小提试剂盒(北京康润诚业生物科技有限公司)提取质粒，并测量浓度，置于-20℃储存备用；挑取红冬孢酵母YM25235单菌落接种于5mL的YPD液体培养基中，于28℃、160rpm振荡培养过夜；将过夜培养的菌液按1％的接种量转接至50mL的YPD液体培养基中28℃、160rpm振荡培养至菌液OD600为0.45，将培养物于4℃、4500rpm离心5min收集菌体；使用事先准备好并放置在冰上的柠檬酸缓冲液(200mL柠檬酸缓冲液含有柠檬酸1.155g、柠檬酸钠4.263g、甘露醇21.909g、NaOH调pH值到5.4)10mL洗涤菌体，于4℃、4500rpm离心5min弃上清，重复该步骤一次，收集菌体并用2mL柠檬酸缓冲液悬浮菌体，置于冰上备用；配制裂解酶液(0.1g蜗牛酶、0.4g溶壁酶，用ddH2O定容至10mL)，用0.22μm无菌滤膜过滤酶液，置于50mL无菌离心管中备用；取4mL酶液与1mL菌液混合后置于28℃、90rpm振荡培养酶解2.5h，将培养物于4℃、1300rpm离心11min收集菌体；用STC(1.2M山梨醇、10mMTris-HCl、100mMCaCl2)于冰上洗涤收集的菌体两次，制成酵母感受态细胞；将酵母感受态细胞按每管100μL分装于5mL无菌离心管中备用；向100μL感受态细胞中加入2.8μg pRHRkAcpa重组质粒并轻轻混匀(通常加入片段体积不应超过10μL)，冰上孵育10min，加入200μL预冷的PTC(50％PEG、10mM Tris-HCl、100mM CaCl2)，冰浴10min，再次加入200μL预冷的PTC冰浴10min，最后加入800μL预冷的PTC，并轻轻混匀，于42℃热激30min，4℃、1500rpm离心11min收集菌体；加入1mL 0.4M蔗糖YPD液体培养基悬浮，28℃、90rpm振荡培养12h复苏菌体；将复苏的菌体于4500rpm离心5min收集菌体，弃上清剩余100μL培养基悬浮菌体，最后涂布于YPD固体培养基(含40μg/mL潮霉素B)上，于28℃倒置培养3d；将涂布后得到的转化子编号，并转接到YPD固体培养基(含150μg/mL潮霉素)上，于28℃倒置培养2d；
以颜色筛选转化子，具体操作为将所得转化子接入5mL YPA液体培养基(酵母粉1％，蛋白胨2％，乙酸钠2％)中，于28℃、160rpm振荡培养120h，以YM25235野生菌株作为对照，观察颜色，筛选出颜色较YM25235更红的转化子；挑取筛选出的转化子，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5所示，从图中可以看出以酵母转化子的基因组为模板通过PCR可扩增出与RkAcpa的cDNA片段大小相同的条带，重组转化子的基因验证正确，说明RkAcpa片段已成功连入酵母的基因组中。
2、RkAcpa基因过表达的红冬孢酵母YM25235中类胡萝卜素含量分析
将含pRHRkAcpa的过表达菌株在YPA液体培养基28℃下培养168h，发酵产物4500rpm离心5min弃上清收集菌体，菌体用蒸馏水悬浮清洗后4500rpm离心5min弃上清收集菌体，将收集到的菌体放置在55℃烘箱中避光烘干，干菌体在研钵中研磨成粉末状，取0.35g干菌体使用丙酮提取2次，收集合并提取液，以原始红冬孢酵母YM25235菌株为对照，利用紫外-可见分光光度计在450nm下测定吸光度，并计算总类胡萝卜素的含量，结果如图6所示；由图可知，过表达菌株YM25235/pRHRkAcpa的总类胡萝卜素合成量较野生型红冬孢酵母YM25235菌株明显提高，野生型红冬孢酵母YM25235菌株的类胡萝卜素合成量为4.11±0.05mg/g，而过表达菌株YM25235/pRHRkAcpa类胡萝卜素合成量为5.07±0.23mg/g，即过表达菌株YM25235/pRHRkAcpa胡萝卜素合成量是对照菌的1.23倍；结果显示在YPA培养基中乙酸渗透酶基因RkAcpa的过表达能引起红冬孢酵母YM25235菌株中总类胡萝卜素含量的增加，RkAcpa基因能够促进总类胡萝卜素的合成。
Claims (2)
1.一种乙酸渗透酶A基因RkAcpa，其核苷酸序列如SEQ ID NO:1所示。
2.权利要求1所述的乙酸渗透酶A基因RkAcpa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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