CN116286899B - 一种NADH激酶基因RkNADHK1及其应用 - Google Patents
一种NADH激酶基因RkNADHK1及其应用 Download PDFInfo
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Abstract
本发明公开了一种分离自红冬孢酵母(Rhodosporidium kratochvilovae)YM25235的NADH激酶基因RkNADHK1，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；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RkNADHK1基因的过表达能够促进红冬孢酵母合成类胡萝卜素；本发明为大规模商业化生产类胡萝卜素提供参考。
Description
技术领域
本发明属于基因工程技术领域，具体涉及一种NADH激酶基因RkNADHK1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
NAD激酶(EC 2.7.1.23)可通过磷酸化供体(ATP和/或无机多磷酸[ploy(P)])催化NAD+生成NADP+，而NADH激酶(EC 2.7.1.86)则可利用ATP和/或[ploy(P)]催化NAD(H)合成NADP(H)，两者构成NADP(H)生物合成的最后一步(McGuinness ET,Butler JR.NAD+kinase--areview.Int J Biochem.1985；17(1):1-11.doi:10.1016/0020-711x(85)90079-5)。在真核模式生物酿酒酵母(Saccharomyces cerevisiae)中存在三种NAD激酶，分别位于胞质的Utr1p、Yef1p和位于线粒体基质的Pos5p，当前的研究显示三者只能利用ATP作为磷酸供体催化NAD(H)合成NADP(H)，均被鉴定为ATP-NADH激酶，对细胞内的NADP(H)的供应起着重要作用(Shi F,Kawai S,Mori S,Kono E,Murata K.Identification of ATP-NADHkinase isozymes and their contribution to supply of NADP(H)in Saccharomycescerevisiae.FEBS J.2005；272(13):3337-3349.doi:10.1111/j.1742-4658.2005.04749.x)。
NADPH(Reduced Nicotinamide Adenine Dinucleotide Phosphate)作为多种生物代谢途径中的重要还原力，是几种高价值物质生物合成的必要辅因子，包括类异戊二烯、脂肪酸和胆固醇等。许多研究显示通过改善细胞内NADPH含量，可有效促进萜类物质的合成，如Zhao等在可产类胡萝卜素的重组酿酒酵母中过表达POS5基因与类胡萝卜素合成途径中的八氢番茄红素脱氢酶基因(CrtI)，通过提高细胞内NADPH的含量，促进了类胡萝卜素合成通路中相关基因的表达，进而使得胞内类胡萝卜素的含量显著提高。
类胡萝卜素(Carotenoids)是一种用途广泛的异戊二烯类(又称萜类)色素，是由植物、真菌、藻类和细菌合成的种类最多的次生代谢物之一。类胡萝卜素存在于细胞膜系统中，与细胞的胁迫应激反应有关，一般呈黄色、橙红色、红色或紫色，具有维生素A原活性、较强的抗氧化性、抗癌能力和调节免疫功能等。因此，类胡萝卜素在医药、保健、食品等领域受到研究者极大的关注。
发明内容
本发明提供了一种从红冬孢酵母(Rhodosporidium kratochvilovae)YM25235中分离获得NADH激酶基因RkNADHK1及其用途，基因RkNADHK1的核苷酸序列如SEQ ID NO:1所示，该基因序列长为2055bp，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，然后反转录合成cDNA，以合成cDNA为模板，采用扩增RkNADHK1基因的特异引物，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pRHRkNADHK1，将重组质粒pRHRkNADHK1转入大肠杆菌中，通过PCR筛选出阳性单克隆，重组质粒pRHRkNADHK1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2055bp的RkNADHK1基因片段；
2、利用PEG介导的原生质体法将重组载体pRHRkNADHK1转化至红冬孢酵母YM25235中，筛选转化子，获得含有pRHRkNADHK1的过表达菌株，含有pRHRkNADHK1的过表达菌株经培养，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。
本发明利用的红冬孢酵母YM25235菌株具有原料成本低、生产周期短、遗传稳定、安全性高等优点，从该菌株中提取的总RNA反转录的cDNA中克隆得到NADH激酶基因RkNADHK1，通过基因工程手段对红冬孢酵母进行改造，红冬孢酵母YM25235中RkNADHK1基因的过表达引起细胞内这个基因转录水平的提高，继而翻译成相应蛋白，引起细胞内NADPH含量的增加，最终使得过表达菌株细胞内类胡萝卜素含量的显著提高。本研究结果有助于阐明红冬孢酵母YM25235中产类胡萝卜素的机制，为揭示微生物提高类胡萝卜素产量的机制提供参考，本发明提供了一种生产类胡萝卜素的新方法，方法简单，操作简便，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，同时为进一步基于辅因子进行的类胡萝卜素高产菌株和工艺化生产类胡萝卜素的研究与应用提供参考。
附图说明
图1为本发明的红冬孢酵母YM25235的RkNADHK1基因的PCR扩增图；1.DNA分子量标记DL5000；2.阴性对照；3.基因RkNADHK1的cDNA片段；
图2为重组质粒pRHRkNADHK1的质粒图谱；
图3为菌落PCR验证电泳图；1.DNA分子量标记DL5000；2.阴性对照；3.基因RkNADHK1的cDNA片段；4-8为转化子；
图4为重组质粒pRHRkNADHK1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EcoRV双酶切；4.重组质粒pRHRkNADHK1的BamHI、EcoRV双酶切；5.基因RkNADHK1的cDNA片段；6.DNA分子量标记DL2000；
图5为重组质粒pRHRkNADHK1转化红冬孢酵母YM25235阳性克隆验证，其中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pRHRkNADHK1扩增的PCR产物；5.以YM25235/pRHRkNADHK1菌株基因组扩增的PCR产物；
图6为过表达菌株YM25235/pRHRkNADHK1与对照菌株YM25235的NADPH含量比较结果；
图7为过表达菌株YM25235/pRHRkNADHK1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中克隆NADH激酶基因RkNADHK1并构建其过表达载体pRHRkNADHK1、转化红冬孢酵母YM25235
1、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)HiScript II 1st Strand cDNA Synthesis Kit(+gDNAwiper)中的操作说明进行反转录合成cDNA，取1μL cDNA为模板进行聚合酶链式反应，根据转录组测序中得到的RkNADHK1序列，设计特异性引物RkNADHK1-F和RkNADHK1-R，以上述得到的cDNA模板，采用引物RkNADHK1-F和RkNADHK1-R，在PCR仪(北京六一生物科技有限公司)上进行PCR扩增，反应所用引物、扩增体系和扩增条件如下：
RkNADHK1-F：5’-ATCACTCACCATGGCGGATCCGATGAGCAGGCAGCTCGG-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)
RkNADHK1-R：5’-CCGGTCGGCATCTACGATATCCTAGTCCCGGTCCGACG-3’(双下划线为下游载体末端同源序列，单下划线为EcoR V酶切位点)；
PCR扩增体系如下(50μL)：Template cDNA 1μL、RkNADHK1-F 2μL、RkNADHK1-R 2μL、dNTPs Mix(10mM each)1μL、Vazyme 2×Phanta Max Buffer 25μL、Vazyme Phanta MaxSuper-Fidelity DNA Polymerase 1μL，ddH2O加至50μL；
扩增条件：95℃预变性3min，再用95℃变性15s，63℃退火15s，72℃延伸2min5s，共30个循环，最后72℃彻底延伸5min；反应后取产物1μL，在浓度1％的琼脂糖凝胶中进行电泳分析，结果如图1所示；扩增得到大小约为2055bp的片段，命名为RkNADHK1；将pRH2034经BamHⅠ、EcoR V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pRHRkNADHK1，连接体系如下(20μL)：ExnaseTMⅡ2μL、RkNADHK1片段82.2ng、线性化pRH2034 200ng、5×CEⅡBuffer 4μL、其余为ddH2O；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。
2、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100μL左右LB培养基轻柔吹打悬浮菌体并涂布于LB固体平板(含有100μg/mL壮观霉素)，于37℃倒置培养12-16h，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；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，收集菌体并提取质粒(OMEGAPlasmid Mini Kit I试剂盒，美国OMEGA公司)，用BamHⅠ、EcoR V对pRHRkNADHK1进行双酶切验证，结果见图4，结果表明，重组质粒pRHRkNADHK1经双酶切后产生了2kb和10kb左右的两个条带(图4第4泳道)，这两个条带分别与RkNADHK1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pRHRkNADHK1构建成功，重组载体pRHRkNADHK1的质粒图谱见图2；将酶切验证正确的质粒送出测序进一步进行验证，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2055bp，与转录组序列一致，核苷酸序列如SEQ ID NO:1所示，命名为RkNADHK1。
3、挑取成功转入正确重组载体pRHRkNADHK1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，提取质粒(OMEGAPlasmid Mini Kit I试剂盒，美国OMEGA公司)，测量浓度，并置于-20℃储存备用。利用PEG介导的原生质体转化法将重组载体pRHRkNADHK1转化至红冬孢酵母YM25235中，具体方法如下：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，将其作为种子液；将种子液按1％的接种量转接至50mL的YPD液体培养基中，于28℃、160rpm振荡培养至菌液OD600为0.45～0.5之间，将菌液于4℃、4500rpm离心5min收集菌体；收集的菌体用事先准备好的柠檬酸缓冲液(30mmol/L柠檬酸、83mmol/L柠檬酸钠、600mmol/L甘露醇、NaOH调pH值到5.4)洗涤两次，继续用800-1000μL柠檬酸缓冲液悬浮菌体，置于冰上备用；配制酶解液(0.075g蜗牛酶、0.03gSigma溶壁酶、0.03g广东微生物溶壁酶，用柠檬酸缓冲液定容至5mL)，并用0.22μm无菌滤膜过滤，置于5mL无菌离心管中备用；取4mL酶液与800-1000μL柠檬酸缓冲液悬浮的菌体混合后置于28℃、90rpm振荡培养2.5-3h进行酶解，取少许菌液用显微镜观察酶解效率，在确认酶解数量足够多的情况下，将培养物于4℃、1300rpm离心10min收集菌体；加入10mL STC缓冲液(1.2mol/L山梨醇、10mmol/L Tris-HCl、100mmol/L CaCl2)于冰上洗涤收集的菌体两次，制成酵母感受态细胞；用800-1000μL的STC缓冲液悬浮菌体，按每管100μL分装到5mL无菌离心管中备用；向100μL感受态细胞中加入10μL、浓度为180.38μg/mL的pRHRkNADHK1重组质粒并轻轻混匀，冰上孵育10min，加入200μL预冷的PTC缓冲液(50％PEG、10mmol/L Tris-HCl、100mmol/L CaCl2)，冰浴10min，再次加入200μL预冷的PTC缓冲液，冰浴10min，最后加入800μL预冷的PTC缓冲液，并轻轻混匀，于42℃水浴30min，水浴结束后4℃、1500rpm离心10min收集菌体，弃去1mL上清，加入1mL 0.4mol/L蔗糖YPD液体培养基悬浮，28℃、90rpm振荡培养24h复苏菌体；将复苏的菌体于1300rpm离心10min收集菌体，弃上清剩余100μL培养基悬浮菌体，最后涂布于添加0.4mol/L蔗糖的YPD固体培养基(含40μg/mL潮霉素B)上，于28℃倒置培养3-4天待固体培养基上长出，将转化子编号，并转接到含150μg/mL潮霉素B的YPD固体培养基上，于28℃倒置培养两天；根据该基因的已知功能及红冬孢酵母YM25235的特性，以颜色作为过表达菌株筛选的依据，具体操作为将所得转化子接菌于5mL含YPD液体培养基的试管中，于28℃、160rpm振荡培养120h，以YM25235野生菌株作为对照，观察颜色，筛选出颜色比YM25235较红的转化子；挑取筛选出的转化子，然后按照DNA提取试剂盒(购买于上海生工生物工程股份有限公司)说明书中的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，从图中可以看出以转化子的基因组为模板通过PCR可扩增出与RkNADHK1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RkNADHK1片段已成功导入酵母转化子的基因组中。
实施例2：过表达菌株YM25235/pRHRkNADHK1的NADPH和类胡萝卜素含量分析
将阳性转化子接种于50mL YPD液体培养基中，28℃、160rpm发酵培养48h后，4500rpm离心6min收集菌体于2mL离心管中，用预冷的ddH2O洗涤两次，最后12000rpm离心5min彻底去除残余水分，随后立即用液氮速冻，存于-80℃。分析时取出，采用冰浴研磨破细胞壁，按照辅酶ⅡNADP(H)含量检测试剂盒(CoenzymeⅡNADP(H)Content Assay KitBC1100，北京索莱宝科技有限公司)说明书对胞内的NADPH进行提取与含量测定。测定结果显示，野生型红冬孢酵母YM25235菌株的NADPH含量为3.72±0.129nmol/g-wet，而过表达菌株YM25235/pRHRkNADHK1的NADPH含量为4.56±0.196mg/g-wet，过表达菌株YM25235/pRHRkNADHK1的胞内NADPH含量是对照组的3.33倍，较野生型红冬孢酵母YM25235菌株明显提高，如图6所示；
将阳性转化子接种于50mL YPD液体培养基中，28℃、160rpm发酵培养168h后，4500rpm离心6min收集菌体于50mL离心管中，用预冷的ddH2O洗涤两次，最后4500rpm离心8min彻底去除上清，轻轻敲击管壁使得菌体均匀黏附在管内壁上，置于烘箱中55℃烘干后把菌体研磨成粉，取0.4g菌粉使用丙酮-甲醇混合液(V(丙酮)：V(甲醇)＝4:1)提取总类胡萝卜素，参照杨万政等的“紫外分光光度法测定沙棘油中总类胡萝卜素方法改进[J].中央民族大学学报(自然科学版),2009,18(03):5-8.”中的方法，利用紫外-可见分光光度计，以野生型红冬孢酵母YM25235菌株为对照，在450nm下测定吸光度并计算总类胡萝卜素的含量，其中野生型红冬孢酵母YM25235菌株的总类胡萝卜素合成量为5.36±0.24mg/g-DCW，而过表达菌株YM25235/pRHRkNADHK1类胡萝卜素合成量为6.57±0.11mg/g-DCW，过表达菌株YM25235/pRHRkNADHK1总胡萝卜素合成量是对照菌的1.23倍，较野生型红冬孢酵母YM25235菌株明显提高，如图7所示。
结果显示NADH激酶基因RkNADHK1的过表达能引起红冬孢酵母YM25235菌株中NADPH和总类胡萝卜素含量的增加，RkNADHK1基因能够促进总类胡萝卜素的合成。
Claims (2)
1.一种NADH激酶基因RkNADHK1，其核苷酸序列如SEQ ID NO:1所示。
2.权利要求1所述的NADH激酶基因RkNADHK1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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