CN115851779A - 一种葡萄糖-6-磷酸脱氢酶基因RkZWF1及其应用 - Google Patents
一种葡萄糖-6-磷酸脱氢酶基因RkZWF1及其应用 Download PDFInfo
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Abstract
本发明公开了一种葡萄糖‑6‑磷酸脱氢酶基因RkZWF1,分离自红冬孢酵母(Rhodosporidium kratochvilovae)YM25235,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RkZWF1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,具体涉及一种葡萄糖-6-磷酸脱氢酶基因RkZWF1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
葡萄糖-6-磷酸脱氢酶(Glucose-6-phosphate dehydrogenase,G6PDH),是磷酸戊糖途径氧化分支的主要调节酶,催化6-磷酸葡糖氧化脱氢生成6-磷酸葡糖酸内酯,同时将NADP+还原为NADPH。G6PDH是由ZWF1基因编码的一个二聚体,每个亚基由两个结构域组成,较小的结构域是一个经典的罗斯曼折叠,参与底物结合,而较大的结构域是一个包含九条反向平行的β-折叠链的β+α结构域,与亚基的聚合有关,该结构突显的保守氨基酸残基包括His-240(从6-磷酸葡糖的C1-OH提取质子)、Asp-177(其羧酸盐在过渡态稳定了在His-240上形成的正电荷)、His178(与6-磷酸葡糖的磷酸部分结合)(Cosgrove MS, Naylor C,Paludan S, Adams MJ, Levy HR. On the mechanism of the reaction catalyzed byglucose 6-phosphate dehydrogenase. Biochemistry. 1998 Mar;37(9) 2759-2767.)。
G6PDH存在于所有类型细胞的胞质中,其主要作用是为细胞内的诸多生化代谢反应提供还原力,支持还原性物质如脂肪酸、类胡萝卜素和胆固醇等的生物合成,同时还通过促进谷胱甘肽循环参与酶促氧化应激进而在保护细胞中起到重要作用。通过磷酸戊糖途径,G6PDH还能够为嘌呤、嘧啶、ATP和CoA等的合成提供前体物质,G6PDH在细胞的生理以及在生物体的整个代谢调控中发挥重要作用。(Park J, Rho HK, Kim KH, Choe SS, LeeYS, Kim JB. Overexpression of glucose-6-phosphate dehydrogenase is associatedwith lipid dysregulation and insulin resistance in obesity. Mol Cell Biol.2005 Jun;25(12):5146-57.)。G6PDH缺乏症是人类最常见的遗传性细胞酶病,G6PDH活性降低,可导致细胞生长和信号传递失调、胚胎发育异常、对病毒易感以及促进退行性疾病的发生。G6PDH与植物的生长发育同样具有紧密的关系,相关研究显示G6PDH的活性在休眠芽中高于萌芽期和恢复生长期;G6PDH参与植物响应外界的生物与非生物胁迫,在高低温、干旱、病毒感染和盐胁迫等方面均有报道,如Scharte J等的研究发现,在抗性烟草源叶中,通过抑制葡萄糖-6-磷酸脱氢酶(G6PDH)或NADPH氧化酶,可以防止烟草疫霉侵染后氧化爆裂和随后形成的过敏性病变。这一观察结果表明,由于细胞质中G6PDH活性的增加,NADPH有效性的提高可能有利于植物的防御。(Scharte J, Schön H, Tjaden Z, Weis E, vonSchaewen A. Isoenzyme replacement of glucose-6-phosphate dehydrogenase in thecytosol improves stress tolerance in plants. Proc Natl Acad Sci U S A. 2009May 12;106(19):8061-6.)。
类胡萝卜素(Carotenoids)是一类脂溶性色素,许多研究发现类胡萝卜素在植物、动物以及微生物之中广泛地存在,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、调节免疫功能、着色功能等。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中分离获得葡萄糖-6-磷酸脱氢酶基因RkZWF1及其用途,基因RkZWF1的核苷酸序列如SEQ IDNO:1所示,该基因序列长为1542bp,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RkZWF1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pRHRkZWF1,将重组质粒pRHRkZWF1转入大肠杆菌中,通过PCR筛选出阳性单克隆,重组质粒pRHRkZWF1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1542bp的葡萄糖-6-磷酸脱氢酶基因RkZWF1;
2、利用PEG介导的原生质体法将重组载体pRHRkZWF1转化至红冬孢酵母YM25235中,筛选转化子,获得含有pRHRkZWF1的过表达菌株,含有pRHRkZWF1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中克隆得到葡萄糖-6-磷酸脱氢酶基因RkZWF1,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RkZWF1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,引起细胞内类胡萝卜素含量的提高;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,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;本发明方法简单,操作简便,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RkZWF1基因的PCR扩增图;1.DNA分子量标记DL5000;2.阴性对照;3.基因RkZWF1的cDNA片段;
图2为重组质粒pRHRkZWF1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RkZWF1的cDNA片段;4-8为转化子;
图4为重组质粒pRHRkZWF1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pRHRkZWF1的BamHI、EcoRV双酶切;5.基因RkZWF1的cDNA片段;6.DNA 分子量标记DL2000;
图5重组质粒pRHRkZWF1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pRHRkZWF1扩增的 PCR产物;5.以YM25235/pRHRkZWF1菌株基因组扩增的PCR产物;
图6过表达菌株YM25235/pRHRkZWF1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中克隆葡萄糖-6-磷酸脱氢酶基因RkZWF1并构建其过表达载体pRHRkZWF1、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 (+gDNA wiper)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RkZWF1序列,设计特异性引物RkZWF1-F和RkZWF1-R,以上述得到的cDNA模板,采用引物RkZWF1-F和RkZWF1-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:
(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)
PCR扩增体系如下(50μL):Template cDNA1μL、RkZWF1-F2μL、RkZWF1-R2μL、dNTPsMix(10mM each) 1μL、Vazyme 2×Phanta Max Buffer25μL、Vazyme Phanta Max Super-Fidelity DNA Polymerase1μL,ddH2O加至50μL;
扩增条件:95℃预变性3min,再用95℃变性15s,67℃退火15s,72℃延伸1min35s,共30个循环,最后72℃彻底延伸5min;反应后取产物2μL,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;扩增得到大小约为1550bp的片段,命名为RkZWF1;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pRHRkZWF1,连接体系如下(20µL):ExnaseTMⅡ2μL、RkZWF1片段61.68ng、线性化pRH2034200ng、5×CEⅡBuffer4μL、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,剩余约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(OMEGA Plasmid Mini Kit I试剂盒,美国OMEGA公司),用BamHⅠ、EcoRⅤ对pRHRkZWF1进行双酶切验证,结果见图4,结果表明,重组质粒pRHRKZW1经双酶切后产生了1.5kb 和10kb左右的两个条带(图4第4泳道),这两个条带分别与RkZWF1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pRHRkZWF1构建成功,重组载体pRHRkZWF1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1542bp,与转录组序列一致,核苷酸序列如SEQID NO:1所示,命名为RkZWF1。
3、挑取成功转入正确重组载体pRHRkZWF1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(OMEGA Plasmid 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pRHRkZWF1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℃、4500 rcf离心5 min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℃、1300rcf离心10min收集菌体;加入10mLSTC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210.36μg/mL的pRHRkZWF1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℃、1500rcf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0.4mol/L蔗糖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RkZWF1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RkZWF1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/pRHRkZWF1的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(mg/g干菌体),其中野生型红冬孢酵母YM25235菌株的总类胡萝卜素合成量为5.535±0.092mg/g,而过表达菌株YM25235/pRHRkZWF1类胡萝卜素合成量为6.735±0.106mg/g,过表达菌株YM25235/pRHRkZWF1总胡萝卜素合成量是对照菌的1.22倍过表达菌株YM25235/pRHRkZWF1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(如图6所示);结果显示葡萄糖-6-磷酸脱氢酶基因RkZWF1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,RkZWF1基因能够促进总类胡萝卜素的合成。
Claims (2)
1.一种葡萄糖-6-磷酸脱氢酶基因RkZWF1,其核苷酸序列如SEQ ID NO:1所示。
2.权利要求1所述的葡萄糖-6-磷酸脱氢酶基因RkZWF1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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CN115011616B (zh) * | 2022-01-26 | 2023-07-21 | 昆明理工大学 | 一种乙醛脱氢酶基因rkaldh及其应用 |
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