CN113621631A - 一种甲羟戊酸激酶基因rkmk及其应用 - Google Patents
一种甲羟戊酸激酶基因rkmk及其应用 Download PDFInfo
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Abstract
本发明公开了一种甲羟戊酸激酶基因RKMK及其用途,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RKMK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
本发明属于生物技术领域和遗传工程技术领域,涉及一种甲羟戊酸激酶基因RKMK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
甲羟戊酸激酶(Mevalonate kinase,MVK)是甲羟戊酸(MVA)途径中三个连续ATP依赖酶中的第一个,是控制整个代谢途径的限速酶之一,甲羟戊酸激酶蛋白N-端包含一段富含Gly/Ser的保守区域,参与结合ATP(三磷酸腺苷)。甲羟戊酸激酶负责将ATP-γ位上的一个磷酸基团转移到甲羟戊酸第5位的羟基上形成甲羟戊酸-5-磷酸并释放ADP。不同生物来源的甲羟戊酸激酶通常是单体或者由相同亚基组成的二聚体,分子量为70-105kDa。所有甲羟戊酸激酶都具有组成酶促活性中心的3个保守区,其中保守区 1与甲羟戊酸底物的结合及催化反应有关,而保守区2和保守区3则与ATP的结合及催化反应有关,由此说明它们的酶促反应机制相似;甲羟戊酸激酶催化的甲羟戊酸磷酸化作用是顺序性的,即甲羟戊酸是第一个与酶结合的底物,然后才是Mg·ATP,而甲羟戊酸-5-磷酸是第一个被释放的底物,然后才释放ADP(王宝莲等.甲羟戊酸激酶基因研究进展[J].中国农业科技导报,2011,13(3):17-25. DOI:10.3969/j.issn.1008-0864.2011.03.03.)。
植物中,该酶主要参与类异戊二烯衍生物的合成,很多植物类异戊二烯具有重要商业价值,如作为食物香料、饮料、维生素A、维生素D、维生素E、天然杀虫剂(如除虫菊素)和橡胶等(王宝莲等.植物甲羟戊酸激酶基因研究究进展[J].山东农业科学,2011(4):12-16.DOI:10.3969/j.issn.1001-4942.2011.04.004.)。研究表明,调控甲羟戊酸激酶的活性能够影响植物的再生与生长,在维持基本生命活动和特殊器官的植物细胞中都有参与作用。动物中,该酶在控制胆固醇生物合成中起重要作用,并与人类遗传病包括甲羟戊酸尿症、超免疫球蛋白D症以及周期性放热综合征等有关,在昆虫中也参与幼虫激素的合成,是杀虫剂的作用靶酶。微生物中,该酶也是类异戊二烯合成途径的关键酶,对筛选有效的酶抑制剂,探索有效的生物农药,为有效解决动植物及真菌病害开辟新途径。
类胡萝卜素具有较强的抗氧化性、维生素A原活性、着色功能及抗癌能力。由于这些多样的生物活性和功能,对人类的健康十分重要,在食品、营养食品、动物饲料和化妆品等领域得到了广泛的研究和应用。
目前未见关于甲羟戊酸激酶基因在促进微生物生产类胡萝卜素中的报道。
发明内容
本发明目的是提供一种甲羟戊酸激酶基因RKMK及其用途,本发明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2679 bp,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RKMK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pRHRKMK,将重组质粒pRHRKMK转入大肠杆菌中,通过PCR筛选出阳性单克隆,重组质粒pRHRKMK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2679bp的甲羟戊酸激酶基因RKMK;
2、利用PEG介导的原生质体法将重组载体pRHRKMK转化至红冬孢酵母YM25235中,筛选转化子,获得含有pRHRKMK的过表达菌株,含有pRHRKMK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。
所述红冬孢酵母(Rhodosporidium kratochvilovae) YM25235具有生产周期短、遗传稳定、生产安全等优点。
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中分离得到甲羟戊酸激酶基因RKMK,红冬孢酵母YM25235中RKMK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;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RKMK基因的PCR扩增图;1.DNA分子量标记DL5000;2.阴性对照;3.基因RKMK的cDNA片段;
图2为重组质粒pRHRKMK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.基因RKMK的cDNA片段;3-7为转化子;
图4为重组质粒pRHRKMK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BamH I和EcoR V双酶切;4.重组质粒pRHRKMK的BamH I、EcoR V双酶切;5.基因RKMK的cDNA片段;6.DNA 分子量标记DL5000;
图5重组质粒pRHRKMK转化红冬孢酵母YM25235阳性克隆验证;1.DNA 分子标量DL5000;2.阴性对照;3.以YM25235基因组扩增的PCR产物;4.以质粒pRHRKMK扩增的 PCR产物;5.以YM25235/pRHRKMK菌株基因组扩增的PCR产物;
图6过表达菌株YM25235/pRHRKMK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中分离甲羟戊酸激酶基因RKMK及过表达载体pRHRKMK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)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,根据转录组测序中发现的RKMK序列,设计特异性引物RKMK-F和RKMK-R(引物1和引物2),以上述得到的cDNA模板,采用引物RKMK-F和RKMK-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:
引物1:RKMK-F:5’-ATCACTCACCATGGCGGATCCTATGACCTCGACCGCGGG-3’
(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)
引物2:RKMK-R:5’-CCGGTCGGCATCTACGATATCCTACGCCCTGACCCAGC-3’(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,64℃退火15s,72℃延伸2min30s,共30个循环,最后72℃彻底延伸5min;反应后取产物2μL,在浓度为0.8%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2700bp的片段,命名为RKMK;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pRHRKMK,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(OMEGA Plasmid Mini Kit I,美国OMEGA公司),用BamHⅠ、EcoRⅤ对pRHRKMK进行双酶切验证;结果见图4,结果表明,重组质粒pRHRKMK经双酶切后产生了2.7kb 和10kb左右的两个条带(图4第4泳道),这两个条带分别与RKMK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pRHRKMK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(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2679bp,序列组成如SEQ ID NO:1所示的核苷酸序列,命名为RKMK,与RKMK基因的cDNA片段大小一致,说明成功构建表达载体pRHRKMK,重组载体pRHRKMK的质粒图谱见图2。
实施例2:RKMK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pRHRKMK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,美国OMEGA公司),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30℃、200rpm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30℃、200rpm振荡培养至菌液OD600为0.5,将培养物于4℃、4500 rpm离心5 min收集菌体;使用事先准备的柠檬酸缓冲液(30mM柠檬酸、83mM柠檬酸钠、600mM甘露醇、NaOH调pH值到5.4)洗涤菌体两次,于4℃,4000 rpm,离心5min收集菌体并用1mL柠檬酸缓冲液悬浮菌体,于4℃、4000 rpm离心5min收集菌体,置于冰上备用;配制裂解酶液(0.156g蜗牛酶、0.08g溶壁酶,用ddH2O定容至5mL),用0.22μm无菌滤膜过滤酶液,置于50mL无菌离心管中备用;取4mL酶液与菌液混合后置于30℃、90rpm振荡培养酶解2.5h,将培养物于4℃、1300rpm离心10min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分装到5mL无菌离心管中备用;向100μL感受态细胞中加入2-5μgpRHRKMK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,再加入800μL预冷的PTC,并轻轻混匀,冰浴10min,4℃、1500rpm离心10min收集菌体;加入1.6mL 0.4M蔗糖YPD液体培养基悬浮,30℃、90rpm振荡培养12h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0.4M蔗糖YPD固体培养基(含130μg/mL潮霉素B)上,于30℃倒置培养2-3d;将涂布后得到的转化子编号,并转接到YPD固体培培养基(含150μg/mL潮霉素)上,于30℃倒置培养2d;根据该基因已知功能,以颜色筛选转化子,具体操作为将所得转化子接入5mL YPD培养基,于30℃、200rpm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RKMK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RKMK片段已成功连入酵母转化子的基因组中。
2、RKMK基因过表达的红冬孢酵母YM25235中类胡萝卜素含量分析
将含pRHRKMK的过表达菌株在28℃条件下培养168h,提取类胡萝卜素,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445nm下测定总类胡萝卜素的含量(mg/g干菌体),含量如图6所示;由图可知,过表达菌株YM25235/pRHRKMK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5.41±0.02mg/g,而过表达菌株YM25235/pRHRKMK类胡萝卜素合成量为6.65±0.11mg/g,即过表达菌株YM25235/pRHRKMK胡萝卜素合成量是对照菌的1.23倍;结果显示甲羟戊酸激酶基因RKMK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,RKMK基因能够促进总类胡萝卜素的合成。
Claims (2)
1.一种甲羟戊酸激酶基因RKMK,其核苷酸序列如SEQ ID NO:1所示。
2.权利要求1所述的甲羟戊酸激酶基因RKMK在促进微生物生产类胡萝卜素中的应用。
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