CN113621630A - 3-酮脂酰-CoA硫解酶基因RkACAA1-1及其应用 - Google Patents
3-酮脂酰-CoA硫解酶基因RkACAA1-1及其应用 Download PDFInfo
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- CN113621630A CN113621630A CN202110895025.2A CN202110895025A CN113621630A CN 113621630 A CN113621630 A CN 113621630A CN 202110895025 A CN202110895025 A CN 202110895025A CN 113621630 A CN113621630 A CN 113621630A
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Abstract
本发明公开了一种3‑酮脂酰‑CoA硫解酶基因RkACAA1‑1,其核苷酸序列如SEQID 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中分离获得的,将该基因转化到红冬孢酵母YM25235中,实验结果显示,3‑酮脂酰‑CoA硫解酶基因RkACAA1‑1的过表达能引起红冬孢酵母YM25235菌株中总类胡萝卜素增加,本发明通过基因工程手段对红冬孢酵母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。
Description
技术领域
本发明属于生物和遗传工程技术领域,涉及一种3-酮脂酰-CoA硫解酶基因RkACAA1-1及其在促进微生物生产类胡萝卜素中的应用。
背景技术
类胡萝卜素(Carotenoid)是一类重要的天然色素,是含有40个碳原子的异戊二烯聚合物。根据类胡萝卜素的分子组成,通常可以将其分为两类,一类是化学结构不含氧元素只含碳氢元素的胡萝卜素类(Carotenes),如α-胡萝卜素、β-胡萝卜素和番茄红素;一类是化学结构含有羟基、酮基、羧基、甲氧基等含氧官能团的叶黄素类(Xanthophylls),如玉米黄质、叶黄素和虾青素。目前,已在自然界中发现了超过800种的天然类胡萝卜素,其广泛的存在于高等植物、藻类、真菌和细菌中。
类胡萝卜素是动植物抗氧化系统中重要的组成,是保护机体免受氧化损伤的有效抗氧化剂。研究表明,人体不仅在正常的生命代谢活动中会生成活性氧,紫外线辐射、环境污染物等也会诱导活性氧的产生。人体内过量的氧自由基,会导致正常的生命大分子被氧化,从而对机体造成损伤。类胡萝卜素分子结构中存在类异戊二烯共轭双键,因此具有较强的抗氧化活性,能够保护细胞和组织免受活性氧破坏。除此之外,类胡萝卜素还是维生素A合成的前体物质,可以促进人类视觉系统的形成;其还具有抑制细胞异常生长,阻止低密度脂蛋白形成的功能,对维持机体健康、预防疾病、抗衰老和预防癌症方面有着重要作用。到目前为止,没有证据证明动物自身能够合成类胡萝卜素,只能依靠从外界摄入。
类胡萝卜素还有着丰富的颜色,随着分子中共轭双键数目、链长和取代基团的变化会呈现出不同的色彩。在食品药品行业,类胡萝卜素作为一种天然着色剂已经被广泛使用;而且得益于其抗氧化性,可以防止食品中的营养成分和风味物质遭到破坏,因此类胡萝卜素不仅可以提供诱人的色彩,还起到了延长食品保质期的作用。除了食品药品领域外,类胡萝卜素在养殖业中也有着重要的作用。向饲料中添加类胡萝卜素,不仅可以降低动物的发病风险、加强动物的免疫力,而且还能够改善肉类的品质,对于蛋类产品,则可以改善其蛋黄的色泽。
随着类萝卜素在医疗、食品、养殖等领域中越来越广泛的应用,需求量也日益增加。虽然已有许多研究者对如何提高类胡萝卜素的产量进行了相当多的研究,但现在类胡萝卜素的产量和质量仍不能满足市场需求。目前,主要的类胡萝卜素生产方法包括植物提取法、化学合成法和生物合成法。植物提取法成本过高,显然不适合商业化、大规模的生产;而化学合成法虽然在产量上可以满足需求,但是也存在环境不友好、化学物质残留、活性不比天然类胡萝卜素的缺点。
发明内容
针对现有技术的不足,本发明提供了一种从红冬孢酵母(Rhodosporidium kratochvilovae)YM25235中分离得到3-酮脂酰-CoA硫解酶基因RkACAA1-1,该基因核苷酸序列如SEQ ID NO:1所示或与SEQ ID NO:1互补的核苷酸序列,该基因序列长为1275bp(碱基),编码的氨基酸序列如SEQ ID NO:2所示的多肽。
本发明另一目的是将上述3-酮脂酰-CoA硫解酶基因RkACAA1-1应用在生产类胡萝卜素中。
上述目的通过以下技术方案实现,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,通过PCR扩增获得目的片段,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,获得重组质粒pRHRkACAA1-1,连接产物转入大肠杆菌中,通过PCR筛选出阳性单克隆,重组质粒pRHRkACAA1-1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1275bp的3-酮脂酰-CoA硫解酶基因RkACAA1-1;利用PEG介导的原生质体法将重组载体pRHRkACAA1-1转化至红冬孢酵母YM25235中,筛选转化子,获得RkACAA1-1过表达菌株,过表达菌株经培养,利用紫外-可见分光光度计测量菌株中总类胡萝卜素的含量。
本发明从红冬孢酵母(Rhodosporidium kratochvilovae)YM25235的总RNA中分离得到3-酮脂酰-CoA硫解酶基因RkACAA1-1,红冬孢酵母YM25235中RkACAA1-1基因的过表达会引起细胞内这个基因的转录水平在一定程度上有一定的提高,说明外源基因在菌体内发生转录,继而翻译成相应蛋白,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,将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。
附图说明
图1为本发明的红冬孢酵母YM25235的RkACAA1-1基因PCR扩增图;1.DNA分子量标记DL2000;2.阴性对照;3.RkACAA1-1的cDNA片段;
图2为重组质粒pRHRkACAA1-1质粒图谱;
图3为菌落PCR验证电泳图;1.DNA分子量标记DL2000;2.阴性对照3. RkACAA1-1的cDNA片段;4-8为转化子;
图4为重组质粒pRHRkACAA1-1限制性酶切分析;其中:1.DNA 分子量标记DL100002.阴性对照;3.空质粒pRH2034的BamHⅠ、EcoRⅤ双酶切;4.重组质粒pRHRkACAA1-1的BamHⅠ、EcoRⅤ双酶切;5.RkACAA1-1的cDNA片段;6.DNA 分子量标记DL2000;
图5重组质粒pRHRkACAA1-1转化红冬孢酵母YM25235阳性克隆验证;1.DNA分子量标记DL2000;2.阴性对照3.野生型菌株特异性基因条带;4.RkACAA1-1cDNA片段;5.转化子验证;
图6过表达菌株YM25235/pRHRkACAA1-1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(Rhodosporidium kratochvilovae)YM25235中分离3-酮脂酰-CoA硫解酶基因RkACAA1的核苷酸序列以及过表达载体pRHRkACAA1-1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)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RkACAA1-1序列,设计特异性引物RkACAA1-1-F和RkACAA1-1-R,以上述得到的cDNA模板在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、组份和扩增条件如下:
RkACAA1-1-F:5’-ATCACTCACCATGGCGGATCCTATGTCTCTCACGAACGCCG -3’(SEQ IDNO: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)
RkACAA1-1-R:5’-CCGGTCGGCATCTACGATATCCTACTGCTCGTTGACGATGA -3’(SEQ IDNO:4)(双下划线为上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5min,再用95℃变性15s,64℃退火15s,72℃延伸1min15s,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1275bp的片段;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进行连接,获得重组质粒pRHRkACAA1-1(图2),连接方式采用ClonExpress II One Step Cloning Kit试剂盒进行,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37℃反应30min;降至4℃或立即置于冰上冷却。
取10µL上面获得的连接产物加到100µL DH5α感受态细胞中,轻轻混匀,冰浴30min,42℃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振荡培养1h,在5000rpm下离心10min后弃900µL上清,剩余约100µL左右培养基悬浮菌体并涂布于含有100µg/mL壮观霉素(Spe+)的LB固体平板,于37℃倒置过夜培养,随机挑取5个平板上生长的白色菌落并编号为1-5号,通过菌落PCR验证阳性克隆,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,提取质粒(OMEGA Plasmid Mini Kit I,美国OMEGA公司),用BamHⅠ、EcoRⅤ两个限制性内切酶进行酶切验证;酶切验证结果见图4,从图中可以看出重组质粒pRHRkACAA1-1经BamHⅠ、EcoRⅤ双酶切后,出现与空质粒pRH2034的BamHⅠ、EcoRⅤ双酶切获得的线性载体片段大小相同的条带,以及与RkACAA1-1基因的cDNA片段大小相同的条带,说明重组质粒成功转入大肠杆菌DH5α菌株;酶切验证后,按相同方法提取质粒,测序(昆明擎科生物科技有限公司)。测序结果显示,所扩增得到的片段大小为1275bp,序列如SEQ ID NO:1所示的核苷酸序列,命名为RkACAA1-1,与RkACAA1-1基因的cDNA片段大小及序列一致,说明成功构建表达载体pRHRkACAA1-1;
实施例2:RkACAA1-1基因过表达对红冬孢酵母YM25235中类胡萝卜素合成的影响
1、转化红冬孢酵母YM25235
挑取成功转入正确重组载体pRHRKACAA1-2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),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,将培养液于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,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,置于无菌50mL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2-5μgpRHRKACAA1-1重组质粒并轻轻混匀(通常加入质粒溶液体积不应超过10μL)冰上孵育10min,加入200μL预冷的PTC(50%PEG3350、10mMTris-HCl、100mMCaCl2),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1.6mL0.4M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,最后涂布于含130μg/mL潮霉素B(HygB+)的0.4M蔗糖YPD固体培养基上,于30℃倒置培养2-3d;将涂布后得到的转化子编号,并转接到含150μg/mL潮霉素(Hyg B+)YPD固体培培养基上,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RkACAA1-1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RkACAA1-1cDNA片段已成功连入酵母转化子的基因组中。
2、RkACAA1-1基因过表达的红冬孢酵母YM25235中类胡萝卜素含量分析
将含pRHRkACAA1-1的过表达菌株在28℃条件下培养168h,提取类胡萝卜素,以野生型红冬孢酵母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/pRHRkACAA1-1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4.87±0.49mg/g,而过表达菌株YM25235/pRHRKACAA1-1类胡萝卜素合成量为7.97±0.16mg/g,即过表达菌株YM25235/pRHRKACAA1-1类胡萝卜素合成量是对照菌的1.636倍;结果显示3-酮脂酰-CoA硫解酶基因RkACAA1-1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,RkACAA1-1基因能够促进总类胡萝卜素的合成。
序列表
<110> 昆明理工大学
<120> 3-酮脂酰-CoA硫解酶基因RkACAA1-1及其应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1275
<212> DNA
<213> 红冬胞酵母YM25235(RhodosporidiumkratochvilovaeYM25235)
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cccatcctcg gcaaggtctg ccataccgcc atcgcgggcg tcgagcccaa gctcatgggc 960
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Claims (2)
1.一种3-酮脂酰-CoA硫解酶基因RkACAA1-1,其核苷酸序列如SEQ ID NO:1所示。
2.权利要求1所述的3-酮脂酰-CoA硫解酶基因RkACAA1-1在促进微生物生产类胡萝卜素中的应用。
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