CN116676327A - 用于合成Forskolin的基因组合及其应用 - Google Patents
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- CN116676327A CN116676327A CN202310649550.5A CN202310649550A CN116676327A CN 116676327 A CN116676327 A CN 116676327A CN 202310649550 A CN202310649550 A CN 202310649550A CN 116676327 A CN116676327 A CN 116676327A
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Abstract
本发明属于植物分子生物学和植物基因工程技术领域。本发明涉及用于合成Forskolin的基因组合及其应用。所述基因组合由TPS2、TPS3、CYP76AH11、CYP76AH15、CYP76AH16和ACT1‑8组成，其核苷酸序列依次如SEQ ID NO.1‑6所示。利用所述的基因组合在体外构建人工代谢途径，将人工代谢途径导入植物，使其整合到植物基因组中；使人工代谢途径中各基因表达合成mRNA和蛋白质，合成Forskolin化合物。本发明在烟草中实现了高价值Forskolin化合物的异源合成，显著超出了现有技术的水平，具有广阔的工业实用前景和规模化发展的潜力。
Description
技术领域
本发明属于植物分子生物学和植物基因工程技术领域。本发明涉及用于合成Forskolin的基因组合及其应用。
背景技术
毛喉鞘蕊花(Coleus forskohlii)是唇形科鞘蕊花属植物，主要分布在印度、斯里兰卡、尼泊尔等国家。我国在云南、四川等地发现野生毛喉鞘蕊花植物，并界定为珍稀植物。毛喉鞘蕊花提取物对支气管哮喘、充血性心力衰竭、多种肿瘤具有明显的药理活性，长期用于治疗心血管疾病、咳嗽和呼吸紊乱等疾病。
研究表明毛喉鞘蕊花中主要有效成分是半日花烷型二萜类化合物毛喉素(Forskolin)，CAS No.:66575-29-9，其具有抗癌、平喘、抗高血压和强心等活性，是具有重要药用价值的天然药物。Forskolin主要存在于毛喉鞘蕊花根部木栓层细胞的油体状结构内，其含量仅占根干重的0.013％。由于毛喉鞘蕊花对生长环境要求较高，生长缓慢，长期采挖和大量人工栽培已造成野生植物资源枯竭和生态环境破坏。虽然目前已在湖北等地进行人工种植，但仍难以解决药材资源缺乏问题。而由于Forskolin化合物结构复杂，现有的化学合成方法步骤繁琐，产率低，难以工业化大量生产。因此目前Forskolin产量无法满足其商业需求，价格高昂。
合成生物学是基于对生物系统运行规律的认识和工程化的原理，设计和改造自然界已有的生命系统，或从头构建自然界没有的人造生命装置或体系，为植物天然产物的研究、开发、植物资源的可持续利用和发展开辟了一条全新而高效的途径。烟草(Nicotianatabacum)是重要的经济作物，生长迅速，生物量大，次生代谢旺盛，叶片和茎秆表面富含腺毛，其中合成、储存和分泌大量萜类、生物碱和糖脂类次生代谢物，因此是理想的植物生物反应器。本发明将来源于毛喉鞘蕊花的基因进行重新组合与优化，整合到烟草基因组中，培育可生产Forskolin的烟草新材料，具有重要生物学和产业意义。
发明内容
本发明的所要解决的技术问题是如何在植物中异源合成Forskolin。
为解决上述技术问题，本发明采用如下技术方案：
第一方面，本发明提供用于合成Forskolin的基因组合，所述基因组合由TPS2、TPS3、CYP76AH11、CYP76AH15、CYP76AH16和ACT1-8组成，其核苷酸序列依次如SEQ ID NO.1-6所示。
第二方面，本发明提供含有所述基因组合的表达载体。
第三方面，本发明提供含有所述基因组合的细胞系。
第四方面，本发明提供含有所述基因组合的宿主菌。
第五方面，本发明提供所述基因组合在转化植物以产生能合成Forskolin的转基因植物中的应用。
进一步的，所述植物包括烟草。
更进一步的，与野生型烟草相比，转基因烟草或其后代的叶组织器官中含有Forskolin化合物。
第六方面，本发明提供一种在植物中异源合成Forskolin的方法，利用所述的基因组合在体外构建人工代谢途径，将人工代谢途径导入植物，使其整合到植物基因组中；使人工代谢途径中各基因表达合成mRNA和蛋白质，合成Forskolin化合物，所述植物包括烟草。
本发明具有如下有益效果：
本发明在生长迅速、生物量大、环境适应能力强的烟草中实现了高价值Forskolin化合物的异源合成，显著超出了现有技术的水平，具有广阔的工业实用前景和规模化发展的潜力。
附图说明
图1为Lv1-TPS2质粒图谱。
图2为Lv1-TPS3质粒图谱。
图3为Lv1-CYP76AH11质粒图谱。
图4为Lv1-CYP76AH15质粒图谱。
图5为Lv1-CYP76AH16质粒图谱。
图6为Lv1-ACT1-8质粒图谱。
图7为为LC-MS检测瞬时表达Forskolin合成途径基因的本氏烟中的Forskolin化合物。
图8为pForskolin质粒图谱。
图9为PCR检测转基因烟草中的Forskolin人工代谢途径基因。
图10为LC-MS检测pForskolin转基因烟草中的Forskolin化合物。
图11为转基因烟草叶片中检测到Forskolin的质谱图。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明，但不应理解为本发明的限制。如未特殊说明，下述实施例中所用的技术手段为本领域技术人员所熟知的常规手段，下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1：Forskolin合成途径基因获得及人工代谢途径载体构建
从NCBI下载毛喉鞘蕊花Forskolin代谢途径基因的核苷酸序列(如表1所示)，由青岛派森诺基因生物技术有限公司进行全基因合成。
表1Forskolin合成途径基因信息
利用TakaRa公司PrimeSTAR Max DNA聚合酶进行PCR扩增，得到目的基因片段，引物信息见表2。
表2PCR扩增引物信息
PCR反应体系为：5μL 10×缓冲液，1μL 10mM dNTP，0.5μL PrimeSTAR Max DNA聚合酶，2μL正向引物，2μL反向引物，2μL模版质粒。PCR条件为：95℃5min；95℃30s，55℃30s，72℃120s，35个循环；72℃延伸5min。PCR产物用1％的琼脂糖凝胶电泳检测并割胶回收。
TPS2、TPS3、CYP76AH11、CYP76AH15、CYP76AH16和ACT1-8的胶回收产物分别与35S启动子、OCS终止子和载体pICH47742(Addgene Plasmid#48001)连接。反应体系为：2μL 10×T4 DNA Ligase缓冲液，1μL T4 ligase，1μL BsaI，1μL pICH41295，1μL胶回收产物。反应条件为16℃24小时。将反应产物转化到大肠杆菌DH5α克隆菌株中，转化条件为：在100μL感受态细胞中加入20μL连接产物，轻轻混匀后冰浴30min；迅速放入42℃水浴中热激90s，立即置于冰上2-3min；加入800μL LB液体培养基，37℃慢摇培养1h；将菌液6000rpm离心1min，弃700μL上清，悬浮菌体，涂布于含有氨苄青霉素(Amp，100mg/L)的LB平板上，37℃倒置暗培养12～16h。采用菌落PCR进行阳性克隆筛选，挑选阳性单克隆菌落，提取质粒后送交测序验证。构建正确的质粒分别命名为Lv1-TPS2、Lv1-TPS3、Lv1-CYP76AH11、Lv1-CYP76AH15、Lv1-CYP76AH16和Lv1-ACT1-8。载体结构如图1-6。
实施例2：本氏烟(Nicotiana bentamiana)瞬时表达验证代谢途径基因功能
将前述载体质粒各5μL分别加入100μL农杆菌GV3101感受态细胞中，液氮速冻2分钟，37℃放置30分钟，加入1mL LB液体培养基，28℃培养3小时，涂布在含25mg/L利福平、25mg/L庆大霉素、50mg/L氨苄青霉素的LB平板上，28℃培养3天。
挑取农杆菌单克隆接种到2mL LB培养基(含25mg/L利福平、25mg/L庆大霉素、50mg/L氨苄青霉素)中于28℃、220rpm培养过夜。菌液于8000rpm离心2分钟，收集沉淀重悬于10mL转化液(10mM MES，10mM MgCl2，0.2mM乙酰丁香酮)中，室温静置3小时后注射本氏烟叶片。温室生长5天后取叶片检测Forskolin化合物。
取100mg本氏烟叶片材料于液氮中充分研磨，转移至1.5mL离心管中，加入1mL乙酸乙酯振荡2分钟，12000rpm离心2分钟。收集上清，氮气吹干，加入1mL甲醇溶解，0.22μm过滤后进行LC-MS检测。采用安捷伦公司HPLC-APCI-MS系统进行检测。色谱柱为Phenomenex C18柱(250mm×4.6mm，5μm)；流动相为水(A)-乙腈(B)梯度洗脱，程序为0-8min，48％ A；13min，34％ A；18min，34％ A；流速1mL/min；柱温40℃；进样5μL。质谱条件为：负离子模式，离子源温度300℃，氮气流速10L/min，碎裂电压180V。
如图7所示，在共转Lv1-TPS2、Lv1-TPS3、Lv1-CYP76AH11、Lv1-CYP76AH15、Lv1-CYP76AH16和Lv1-ACT1-8农杆菌的本氏烟叶片提取物中，检测到Forskolin化合物，其色谱保留时间与Forskolin标准品一致，说明在本氏烟中瞬时表达代谢途径基因可以合成Forskolin化合物。
实施例3：产Forskolin转基因烟草培育
将Lv1-TPS2、Lv1-TPS3、Lv1-CYP76AH11、Lv1-CYP76AH15、Lv1-CYP76AH16和Lv1-ACT1-8质粒构建成人工代谢途径载体。反应体系为：2μL 10×T4 DNA Ligase缓冲液，1μLT4 ligase，1μL BsaI，1μL pICH41722(Addgene Plasmid#48016)，1μL各质粒。反应条件为16℃24小时。将反应产物转化到大肠杆菌DH5α克隆菌株中，转化条件为：在100μL感受态细胞中加入20μL连接产物，轻轻混匀后冰浴30min；迅速放入42℃水浴中热激90s，立即置于冰上2-3min；加入800μL LB液体培养基，37℃慢摇培养1h；将菌液6000rpm离心1min，弃700μL上清，悬浮菌体，涂布于含有卡那霉素(50mg/L)的LB平板上，37℃倒置暗培养12～16h。采用菌落PCR进行阳性克隆筛选，挑选阳性单克隆菌落，提取质粒后送交测序验证。构建正确的质粒命名为pForskolin(图8)。
将pForskolin载体质粒5μL加入100μL农杆菌LBA4404感受态细胞中，液氮速冻2分钟，37℃放置30分钟，加入1mL LB液体培养基，28℃培养3小时，涂布在含25mg/L利福平、25mg/L链霉素、50mg/L卡那霉素的LB平板上，28℃培养3天。
挑取农杆菌单克隆接种到50ml LB培养基(含25mg/L利福平、25mg/L链霉素、50mg/L卡那霉素)中于28℃、220rpm培养过夜。菌液于8000rpm离心2分钟，收集沉淀重悬于1/2MS液体培养基中，调整OD600＝0.6，作为感染液备用。
无菌培养的烟草(Nicotiana tabacum，K326)叶片切成1×1cm的小片，在农杆菌感染液中浸泡15分钟，转移到共培培养基(MS培养基+30g/L蔗糖+2mg/L 6-BA+8g/L Agar)上避光培养48小时，转移到筛选培养基(MS培养基+30g/L蔗糖+2mg/L 6-BA+50mg/L卡那霉素+8g/L Agar)上于25℃、16h光/8h暗条件下培养4-8周。切取抗性芽继代到生根培养基(MS培养基+30g/L蔗糖+8g/LAgar)中培养2周。再生苗移栽到花盆中，置于人工气候室生长(25℃、16h光/8h暗)。
实施例4：产Forskolin烟草筛选和检测
取烟草材料(100mg)于液氮中充分研磨，转移至1.5ml离心管中，加入1ml DNA提取液(100mM Tris，2M NaCl，2％ CTAB，2％ PVP)混匀，65℃放置15min，12000rpm离心10min，弃沉淀。上清中加入0.5ml三氯甲烷，混匀，12000rpm离心10min，取上清加入0.5ml异丙醇沉淀DNA。12000rpm离心10min，沉淀溶于0.1ml水。
使用2×Taq Master Mix(Vazyme)进行PCR检测。利用引物SEQ ID NO.7-18检测TPS2、TPS3、CYP76AH11、CYP76AH15、CYP76AH16和ACT1-8。PCR反应体系为：2×Taq MasterMix 10μL，1μL正向引物，1μL反向引物，1μL DNA。PCR条件为：95℃5min；95℃30s，55℃30s，72℃120s，35个循环；72℃延伸5min。PCR产物用1％的琼脂糖凝胶电泳检测。
如图9所示，大多数转基因植物基因组中均已整合Forskolin途径基因。
取100mg本氏烟叶片材料于液氮中充分研磨，转移至1.5mL离心管中，加入1mL乙酸乙酯振荡2分钟，12000rpm离心2分钟。收集上清，氮气吹干，加入1mL甲醇溶解，0.22μm过滤后进行LC-MS检测。采用安捷伦公司HPLC-APCI-MS系统进行检测。色谱柱为Phenomenex C18柱(250mm×4.6mm，5μm)；流动相为水(A)-乙腈(B)梯度洗脱，程序为0-8min，48％ A；13min，34％ A；18min，34％ A；流速1mL/min；柱温40℃；进样5μL。质谱条件为：负离子模式，离子源温度300℃，氮气流速10L/min，碎裂电压180V。
如图10所示，在转pForskolin载体的烟草叶片提取物中，检测到Forskolin化合物，其色谱保留时间和质谱图谱(图11)与Forskolin标准品一致，说明该转基因烟草可以合成Forskolin化合物。
尽管已描述了本发明的优选实施例，但本领域内的技术人员一旦得知了基本创造性概念，则可对这些实施例作出另外的变更和修改。所以，所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。
Claims (9)
1.用于合成Forskolin的基因组合，其特征在于，所述基因组合由TPS2、TPS3、CYP76AH11、CYP76AH15、CYP76AH16和ACT1-8组成，其核苷酸序列依次如SEQ ID NO.1-6所示。
2.含有权利要求1所述基因组合的表达载体。
3.含有权利要求1所述基因组合的细胞系。
4.含有权利要求1所述基因组合的宿主菌。
5.权利要求1所述基因组合在转化植物以产生能合成Forskolin的转基因植物中的应用。
6.根据权利要求5所述的应用，其特征在于，所述植物包括烟草。
7.根据权利要求6所述的应用，其特征在于，与野生型烟草相比，转基因烟草或其后代的叶组织器官中含有Forskolin化合物。
8.一种在植物中异源合成Forskolin的方法，其特征在于，利用权利要求1所述的基因组合在体外构建人工代谢途径，将人工代谢途径导入植物，使其整合到植物基因组中；使人工代谢途径中各基因表达合成mRNA和蛋白质，合成Forskolin化合物。
9.根据权利要求8所述的一种在植物中异源合成Forskolin的方法，其特征在于，所述植物包括烟草。
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