CN105087600B - 一种珠子参转录因子基因PjbHLH1的应用 - Google Patents
一种珠子参转录因子基因PjbHLH1的应用 Download PDFInfo
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- CN105087600B CN105087600B CN201510561852.2A CN201510561852A CN105087600B CN 105087600 B CN105087600 B CN 105087600B CN 201510561852 A CN201510561852 A CN 201510561852A CN 105087600 B CN105087600 B CN 105087600B
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- Prior art keywords
- panax japonicus
- japonicus majoris
- pjbhlh1
- saponin
- gene
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Abstract
本发明公开了一种珠子参转录因子基因PjbHLH1的用途，即在提高珠子参皂苷生物合成关键酶基因表达量和增加珠子参愈伤组织中皂苷含量的应用，PjbHLH1基因核苷酸序列如SEQ ID NO︰1所示，编码bHLH类转录因子；本发明采用功能基因组学和代谢工程相关技术证明珠子参PjbHLH1转录因子具有正调控珠子参皂苷生物合成的功能，将本发明所述的珠子参PjbHLH1转录因子基因构建到植物表达载体上并转入珠子参愈伤组织中使其过量表达，增强了珠子参皂苷合成途径关键酶基因的表达量，提高了珠子参皂苷的产量。
Description
技术领域
本发明涉及分子生物学以及基因工程领域，尤其是一种珠子参皂苷生物合成转录因子基因PjbHLH1的应用。
背景技术
珠子参Panax japonicus C. A. Mey.var. major (Burk.) C. Y. Wu et K. M.Feng为五加科Araliaceae人参属Panax植物，因根茎节间纤细，节膨大成球形念珠状而得名，其根茎入药。珠子参作为药材，始载于《滇南本草》，为历版《中国药典》收载品种。药用珠子参主产于云南，属名贵常用中药材，是彝族、纳西族、白族、藏族、傈僳族等少数民族的传统用药。目前，野生珠子参主要分布于滇西北、滇东北地区，常见于海拔达2500-4000 m的亚高山针叶林及阔叶林下。珠子参性味苦、甘、微寒，归肝、肺、胃经，具有补肺养阴、祛瘀止痛，止血之功效，临床上应用于气阴两虚，烦热口渴，虚痨咳嗽，跌打损伤，关节疼痛，咳血，吐血，外伤出血等。
珠子参皂苷(Panax japonicus saponins, PJS)为珠子参的主要活性成分，包括达玛烷型、齐墩果烷型三萜皂苷。目前，已从珠子参根茎及叶中分离出了30多种皂苷成分，主要包括竹节参皂苷IVa、竹节参皂苷IV、竹节参皂苷V、人参皂苷Ro、人参皂苷Re、人参皂苷Rd、人参皂苷Rb1等。人参属的代表物种“人参”(Panax ginseng)主要含达玛烷型皂苷，齐墩果烷型皂苷仅发现含量极微的人参皂苷Ro；另一种人参属著名药材“三七”(Panax notoginseng)，只含达玛烷型皂苷，不含齐墩果烷型皂苷。珠子参所含皂苷组分与同属“人参”、“三七”相比，在成分种类以及各组分含量上均存在明显差异，因其含有大量齐墩果烷型皂苷，导致临床上的特殊用途。
珠子参为多年生草本植物，需生长6年以上方能入药。目前，珠子参药材多为野生品，无序采挖导致珠子参资源日渐枯竭，生境受到破坏。近些年，以珠子参为组分的药品市场迅速扩大，导致珠子参药材需求增长，供需矛盾突出。鉴于人工栽培时间长，化学合成机理和路线不够清晰等弊端，利用生物工程技术和基因调控的方法来生产珠子参皂苷逐渐成为研究热点。
转录因子对基因的转录激活是植物次生代谢过程重要的调控环节。利用转录因子作为改造植物代谢途径的工具，以其独有的“多点调控”优势，弥补了代谢工程操作中单个关键酶基因作用不足和多个关键酶基因可能产生组成性致死表达的情况，成为一种新的策略。次生代谢途径中多个功能相关的酶基因常被同一转录因子正调控或负调控，对转录因子进行基因修饰显然比多基因操作更容易改良目标次生代谢途径。
含碱性/螺旋-环-螺旋结构(basic/helix-loop-helix，bHLH)的转录因子广泛存在于生物中，是真核生物中一类含有众多成员的重要转录因子。越来越多的研究表明该类转录因子对真核生物的次生代谢过程具有重要调控作用。例如青蒿（Artemisia apiacea）AabHLH1转录因子可以提高青蒿素生物合成相关的紫穗槐4,11-二烯合酶（ADS）和细胞色素P450单加氧酶（CYP71AV1）基因的表达量，正调控青蒿素的合成；红豆杉（Taxus chinensis）bHLH类转录因子TcJAMYC可与紫杉醇生物合成相关基因启动子的E-box结合，激活这些关键酶基因的表达，提高紫杉醇的合成量。
随着对植物次生代谢网络的深入解析和调控机制的阐明，特别是调节特定次生代谢物合成的转录因子的分离和鉴定，基于转录因子的基因工程将为开发利用植物次生代谢物提供更加有效的手段。本发明以体外培养的珠子参愈伤组织为研究对象，克隆PjbHLH1转录因子基因，并对该转录因子进行分析和功能鉴定，明确PjbHLH1转录因子在珠子参皂苷生物合成过程中的地位和作用，为获得高效、稳定的珠子参皂苷合成调控技术和同源或异源高效表达系统的建立提供理论参考和依据。
发明内容
本发明的目的是提供一种珠子参转录因子基因PjbHLH1的用途，即珠子参转录因子基因PjbHLH1在提高珠子参皂苷生物合成关键酶基因表达量和增加珠子参愈伤组织中皂苷含量中的应用；本发明从珠子参中克隆获得可调控珠子参皂苷生物合成的转录因子基因PjbHLH1以及明确该转录因子的应用。
本发明基于同源克隆的原理，从珠子参中克隆得到bHLH类转录因子基因的cDNA并对其编码蛋白进行功能鉴定。发明人将这个基因命名为PjbHLH1，基因登录号为KP890785，其中所述cDNA如SEQ ID NO︰1所示。对该基因进行序列分析，表明PjbHLH1 cDNA大小为966bp，正好为开放阅读框（Open reading frame，ORF），编码含有321个氨基酸的蛋白质，氨基酸序列如SEQ ID NO︰2所示。利用植物表达载体，通过农杆菌介导法将本发明的PjbHLH1转录因子基因导入珠子参愈伤组织中，可提高珠子参皂苷合成途径关键酶基因的表达量，使珠子参皂苷的产量增加。
上述转录因子基因可应用于正调控珠子参皂苷的生物合成，具体操作如下：
（1）基因的获得：提取珠子参总RNA，反转录合成cDNA第一链；通过RT-PCR扩增出PjbHLH1的全长编码区，然后将其连接到pGEM-T easy载体上，经测序验证获得具有目的基因的克隆；
（2）植物表达载体构建与遗传转化：用限制性内切酶XbaI和PstI酶切pGEM-T-PjbHLH1质粒，通过胶回收获得目的基因片段。用同样的内切酶消化植物表达载体pCAMBIA2300S，胶回收获得载体大片段。将目的基因片段与pCAMBIA2300S载体片段连接，构建植物超表达载体pCAMBIA2300S-PjbHLH1。通过液氮冻融法将pCAMBIA2300S-PjbHLH1质粒导入农杆菌菌株EHA105中。利用农杆菌介导的遗传转化法，将PjbHLH1导入珠子参愈伤组织中使其过量表达。通过抗生素筛选和qRT-PCR筛选阳性转基因细胞系；
（3）转基因细胞系皂苷含量检测：提取珠子参转基因和非转基因细胞系中的皂苷，分析转基因和非转基因细胞系间皂苷含量的差异。
本发明为提高珠子参中皂苷的含量提供了一种新方法，利用生物工程技术和基因调控的方法可更高效率合成珠子参皂苷，克服了人工栽培周期长、化学合成机理和路线不够清晰等缺点。将转录因子PjbHLH1基因导入珠子参细胞中表达，使珠子参皂苷生物合成途径关键酶基因的表达量提高，增加了珠子参皂苷的产量，为大规模产业化生产珠子参皂苷提供了理论参考和科学依据。
附图说明
图1为本发明中珠子参总RNA电泳图谱；
图2为本发明中PjbHLH1 RT-PCR检测结果，其中M为DL2000 DNA Marker，1为PjbHLH1的RT-PCR产物；
图3为本发明中PjbHLH1的三维结构预测；
图4为本发明中qRT-PCR结果分析图，表示珠子参皂苷合成途径中受PjbHLH1调控的FPS基因在野生型和转基因细胞系中的表达水平，其中C为野生型细胞系，1-3为转基因细胞系；
图5为本发明中珠子参皂苷的含量测定结果，其中C为野生型细胞系，1-3为转基因细胞系。
具体实施方式
下面通过附图和实施例对本发明进一步说明，但本发明保护范围不局限于所述内容，实施例中方法如无特殊说明均为常规方法，使用的试剂如无特殊说明均为常规市售试剂或按常规方法配制的试剂。
实施例1：PjbHLH1基因的克隆以及生物信息学分析
因珠子参中含有较多的次生代谢物质，需采用两步法进行总RNA的提取。先采用改良的异硫氰酸胍法取进行粗提，接着使用DNA酶进行消化并利用氯仿抽提得到较纯的珠子参总RNA（图1）。采用逆转录酶M-MLV (promega)以珠子参总RNA为模板合成cDNA 第一链，反应体系和操作过程为：取5 μg Total RNA，依次加入50 ng oligo(dT) 15、2 μL dNTP (2.5mM each)、DEPC水至反应体积为13.5 μL；混匀后，70℃加热变性5 min后迅速在冰上冷却5min，然后依次加入4 μL 5×First-stand buffer、0.5 μL RNasin (200U)、1 μL M-MLV(200 U)，混匀并瞬时离心，42 ℃温浴1.5 h，取出后70 ℃加热10 min，终止反应。cDNA第一链合成后置于-20 ℃保存备用。
以合成的第一链cDNA为模板，根据三七中与调控三七皂苷生物合成相关的bHLH类转录因子基因cDNA序列设计特异性引物，扩增目的基因PjbHLH1，所用引物序列分别为5’-TCTAGAATGGAGGATCCTTATTCCAATATCC-3’以及5’-CTGCAGTCACATTAACTGTT TGAGAGCTGCG-3’。采用AdvantageTM 2 PCR Enzyme (Clontech)扩增出目的基因。PCR反应条件：94℃ 5 min；94 ℃ 30 s，54.5 ℃ 30 s，72 ℃ 1 min，32 cycles；72 ℃ 10 min。反应体系(50 μL)为4μL 第一链cDNA、5 μL 10×Buffer、1 μL 50×dNTP Mix、0.5 μL 正向引物(10 μM)、0.5 μL反向引物(10 μM)、1 μL AdvantageTM 2 PCR Enzyme、38 μL PCR-Grade Water。PCR结束后，取5 μL用于琼脂糖凝胶电泳，检测扩增产物的特异性以及大小（图2），其余PCR产物进行胶回收。将目的片段胶回收产物进行TA克隆，反应体系和操作过程为：取3.6 μL 胶回收产物，依次加入0.7 μL pGEM-T easy vector (50 ng/μL) 、5 μL 2×Rapid Ligation Buffer，和0.7 μL T4 DNA Ligase混匀置于8 ℃过夜反应。采用热激转化法将连接产物转入大肠杆菌Trans1-T1中。用含有氨苄青霉素(ampicillin，Amp)的LB固体培养基筛选阳性克隆。挑选若干个白色菌落，摇菌后用扩增PjbHLH1的特异引物鉴定出多克隆位点插入PjbHLH1的克隆，将鉴定的克隆进行测序。
最终获得的PjbHLH1 cDNA大小为966 bp，通过NCBI ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html)分析发现其正好为开放阅读框(见序列表)。PjbHLH1编码蛋白的分子量约为36.3 KD，等电点为8.36，不稳定系数为39.92，预测PjbHLH1编码的蛋白质稳定。生物信息学预测PjbHLH1不包含跨膜区，不含信号肽，具有一个bHLH转录因子特征保守结构域。通过在线工具iPSORT预测PjbHLH1可能定位于细胞核。借助SWISS-MODEL工具，PjbHLH1以Myc原癌基因蛋白为模板进行三维结构建模（图3），结果表明PjbHLH1与Myc原癌基因蛋白具有31.03%的序列相似性的空间结构。
实施例2：植物表达载体构建
采用SanPrep柱式质粒DNA小量抽提试剂盒(上海生工)提取插入PjbHLH1的大肠杆菌质粒pGEM-T- PjbHLH1以及植物表达载体pCAMBIA2300S的质粒，取1 μL用于琼脂糖凝胶电泳以检测所提取质粒的完整性和浓度高低。用XbaI (TaKaRa)和PstI (TaKaRa)分别对质粒pGEM-T- PjbHLH1和pCAMBIA2300S进行双酶切(100μL体系)，反应体系和操作过程为：取20 μL pGEM-T- PjbHLH1或pCAMBIA2300S质粒，依次加入10 μL 10×M buffer、5 μL Xba I、5 μL PstI、60 μL ddH2O，混匀后短时离心，置于37 ℃过夜反应。将所有酶切产物点于琼脂糖凝胶中进行电泳，然后对PjbHLH1片段和pCAMBIA2300S大片段分别进行胶回收，整个过程使用SanPrep柱式DNA胶回收试剂盒(上海生工)。取1 μL回收产物通过琼脂糖凝胶电泳检测回收片段的大小以及浓度，置于-20 ℃保存备用。
利用T4 DNA Ligase (TaKaRa)，将回收的PjbHLH1DNA片段和pCAMBIA2300S载体片段连接起来，反应体系(20 μL)和操作过程为：取10 μL PjbHLH1DNA片段依次加入2 μLpCAMBIA2300S载体DNA、2 μL 10×T4 DNA Ligase Buffer、1 μL T4 DNA Ligase、5 μLddH2O，混匀后短时离心，置于16 ℃水浴过夜反应。接着采用热激转化法将连接产物转入大肠杆菌Trans1-T1中，用含有50 mg/L卡那霉素(kanamycin，Km)的固体培养基筛选阳性克隆。挑选单菌落摇菌，以菌液为模板用扩增PjbHLH1的特异引物进行PCR，挑选出PjbHLH1与pCAMBIA2300S成功连接的克隆，所检测的菌株若为阳性，加入20%甘油混匀后置于-80 ℃保存备用。
用试剂盒提取并纯化上述大肠杆菌中的pCAMBIA2300S-PjbHLH1质粒。制备农杆菌EHA105菌株的感受态细胞并分装于1.5 mL离心管中，每管150 μL，液氮速冻后置于-80 ℃保存备用。采用液氮冻融法将上述构建的植物表达载体pCAMBIA2300S-PjbHLH1转入所制备的农杆菌EHA105感受态细胞中。操作步骤为：取3 μg pCAMBIA2300S-PjbHLH1质粒加入含有150 μL感受态细胞的离心管中，轻轻混匀后冰浴30 min，接着转入液氮中速冻5 min，然后迅速置于37 ℃水浴5 min，之后立即冰浴2 min，加入600 μL LB液体培养基，28 ℃振荡培养4 h。将活化后的农杆菌涂于含有50 mg/L Km的LB固体培养基上，28 ℃静置培养。挑选单菌落摇菌，用扩增PjbHLH1的特异引物进行PCR，检测pCAMBIA2300S-PjbHLH1是否转入农杆菌中。对于阳性克隆，加入甘油后置于-80 ℃保存备用。
实施例3：农杆菌介导的珠子参遗传转化
从-80 ℃冰箱中取出保存的含有pCAMBIA2300S-PjbHLH1质粒的农杆菌EHA105菌种，接种于5 mL含有50 mg/L Km和25 mg/L利福平的LB液体培养基中，28 ℃培养至浑浊。吸取1 mL浑浊的菌液至含有50 mg/L Km的LB固体培养基上，28 ℃培养48 h。将LB固体培养基上的农杆菌刮下适量接种于MGL液体培养基中，附加40 mg/L的乙酰丁香酮，28 ℃振荡培养至OD600为0.6时停止摇菌，所得菌液用于侵染。
将生长状态较好的珠子参愈伤组织接到MS预培养基（含35 mg/L乙酰丁香酮）上进行预培养3天。预培养完成后，将愈伤组织完全浸泡于上述农杆菌菌液中进行振荡培养。浸染完毕，除去菌液，用无菌的滤纸吸取愈伤组织表面的菌液，最后将愈伤组织接种到共培养基中，于25 ℃进行暗培养，时间为三天。浸染后的愈伤组织接种于MS共培养基（含35 mg/L乙酰丁香酮）中，暗培养3天，然后用无菌水对愈伤组织进行洗涤，再转接于含400 mg/L头孢霉素的MS培养基上进行除菌培养，于25 ℃暗培养15天，防止农杆菌过度生长。最后将愈伤组织转接到筛选培养基中，每45天继代一次。经过5次筛选，最终分离出具有Km抗性的增值速度快的纯的细胞系，用于后续检测。
实施例4：PjbHLH1基因超表达对珠子参皂苷合成途径关键酶基因FPS表达量的影响
选取25天左右、生长状态良好的珠子参转基因细胞系和野生型细胞系，分别提取RNA，然后按照GoTaq® 2-Step RT-qPCR System试剂盒说明书合成cDNA，反应体系和操作过程为：在离心管中加入4 µg 总RNA和1 µL Oligo(dT)15，用Nuclease-free Water补齐至10µL，将反应体系放于70 ℃变性5 min，然后置于冰上5 min。随后将离心管在离心机中短暂离心，使反应液收集于管底，再向其中加入4 µL GoScriptTM 5×Reaction Buffer、2 µLMgCl2 (25 mM)、1 µL PCR Nucleotide Mix(10 mM)、0.5 µL Recombinant RNasinRibonuclease Inhibitor和1 µL GoScriptTM Reverse Transcriptase，将整个反应体系漩涡混匀，离心收集到管底，将反应物置于42 ℃恒温水浴锅中反应1 h，再于70 ℃水浴中维持15 min以终止反应，最后将合成的cDNA置于-20 ℃冰箱中保存备用。
以该cDNA为模板，根据珠子参18S rRNA基因（登录号：AB088018.1）和法尼基焦磷酸合成酶（Farnesyl diphosphate synthase, FPS）基因（登录号：KP684141）设计引物，依据GoTaq® 2-Step RT-qPCR System试剂盒说明书进行荧光半定量PCR扩增珠子参内参基因和FPS基因。所用引物序列为18S F：5’-GTTGTTGCAGTTAAAAAGCTCGTAG-3’，18S R：5’-ACCTCTGACTATGAAATACGAATGC-3’；FPS F：5’-AGAATGAGCGATCTGAAGACG AG-3’；FPS B：5’-ACAGACAACAACTTCCCCTCCAT-3’。具体反应体系和操作过程为：在PCR管中加入20 ng cDNA、25 µL GoTaq® qPCR Master Mix (2×)和0.2 µL qPCR Primers(18S F /18S R, FPS B /FPS F, 10 mM)，用Nuclease-Free Water补齐至50 µL。将反应体系漩涡混匀后，离心将其收集到管底，随后将其置于荧光定量PCR仪中进行反应，采用两步法进行荧光定量PCR，反应参数如下：热启动 95 ℃ 2 min；变性 95 ℃ 15 s，退火/延伸 60 ℃ 1 min，共45个循环。每个样品对应的每个基因重复检测2次。
qRT-PCR结果显示，转PjbHLH1基因珠子参细胞中FPS基因的表达量要比野生型的高（图4），说明PjbHLH1作为转录因子，能够促进珠子参皂苷合成代谢途径中关键酶基因FPS的表达，图中，C表示对照组野生型细胞系，1、2和3分别表示不同的转基因细胞系实验组。
实施例5：PjbHLH1基因超表达对珠子参皂苷合成量的影响
选取生长35天左右的转基因细胞系和野生型细胞系，分别置于洗净的100 mL三角瓶中，加入20 mL的甲醇溶液浸泡过夜后，常温超声波处理1 h。过滤，收集滤液，将滤液挥干后再用甲醇溶解，定容至25 mL，得粗提液。将残渣在50 ℃下烘至恒重，称重。精密吸取粗提液5 mL，置50 mL烧杯中水浴蒸干。蒸干后用4倍体积的蒸馏水分次溶解，充分溶解后过滤，滤液全部转移至已处理好的Hsp100大孔树脂柱内，先用2个柱体积的蒸馏水慢慢洗去糖类等杂质。Molish反应检测糖类杂质是否去除干净，如果结果呈阳性，继续用蒸馏水洗至阴性，然后用75%乙醇溶液洗脱2个柱体积，收集醇液，水浴蒸干，残渣用甲醇溶液溶解，定容至25 mL。
精确吸取此样品150 μL至带塞的10 mL试管中（设3个重复），挥干溶剂，加入新配制的5%香草醛-冰醋酸溶液0.2 mL，高氯酸0.8 mL，混匀后60 ℃水浴加热15 min，立即用冰水冷却，加入5 mL冰醋酸，混匀静止10 min后554 nm测吸光度，参照标准曲线计算PJS含量。结果显示，转PjbHLH1基因珠子参细胞中皂苷含量明显高于野生型细胞中皂苷含量（图5），说明PjbHLH1转录因子参与了珠子参皂苷的合成代谢调控，有助于皂苷产量的提高。图中，C表示对照组野生型细胞系，1、2和3分别表示不同的转基因细胞系实验组。
序列表
<110> 昆明理工大学
<120> 一种珠子参转录因子基因PjbHLH1的应用
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<170> PatentIn version 3.5
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Ser Trp Ser Ser Glu Arg Thr Asp His Thr Ser Asn Thr Gln Ala Thr
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Tyr Ser Asn Tyr Thr Asn Gln Phe Gly Asn Val Lys Pro Lys Asp Glu
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Ser Gln Gly Ser Phe Glu Asn Gln Asn Tyr Val Phe Lys Ala Ser Gln
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Claims (2)
1.一种珠子参转录因子基因PjbHLH1在提高珠子参皂苷生物合成关键酶基因FPS表达量和增加珠子参愈伤组织中皂苷含量的应用，其特征在于：所述珠子参转录因子基因PjbHLH1的核苷酸序列如SEQIDNO︰1所示。
2.根据权利要求1所述珠子参转录因子基因PjbHLH1在提高珠子参皂苷生物合成关键酶基因FPS表达量和增加珠子参愈伤组织中皂苷含量的应用，其特征在于，具体操作如下：
（1）将珠子参转录因子基因PjbHLH1与植物超表达载体pCAMBIA2300S连接，构建植物超表达载体；
（2）将上述构建的表达载体通过农杆菌介导转入珠子参愈伤组织中；
（3）通过抗生素筛选珠子参转基因细胞系，qRT-PCR检测转基因细胞系中珠子参皂苷合成途径关键酶基因表达量；
（4）提取珠子参转基因和非转基因细胞系中的皂苷，分析转基因和非转基因细胞系间皂苷含量的差异，最后筛选出皂苷含量得到提高的阳性转基因细胞系。
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