CN105441462A - 一种三七转录因子基因PnERF1及其应用 - Google Patents
一种三七转录因子基因PnERF1及其应用 Download PDFInfo
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- CN105441462A CN105441462A CN201610001934.6A CN201610001934A CN105441462A CN 105441462 A CN105441462 A CN 105441462A CN 201610001934 A CN201610001934 A CN 201610001934A CN 105441462 A CN105441462 A CN 105441462A
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Abstract
本发明公开了一种三七转录因子基因<i>PnERF1</i>及其用途，即在提高三七皂苷生物合成关键酶基因表达量和增加三七愈伤组织中总皂苷与单体皂苷含量的应用，<i>PnERF1</i>基因核苷酸序列如SEQ？ID？NO︰1所示，编码AP2/ERF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三七PnERF1转录因子具有正调控三七皂苷生物合成的功能。将本发明所述的三七<i>PnERF1</i>转录因子基因构建到植物表达载体上并转入三七愈伤组织中使其过量表达，增强了三七皂苷合成途径关键酶基因的表达量，提高了三七总皂苷与单体皂苷的产量。
Description
技术领域
本发明涉及分子生物学以及基因工程领域，尤其是一种三七皂苷生物合成相关转录因子基因PnERF1及应用。
背景技术
三七Panax notoginseng (Burk.) F. H. Chen为五加科人参属植物，根和根茎入药，是云南的道地药材。三七作为传统中药已有悠久的应用历史，清代药学著作《本草纲目拾遗》中记载“人参补气第一，三七补血第一，味同而功亦等，故称人参三七，为中药之最珍贵者”。三七具有活血和止血的双重功效，是驰名中外的“云南白药”的重要成分。三七主产于我国云南省文山州，均为人工栽培品，全世界98%的三七原药材产于云南，三七已经成为云南省最重要的药材资源。
三七皂苷(saponins of Panax notoginseng, PNS)是三七的主要药用成分，由多种四环三萜皂苷组成。目前，已从三七的根块、根茎（剪口）、茎、叶和花等部位分离和鉴定出70余种达玛烷型四环三萜皂苷，如人参皂苷Rb1、Rg1、Rh1、Rd、Re和F1等，这些单体皂苷大多数为达玛烷20(S)-原人参二醇型[20(S)-protopanaxadiol]和20(S)-原人参三醇型[20(S)-protopanaxatriol]，未发现含有齐墩果酸型皂苷，这与人参和西洋参有显著差异。三七皂苷具有扩张冠状动脉和外周血管、增加脑血流量的作用；还有抑制血小板凝聚、降低血液粘稠度、抑制血栓形成的功效；同时，兼具降血脂、抗疲劳、耐缺氧，提高和增强巨噬细胞功能等作用。
三七为多年生草本植物，一般需生长3-7年方能入药，故名“三七”。三七对生境要求苛刻，适合种植三七的土地面积有限；同时，三七生长过程中病虫害严重，土壤次生盐渍化和酸化突出，导致三七种植需要“轮作”。这种生长周期长，土地利用率低下的现状，制约了三七产业的可持续发展。近些年，以三七为组分的药品市场迅速扩大，导致三七药材需求增长，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三七皂苷逐渐成为研究热点。
茉莉酸甲酯(MeJA)是植物重要次级代谢信号分子，在植物抗逆自我保护过程中扮演重要角色，能够引起细胞次生代谢物的合成。目前，有关茉莉酸甲酯介导的植物次生代谢合成过程已经从研究重要基因的表达逐步过渡到相关转录因子的研究。越来越多的证据表明，控制重要萜类次生代谢物合成的转录因子大多能被茉莉酸甲酯诱导表达。
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。
AP2/ERF(APETALA2/ETHYLENE
Responsive Factor)转录因子是植物所特有的一类转录因子，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。例如长春花(Catharanthus roseus)AP2/ERF类转录因子ORCA3可调控参与萜类吲哚生物碱生物合成的异胡豆苷合成酶基因的表达；青蒿(Artemisia
apiacea)AaERF1和AaERF2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；此外，红豆杉(Taxus
chinensis) TcAP2转录因子可调控紫杉醇生物合成关键酶基因的表达。
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三七愈伤组织为研究对象，克隆PnERF1转录因子基因，并对该转录因子进行分析和功能鉴定，明确PnERF1转录因子在三七皂苷生物合成过程中的地位和作用，为获得高效、稳定的三七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
本发明的目的是从三七中克隆获得可调控三七皂苷生物合成的转录因子基因PnERF1以及明确该转录因子的应用，即在提高三七皂苷合成代谢途径中关键酶基因表达量和增加三七愈伤组织中总皂苷与单体皂苷含量的应用。
本发明根据基因的保守结构域和cDNA末端快速扩增技术从三七中克隆到AP2/ERF类转录因子基因并对其编码蛋白进行功能鉴定。发明人将这个基因命名为PnERF1，其中所述的cDNA全长如SEQ ID NO︰1所示。对该基因进行序列分析，表明PnERF1全长cDNA大小为1113 bp，PnERF1编码区是序列表SEQ ID NO︰1中第127-927位所示的核苷酸序列，具有801 bp的开放阅读框(Open reading
frame, ORF) 、126 bp的5’非翻译区和186 bp的3’非翻译区，编码含有266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PnERF1转录因子基因导入三七愈伤组织中，可提高三七皂苷合成途径关键酶基因的表达量，使三七皂苷的产量增加。
上述转录因子基因可应用于正调控三七皂苷的生物合成，具体操作如下：
（1）基因全长cDNA的获得：首先根据长春花ORCA基因家族的保守结构域设计简并引物，扩增出AP2/ERF类转录因子基因的5’核心序列，再利用DNA末端快速扩增技术(3’RACE)获得PnERF1的全长cDNA，设计引物扩增PnERF1的ORF框，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SmaI酶切pGEM-T-PnERF1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-PnERF1；通过液氮冻融法将pCAMBIA2300S-PnERF1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PnERF1导入三七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）转基因细胞系总皂苷含量检测：提取三七非转基因和转基因细胞系中的皂苷，分析非转基因和转基因细胞系间总皂苷含量的差异；
（4）转基因细胞系部分重要单体皂苷含量检测：制备三七非转基因和总皂苷含量得到提高的转基因细胞系的皂苷溶液，利用HPLC法对非转基因和转基因细胞系中的部分重要单体皂苷含量进行测定，分析非转基因和转基因细胞系间单体皂苷含量的差异。
本发明为提高三七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三七皂苷，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PnERF1基因导入三七细胞中表达，使三七皂苷生物合成途径关键酶基因的表达量提高，增加了三七皂苷的产量，为大规模产业化生产三七皂苷提供了理论参考和科学依据。
附图说明
图1为本发明中三七总RNA电泳图谱；
图2为本发明中纯化mRNA电泳图谱，其中M为DL2000 DNA
Marker，1为纯化的mRNA；
图3为本发明中PnERF1全长cDNA扩增结果；其中M为DL2000 DNA
Marker，1为PnERF1全长cDNA扩增产物；
图4为本发明中PnERF1的三维结构预测；
图5为本发明中PnERF1基因ORF框扩增结果，其中M为DL2000 DNA Marker，1为PnERF1 ORF框扩增产物；
图6为本发明中qRT-PCR结果分析图，表示三七皂苷合成途径中受PnERF1调控的FPS、HMGR和SE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7为本发明中三七总皂苷含量测定结果，其中C为野生型细胞系，1-3为转基因细胞系；
图8为本发明三七细胞中部分重要单体皂苷含量测定结果，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：PnERF1基因的克隆以及生物信息学分析
首先根据长春花ORCA基因家族的保守结构域设计简并引物，从三七中扩增出PnERF1转录因子基因的5’核心序列。采用改良的异硫氰酸胍法提取经茉莉酸甲酯处理3-6 h的三七细胞总RNA（图1），参照NucleoTeap® mRNA
(MACHERGY-NAGEL)试剂盒进行mRNA的分离（图2），再利用SMARTerTM RACE
Cdna Amplification试剂盒制备RACE-Ready
cDNA。以合成的3’-RACE-Ready cDNA文库为模板，通过使用cDNA末端快速扩增技术(3’RACE)得到了PnERF1基因的3’片段，其引物为5’-CAGCGTCCATGGGGGAAATTCGCGGCGG-3’；再根据PnERF1基因的5’和3’末端序列设计引物，用于扩增PnERF1基因的cDNA全长，分别为PnERF1 F：5’-CATGGGGTCTCATCAAAAACAAATC-3’；PnERF1 R：5’- GATTTGATAAGTTG
TTCCCCTAAGC-3’；PCR反应条件：94℃，5 min；94℃，30 s；54℃，30 s；72℃，70 s，32个循环；72℃，10 min。将PCR产物用1.2%的琼脂糖凝胶分离（图3）、胶回收并连接到pGEM
T-easy载体上，转化大肠杆菌感受态，挑单克隆摇菌，菌液PCR检测后送测序。
最终获得的PnERF1全长cDNA大小为1113 bp，通过NCBI ORF finder
(http:// www.ncbi.nlm.nih.gov/gorf/gorf.html)分析发现其包含一个801 bp的ORF（见序列表）。PnERF1编码蛋白的分子量约为30.1
KD，等电点为6.09，不稳定系数为44.07，预测PnERF1编码的蛋白质不稳定。生物信息学预测PnERF1不包含跨膜区，不含信号肽，具有一个由64个氨基酸组成的AP2/ERF转录因子特征保守结构域。借助SWISS-MODEL对PnERF1进行三维结构预测分析（图4），结果表明，PnERF1有三个反向平行的β-折叠和一个α-螺旋，且在71-136位氨基酸处的结构与拟南芥AtERF1转录因子晶体结构的相似率达71.93%，这进一步说明PnERF1属于AP2/ERF类转录因子。
实施例2：植物表达载体构建
根据PnERF1基因ORF框的5’和3’末端序列设计引物，用于扩增PnERF1基因的ORF，分别为上游引物：5’-TCTAGAGTGCTCTTTAAACGACATCGTCGAA-3’；下游引物：5’-GGGCCCATGTGTGGAGGTGCAATCCTAGGTG-3’。PCR反应条件：94℃，5 min；94℃，30 s；58 ℃，30 s；72 ℃，50 s，32个循环；72 ℃，10 min。将PCR产物用1.2%的琼脂糖凝胶分离（图5）、胶回收并连接到pGEM
T-easy载体上，转化大肠杆菌感受态，挑单克隆摇菌，菌液PCR检测后送测序。
采用SanPrep柱式质粒DNA小量抽提试剂盒(上海生工)提取经测序正确的大肠杆菌质粒pGEM-T-PnERF1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SmaI (TaKaRa)分别对质粒pGEM-T-PnERF1和pCAMBIA2300S进行双酶切(100 μL体系)，反应体系和操作过程为：取20 μL pGEM-T-PnERF1或pCAMBIA2300S质粒，依次加入10 μL 10×M buffer、5 μL Xba I、5 μL SmaI、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PnERF1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PnERF1 DNA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 PnERF1 DNA片段依次加入2 μL pCAMBIA2300S载体DNA、2 μL 10×T4 DNA Ligase Buffer、1 μL T4 DNA Ligase、5 μL ddH2O，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PnERF1的特异性引物进行PCR，挑选出PnERF1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-PnERF1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-PnERF1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-PnERF1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PnERF1的特异引物进行PCR，检测pCAMBIA2300S-PnERF1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：农杆菌介导的三七遗传转化
从-80 ℃冰箱中取出保存的含有pCAMBIA2300S-PnERF1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。
将生长状态较好的三七愈伤组织接种到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，再将愈伤组织接种到MS共培养基（含35 mg/L乙酰丁香酮）上进行共培养3天。共培养完成后，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：PnERF1基因超表达对三七皂苷合成途径关键酶基因FPS、HMGR和SE表达
量的影响
选取25天左右、生长状态良好的三七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10 µL，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 µL MgCl2 (25 mM)、1 µL PCR
Nucleotide Mix(10 mM)、0.5
µL Recombinant RNasin Ribonuclease
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，根据三七GAPDH基因（登录号：KF815711.1）、法尼基焦磷酸合成酶(Farnesyl diphosphate synthase, FPS)基因（登录号：DQ059550.1）、3-羟基-3-甲基戊二酰辅酶A还原酶(3-hydroxy-3-methylglutaryl-CoA reducetase,
HMGR)基因（登录号：KJ578757.1）以及鲨烯环氧酶(Squalene epoxidase, SE)基因（登录号：KC422651.1）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扩增三七内参基因和皂苷合成途径关键酶基因。所用引物序列为GAPDH F：5’-
CTACCAACTGTCTTGCTCCCCT-3’，GAPDH R：5’-TGATGCAGCTCTTCCACCTCTC-3’；FPS F：5’-CGGATGCTGGACTATAATG
TG-3’，FPS R：5’-ATTTACGGCAATCATACCAACC-3’；HMGR F：5’-GGCAGGACCCAGCAC
AAAATA-3’，HMGR R：5’-ACACCCAGAAGGTTCAAGCAA-3’； SE F：5’-TGGTTGATTTGC
CTGGAC-3’，SE R：5’-AATTGGACGCGGGTTTAG-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(GAPDH F
/ GAPDH R, FPS F / FPS R,
HMGR F / HMGR R, SE F /
SE R, 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PnERF1基因三七细胞中FPS、HMGR和SE基因的表达量都比野生型的高（图6），说明PnERF1作为转录因子，能够促进三七皂苷合成代谢途径中关键酶基因FPS、HMGR和SE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：PnERF1基因超表达对三七总皂苷合成量的影响
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PNS含量。结果显示，转PnERF1基因三七细胞中总皂苷含量高于野生型细胞中总皂苷含量（图7），结合qRT-PCR结果，表明PnERF1转录因子参与了三七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。
实施例6：PnERF1基因超表达对三七单体皂苷合成量的影响
利用HPLC法测定三七细胞系中部分重要单体皂苷（Rb1、Rg1、Rh1、Rd、Re和F1）的含量。高效液相色谱条件为：高效液相色谱仪（戴安ULTIMATE
3000 LPG-3400A四元梯度泵，WPS-3000SL自动进样器，PDA-3000二极管阵列检测器，TCC-3000柱温箱），Waters symmertry C18色谱柱(4.6×250mm, 5 µm)，采用乙腈(A)：水(B)为流动相进行线性梯度洗脱(v/v)，检测柱温30℃，检测波长为203 nm，流速设定为1.0 mL/min。
精确称取适量的单体皂苷Rb1、Rg1、Rh1、Rd、Re和F1标准品，加入1 mL甲醇溶液，制成浓度分别为340，300，260，320，280，300 µg/mL的标准品混合溶液。分别吸取4，6，8，10，15，20，25，30 µL的混合标准品溶液注入高效液相色谱仪，根据上述色谱条件进行测定。以进样量(µg)为横坐标(x)，色谱峰的面积为纵坐标(y)，绘制得出各单体皂苷的线性回归方程。
分别称取0.1 g非转基因和总皂苷含量得到提高的转基因三七细胞粉末于干净的50 mL三角瓶中，各自加入10 mL 70%甲醇溶液，过夜浸泡后用超声破碎仪处理90
min(60 w，4 s/5 s)。超声结束后，去滤渣留滤液，将滤液放置在50 ℃烘箱中过夜烘干。加10 mL蒸馏水溶解烘干后的残余物，之后用相同体积的水饱合正丁醇（勿使用未水饱合过的正丁醇）萃取2-3次，将最终收集到的萃取液放置在50 ℃烘箱中过夜烘干。用适量100%甲醇溶解烘干后的残余物，之后将溶液定容至5 mL，混合均匀后经0.45 μm滤膜过滤，利用高效液相色谱测定皂苷溶液中部分重要单体皂苷的含量。结果显示，非转基因与转基因三七细胞系均检测到了Rb1、Rg1、Rh1、Rd、Re和F1这六种单体皂苷，转基因细胞系与非转基因细胞系相比，这六种单体皂苷均出现不同幅度增长（图8）。目前普遍认为，T-DNA插入到宿主基因组上的位点具有随机性，但本实验结果证明这种随机性当中可能蕴含着某种定向（趋向）性。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> 一种三七转录因子基因PnERF1及其应用
<160> 15
<170> PatentIn version 3.5
<210> 1
<211> 1113
<212> DNA
<213> Panax notoginseng
<220>
<221> mRNA
<222> (1)..(1113)
<220>
<221> 5'UTR
<222> (1)..(126)
<220>
<221> CDS
<222> (127)..(927)
<220>
<221> 3'UTR
<222> (928)..(1113)
<400> 1
acatggggtc tcatcaaaaa caaatctatt tccaaacaat tccctcaagt
tttgcctcaa 60
atattattac catcatttag ttcaagttct ctcatatcaa gcaaagattt
cattttctga 120
ttcaaaatgt gtggaggtgc aatcctaggt gatcttaccg ctcgaaattt
caaccgccgt 180
gtctccgccg ctgacttctg gcccacatct ctctccgaaa aactcgacaa
tttccagtcc 240
gaatttaatc atttccctca agaggagatt cgaaccctca aaagagcgct
gcctaattca 300
ggtggtgtac ctctcggaaa gacagcaaag aggcagagga agaatatgta
cagaggtata 360
aggcagcgtc catgggggaa atgggcagcg gagattaggg atccgagaaa
aggagtgagg 420
gtttggctgg gtactttcaa cacggctgag gaggctgcca gagcctacga
caaagaagct 480
cgcaagatta gaggaaacaa agccaaagtt aacttcccaa atgaggactg
ctttaatcaa 540
ttcaatgtca aaaatatgaa tcaatttggg tcaaattctc gttctgggtt
ttctgcattg 600
aacagggagg accaatcacc tttggatttt acctgtctga aaaatggaag
cgatggactt 660
agtattgctg agcaggtggt gaaggtaaaa gaagaaaaag aggagagaga
aaataaagag 720
agtgtgattg atcaagtgga ggaacagaac gaactgcaga agctctcgga tgagctaatg
780
gcctacgaat cttacatgaa attttatgaa attccgtatc ttgatggcca
gtcagcgacg 840
gcggcagcag ctccgacaag tgcggtgctg gaaaacgtcg tggacggtgg
tttgctaaat 900
ctttggagct tcgacgatgt cgtttaaaga gccctgtctg ttaaattgac
aacaacattg 960
ttttgctttc tattttaatt ttattaatgg gtagctgtaa attcgttgga
ataattattt 1020
gtaagactga agttttaata gcttagggga acaacttatc aaatcaattt
attatttatt 1080
ttttgaaaaa aaaaaaaaaa aaaaaaaaaa
aaa
1113
<210> 2
<211> 266
<212> PRT
<213> Panax notoginseng
<400> 2
Met Cys Gly Gly Ala Ile Leu Gly Asp Leu Thr Ala Arg Asn Phe Asn
1
5
10
15
Arg Arg Val Ser Ala Ala Asp Phe Trp Pro Thr Ser Leu Ser Glu Lys
20 25
30
Leu Asp Asn Phe Gln Ser Glu Phe Asn His Phe Pro Gln Glu Glu Ile
35
40
45
Arg Thr Leu Lys Arg Ala Leu Pro Asn Ser Gly Gly Val Pro Leu Gly
50
55
60
Lys Thr Ala Lys Arg Gln Arg Lys Asn Met Tyr Arg Gly Ile Arg Gln
65
70
75
80
Arg Pro Trp Gly Lys Trp Ala Ala Glu Ile Arg Asp Pro Arg Lys Gly
85
90
95
Val Arg Val Trp Leu Gly Thr Phe Asn Thr Ala Glu Glu Ala Ala Arg
100
105
110
Ala Tyr Asp Lys Glu Ala Arg Lys Ile Arg Gly Asn Lys Ala Lys Val
115 120
125
Asn Phe Pro Asn Glu Asp Cys Phe Asn Gln Phe Asn Val Lys Asn Met
130
135
140
Asn Gln Phe Gly Ser Asn Ser Arg Ser Gly Phe Ser Ala Leu Asn Arg
145 150
155
160
Glu Asp Gln Ser Pro Leu Asp Phe Thr Cys Leu Lys Asn Gly Ser Asp
165
170
175
Gly Leu Ser Ile Ala Glu Gln Val Val Lys Val Lys Glu Glu Lys Glu
180
185
190
Glu Arg Glu Asn Lys Glu Ser Val Ile Asp Gln Val Glu Glu Gln Asn
195
200
205
Glu Leu Gln Lys Leu Ser Asp Glu Leu Met Ala Tyr Glu Ser Tyr Met
210
215
220
Lys Phe Tyr Glu Ile Pro Tyr Leu Asp Gly Gln Ser Ala Thr Ala Ala
225
230
235
240
Ala Ala Pro Thr Ser Ala Val Leu Glu Asn Val Val Asp Gly Gly Leu
245
250
255
Leu Asn Leu Trp Ser Phe Asp Asp Val Val
260
265
<210> 3
<211> 28
<212> DNA
<213> 人工序列
<400> 3
cagcgtccat gggggaaatt
cgcggcgg
28
<210> 4
<211> 25
<212> DNA
<213> 人工序列
<400> 4
catggggtct catcaaaaac
aaatc
25
<210> 5
<211> 25
<212> DNA
<213> 人工序列
<400> 5
gatttgataa gttgttcccc
taagc
25
<210> 6
<211> 31
<212> DNA
<213> 人工序列
<400> 6
tctagagtgc tctttaaacg acatcgtcga
a
31
<210> 7
<211> 31
<212> DNA
<213> 人工序列
<400> 7
gggcccatgt gtggaggtgc aatcctaggt
g
31
<210> 8
<211> 22
<212> DNA
<213> 人工序列
<400> 8
ctaccaactg tcttgctccc
ct
22
<210> 9
<211> 22
<212> DNA
<213> 人工序列
<400> 9
tgatgcagct cttccacctc
tc
22
<210> 10
<211> 22
<212> DNA
<213> 人工序列
<400> 10
cggatgctgg actataatg tg
22
<210> 11
<211> 22
<212> DNA
<213> 人工序列
<400> 11
atttacggca atcataccaa
cc
22
<210> 12
<211> 21
<212> DNA
<213> 人工序列
<400> 12
ggcaggaccc agcacaaaat
a
21
<210> 13
<211> 21
<212> DNA
<213> 人工序列
<400> 13
acacccagaa ggttcaagca
a
21
<210> 14
<211> 18
<212> DNA
<213> 人工序列
<400> 14
tggttgattt
gcctggac
18
<210> 15
<211> 18
<212> DNA
<213> 人工序列
<400> 15
aattggacgc
gggtttag
18
Claims (2)
1.一种三七转录因子基因PnERF1，其特征在于：其核苷酸序列如SEQ ID NO︰1所述。
2.权利要求1所述的三七转录因子基因PnERF1在提高三七皂苷合成代谢途径中关键酶基因表达量和增加三七愈伤组织中总皂苷与单体皂苷含量的应用。
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| CN110484562A (zh) * | 2019-09-25 | 2019-11-22 | 福建农林大学 | 一种利用人参转录因子提高水稻甾醇含量的方法 |
| CN112831504A (zh) * | 2021-03-16 | 2021-05-25 | 昆明理工大学 | 三七WRKY转录因子基因PnWRKY9及其应用 |
| CN115725620A (zh) * | 2022-09-12 | 2023-03-03 | 昆明理工大学 | 一种在三七细胞中合成竹节参皂苷的方法 |
| CN116218877A (zh) * | 2023-04-28 | 2023-06-06 | 昆明理工大学 | 三七WRKY转录因子PnWRKY12的应用 |
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| CN109627303A (zh) * | 2018-12-11 | 2019-04-16 | 昆明理工大学 | 三七病程相关蛋白PnPR3的基因及其应用 |
| CN109627303B (zh) * | 2018-12-11 | 2021-11-12 | 昆明理工大学 | 三七病程相关蛋白PnPR3的基因及其应用 |
| CN110484562A (zh) * | 2019-09-25 | 2019-11-22 | 福建农林大学 | 一种利用人参转录因子提高水稻甾醇含量的方法 |
| CN112831504A (zh) * | 2021-03-16 | 2021-05-25 | 昆明理工大学 | 三七WRKY转录因子基因PnWRKY9及其应用 |
| CN112831504B (zh) * | 2021-03-16 | 2023-03-24 | 昆明理工大学 | 三七WRKY转录因子基因PnWRKY9及其应用 |
| CN115725620A (zh) * | 2022-09-12 | 2023-03-03 | 昆明理工大学 | 一种在三七细胞中合成竹节参皂苷的方法 |
| CN115725620B (zh) * | 2022-09-12 | 2023-09-15 | 昆明理工大学 | 一种在三七细胞中合成竹节参皂苷的方法 |
| CN116218877A (zh) * | 2023-04-28 | 2023-06-06 | 昆明理工大学 | 三七WRKY转录因子PnWRKY12的应用 |
| CN116218877B (zh) * | 2023-04-28 | 2023-11-24 | 昆明理工大学 | 三七WRKY转录因子PnWRKY12的应用 |
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