CN113265408A - 一种三七DOF转录因子基因PnDof1及其应用 - Google Patents
一种三七DOF转录因子基因PnDof1及其应用 Download PDFInfo
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- CN113265408A CN113265408A CN202110582118.XA CN202110582118A CN113265408A CN 113265408 A CN113265408 A CN 113265408A CN 202110582118 A CN202110582118 A CN 202110582118A CN 113265408 A CN113265408 A CN 113265408A
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Abstract
本发明公开了一种三七DOF转录因子基因PnDof1,其核苷酸序列如SEQ ID NO:1所述,编码如SEQ ID NO:2所示氨基酸序列的蛋白质;本发明通过功能基因组学相关技术研究证实PnDof1基因具有促进三七细胞系中皂苷合成的功能,将本发明DOF转录因子基因PnDof1构建到植物表达载体上并转入三七细胞中过量表达,增加了三七细胞系中皂苷的含量并调控了三七皂苷合成途径中相关酶基因的表达,PnDof1基因的过表达提高了三七细胞中几种重要单体皂苷的含量以及总皂苷的含量。
Description
技术领域
本发明涉及分子生物学以及基因工程技术领域,特别是一种能够促进三七细胞中皂苷合成的三七DOF转录因子基因PnDof1及应用。
背景技术
三七[Panaxnotoginseng(Burk.) F. H. Chen]是五加科人参属多年生药用草本植物,具有定痛消肿、止血散淤、抗癌、抗高血压、提高免疫力和保护心脑血管等多种功效,被广泛用作临床药物和滋补产品。三七皂苷(PNS)是三七中具有多种生物活性的一类重要植物次生代谢产物。其合成受多种因素的影响,包括环境及三七皂苷合成通路中催化酶基因的活性等。因为田间环境时常不可控,所以研究三七基因对三七皂苷积累的影响成为重要研究方向。在三七中,皂苷在主根与剪口中积累量最多,三七皂苷在体内的合成受多种催化酶和转录因子共同调控。三七皂苷主要通过甲羟戊酸(mevalonate,MVA)途径合成,包括异戊烯基二磷酸异构酶(isopentenylallyldiphosphate,IPP)、二甲基烯丙基焦磷酸酯(dimethylallyldiphosphate,DMAPP)的合成及2,3-氧化鲨烯(2,3-oxidosaqualene)的合成、环化、羟基化和糖基修饰等过程。还涉及乙酰辅酶A酰基转移酶(Acetyl-CoAacetyltransfeaase,AACT)、鲨烯环氧酶(squaleneepoxidase,SE)、达玛烯二醇合成酶(dammarenediol synthase,DS)等多种关键合成酶。
DOF是植物特异性转录因子家族中的一员,具有一个特别的单锌指保守DNA结合域,能与特定的DNA相结合。Yan等人在玉米(Zea mays)的叶、茎和根中首次发现Dof基因,并且发现其参与玉米的组织特异性表达,是一种转录激活因子(Yanagisawa, S.Involvement of maize dofzinc finger proteins in tissue-specific and light-regulated gene expression. Plant Cell, 1998, 10(1): 75-89)。之后陆续在小麦(Triticumaestivum)、番茄(Lycopersiconesculentum)、拟南芥(Arabidopsis thaliana)等多种作物中都有Dof基因参与生长发育调控的报道。研究结果表明其在植物维管发育、光敏色素信号传导、种子萌发、氮同化、光合过程和对非生物胁迫的抗性等方面都有广泛的参与(Ahmad M, Rim Y, Chen H,et al. Functional characterization ofarabidopsisDof transcription factor AtDof4.1.Russian Journal of PlantPhysiology, 2013,60(1):116-123)。
迄今为止,关于三七Dof基因的研究还未见报道。因此,对三七Dof基因进行功能研究具有重要的意义。
发明内容
本发明目的是提供一种从三七中克隆的DOF转录因子的全长基因PnDof1,DOF转录因子基因PnDof1核苷酸序列如SEQ IDNO:1所示,该基因cDNA全长序列为1057bp,包含一个729bp的开放阅读框、240bp的5’非翻译区、88bp的3’非翻译区,编码如SEQ IDNO:2所示氨基酸序列的蛋白质。
本发明中PnDof1基因的编码区是序列SEQ IDNO:1中第241-969位所示的核苷酸序列。
本发明分离三七的一个调控皂苷合成相关基因的完整cDNA片段,利用根癌农杆菌(Agrobacterium tumefaciens)介导将目的基因转入三七细胞系中过量表达,通过进一步实验验证该基因是否具有促进三七皂苷合成的功能,为后期利用该基因改良三七合成皂苷的能力奠定基础,发明人将这个基因命名为PnDof1。
本发明涉及分离包含PnDof1的DNA片段并鉴定其功能,其中所述DNA片段如序列表SEQ IDNO:1所示。对该基因进行序列分析,发现PnDof1全长cDNA为1057bp,包含一个729bp的开放阅读框(ORF)、240bp的5’非翻译区(untranslated region,UTR)、88bp的3’UTR,其中ORF编码一个具有242个氨基酸的蛋白质,PnDof1编码蛋白具有DOF蛋白的保守结构域,表明其属于三七中的DOF转录因子。
将上述PnDof1基因应用于提高三七皂苷的积累,以及调控三七皂苷合成途径中相关酶基因的表达,具体操作如下:
(1)采用异硫氰酸胍法提取三七根的总RNA,以提取的RNA为模板,以oligo(dT)18为反转录引物,通过逆转录-聚合酶链式反应(reverse transcription-polymerase chainreaction,RT-PCR)扩增出PnDof1的编码区,然后将其连接到pGEM-T载体上,经测序获得具有目的基因的克隆;
(2)用限制性内切酶KpnⅠ和HindⅢ酶切pGEM-T-PnDof1,通过胶回收得到目的基因片段,用同样的内切酶酶切植物表达载体pCAMBIA2300s,胶回收获得所需载体大片段,再将所获得PnDof1基因片段与pCAMBIA2300s片段连接,构建植物超表达载体,之后将所构建的重组载体通过根癌农杆菌介导转入三七细胞中表达;
(3)以重组载体pCAMBIA2300s-PnDof1上具有的抗性标记筛选转化子,并通过PCR检测得到真正的转基因细胞系,分析转基因细胞的PnDof1表达水平和三七中皂苷的含量;
本发明为提高三七皂苷的含量提供了一种新的方法,通过利用基因工程技术更高效率合成三七皂苷,克服了三七人工栽培育种周期长、性状不稳定难以筛选等缺点。将DOF转录因子PnDof1基因导入三七细胞中,使其在三七细胞系中过量表达,增加了三七细胞中皂苷的含量并调控了皂苷合成途径中相关酶基因的表达;这些结果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,因此本发明具有广阔的市场应用前景。
附图说明
图1是本发明部分PnDof1转基因三七细胞系基因组DNA的PCR检测结果,其中Marker为DL15000 DNA Marker(大连宝生物),阴性对照是以无菌水为模板的PCR反应,阳性对照是以pGEM-T-PnDof1质粒为模板的PCR反应;
图2是本发明阳性PnDof1转基因三七细胞系中PnDof1转录水平的表达分析结果图;其中O-1~O-8为过表达PnDof1基因的转基因三七细胞系;N-1、N-2为非转基因三七细胞系;a~g等不同的字母代表差异达到显著水平(P<0.05);
图3是在转基因三七细胞系中皂苷合成途径中相关酶基因的表达水平图;其中O-2、O-5、O-7与O-8为筛选得到的四个过表达PnDof1基因的转基因三七细胞系;N-1为非转基因三七细胞系;*表示与非转基因三七细胞相比差异显著(P<0.05),**表示与非转基因三七细胞相比差异极显著(P<0.01);图中所涉及的三七皂苷合成途径中的关键酶包括:达玛烯二醇合成酶(Dammarenediol synthase,DS)、环阿屯醇合成酶(Cycloartenol synthase,CAS)、鲨烯环氧酶(Squaleneepoxidase,SE)、乙酰辅酶A酰基转移酶(Acetyl-CoAacetyltransfeaase,ACAT)、异戊烯基二磷酸异构酶(Isopentenyldiphosphateisomerase,IDI)、羟甲基戊二酰辅酶A还原酶 (3-hydroxy-3-methylglutarylcoenzyme-A reductase,HMCAR)、甲羟戊酸激酶(Mevalonate kinase,MVK);
图4是本发明过表达三七细胞系中几种单体皂苷(a图)和总皂苷(b图)的含量图;其中a、b图示中的N-1为非转基因三七细胞,O-2、O-5、O-7与O-8为筛选得到的四个过表达PnDof1基因的转基因三七细胞系;*表示与非转基因三七细胞相比差异显著(P<0.05),**表示与非转基因三七细胞相比差异极显著(P<0.01)。
具体实施方式
下面通过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:PnDof1全长基因克隆以及序列分析
取三七根提取总RNA,用液氮将三七须根研磨成粉末,然后转入离心管中,采用异硫氰酸胍法提取总RNA,采用逆转录酶M-MLV(promega)以总RNA为模板合成cDNA第一链,反应体系和操作过程为:取5μg Total RNA,依次加入50ng oligo(dT),2μL dNTP(2.5mMeach)、DEPC水至反应体积为14.5μL;混匀后,70℃加热变性5min后迅速在冰上冷却5min,然后依次加入4μL 5×First-stand buffer、0.5μL RNasin(200U)、1μL M-MLV(200U),混匀并短时离心,42℃温浴1.5 h,取出后70℃加热10min,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,扩增目的基因PnDof1;所用上下游引物序列分别为5’ATGTCATCGGAGTCCGGC3’及5’AGTTGACACCATTGCCTGGA3’。采用AdvantageTM 2 PCR Enzyme(Clontech)扩增出目的基因;PCR反应条件:94℃、5min;94℃、30s,55℃、30s,72℃、32s,32个循环;72℃、7 min;反应体系(10μL)为1μL cDNA、1μL10×Advantage 2 PCR Buffer、0.5μL 50×dNTP Mix (10mM each)、0.2μL 正向引物(10μM)、0.2μL反向引物(10μM)、0.2μLAdvantage 2 PCR Polymerase Mix、6.9μL PCR-Grade water;PCR结束后,取5μL用于琼脂糖凝胶电泳,以检测扩增产物的特异性以及大小。
对PCR产物进行TA克隆,使用的试剂盒为pGEM-T Vector SystemⅠ(Promega),反应体系和操作过程为:取1.5μL PCR产物,依次加入1μLpGEM-T Vector(50ng/μL)和2.5μL 2×Ligation solution I,混匀后置于16℃过夜反应。采用热激转化法将连接产物转入大肠杆菌DH5α中;使用含有氨苄青霉素(ampicillin,Amp)的LB固体培养基筛选阳性克隆,挑选若干个单菌落,摇菌后用扩增PnDof1的特异引物鉴定出多克隆位点插入PnDof1的克隆,将所鉴定的克隆进行测序,最终获得的PnDof1全长cDNA为1057bp,通过NCBI ORF finder(http://www.ncbi.nlm.nih.gov/gorf/gorf.html)分析发现其包含一个729bp的开放读码框,PnDof1编码一个含242个氨基酸的蛋白质,其分子量约为24.90KDa,等电点约为8.41。PnDof1编码的蛋白质序列具有DOF转录因子中一个特别的单锌指保守DNA结合域,这表明其属于三七中的Dof基因。
实施例2:植物超表达载体构建
采用SanPrep柱式质粒DNA小量抽提试剂盒(上海生工)提取插入PnDof1的大肠杆菌质粒pGEM-T-PnDof1以及植物表达载体pCAMBIA2300s的质粒,取1μL用于琼脂糖凝胶电泳以检测所提取质粒的完整性及浓度高低;用限制性内切酶KpnⅠ(TaKaRa)和HindⅢ(TaKaRa)分别对质粒pGEM-T-PnDof1和pCAMBIA2300s进行双酶切(50μL体系);反应体系和操作过程为:取5μgpGEM-T-PnDof1和pCAMBIA2300s质粒,依次加入5μL 10×M buffer、2.5μLKpnI、2.5μL HindⅢ,补齐ddH2O至总体积为50μL,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PnDof1片段和pCAMBIA2300s载体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;取1μL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PnDof1片段和pCAMBIA2300s载体片段连接起来,反应体系(10μL)和操作过程为:取6μL PnDof1片段依次加入2μL pCAMBIA2300s载体DNA、1μL 10×T4 DNA Ligase Buffer、1μL T4 DNA Ligase,混匀后短时离心,然后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DH5α中,用含有50mg/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PnDof1的特异引物进行PCR,挑选出PnDof1与pCAMBIA2300s成功连接的克隆,所检测的菌株若为阳性,加入甘油并置于-80℃保存备用。
提取并纯化上述大肠杆菌中的pCAMBIA2300s-PnDof1质粒;随后用液氮冻融法将上述构建的植物表达载体pCAMBIA2300s-PnDof1转入根癌农杆菌LBA4404感受态细胞中。操作步骤为:取2μg pCAMBIA2300s-PnDof1质粒加入含有200μL感受态细胞的离心管中,轻轻混匀后冰浴5min,随后转入液氮中冷冻1min,然后迅速置于37℃水浴5min,之后立即冰浴2min,加入800μL LB液体培养基于28℃振荡培养4h;将活化后的农杆菌涂于含有50mg/L Km的LB固体培养基上,28℃静置培养。挑选单菌落摇菌,再用扩增PnDof1的特异性引物进行PCR,检测pCAMBIA2300s-PnDof1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:农杆菌介导的遗传转化以及转基因三七细胞系筛选
从-80℃冰箱中取出保存的含有pCAMBIA2300s-PnDof1质粒的农杆菌LBA4404菌种,接种于含有50mg/L Km和25mg/L利福平(rifampicin,Rif)的LB固体培养基中,28℃培养48h;随后挑取单克隆于600μL含有50mg/LKm和25mg/LRif等两种抗生素的LB液体培养基中,并在28℃、180 rpm条件下培养至菌液浑浊。进行菌液PCR筛选阳性克隆;吸取适量的阳性菌液,均匀涂布于含有50mg/LKm和25mg/LRif等两种抗生素的LB固体培养基中,28℃培养直至平板长满菌苔;用无菌接种环轻轻刮取适量菌苔,移至含有40mg/L乙酰丁香酮(acetosyringone,AS)的MGL培养基中,在28℃、180 rpm条件下培养至菌液浑浊,以活化农杆菌用于三七细胞的遗传转化。
选择生长状态较好的三七细胞作为转化材料,在25℃条件下于三七细胞预培养基中暗培养3~4天;接着把预培养后的三七细胞完全浸没于含有活化农杆菌的MGL培养基中,在28℃、120rpm条件下培养20 min,进行三七细胞的浸染;浸染后,用布氏漏斗进行抽滤,并用已灭菌的干燥滤纸吸干三七细胞表面残余的菌液,然后将细胞转移至三七共培养培养基(先在培养基表层平铺一层滤纸,防止农杆菌生长过快),于28℃暗培养3~5天;对于长出农杆菌的细胞,使用含400mg/L头孢噻肟钠(cefotaxime sodium salt,Cef)的无菌水进行清洗和抽滤,并充分吸干三七细胞表面残留液体,之后将细胞转移至除菌培养基中继续培养;除菌培养结束后,将三七细胞转移至筛选培养基中,根据细胞生长情况进行继代培养,约30天继代一次。
采用CTAB法提取转基因三七细胞系的基因组DNA,将提取的基因组DNA取1μL通过琼脂糖凝胶电泳检测其完整性和浓度,以转基因三七细胞系的基因组DNA为模板用扩增PnDof1的特异引物进行PCR,PCR结束后,取8μL产物用于琼脂糖凝胶电泳以检测阳性三七细胞系,如图1所示,PnDof1转基因三七细胞系共筛选到8个阳性转基因三七细胞系。
实施例4:PnDof1调控皂苷合成途径中的关键酶基因
提取阳性转基因三七细胞系(O-1~O-8)和未转基因三七细胞总RNA,并将总RNA逆转录为cDNA。设计三七皂苷合成途径中关键酶基因(DS、SE、CAS、ACAT、IDI、HMCAR、MVK)的定量PCR引物,引物序列见SEQ IDNO:5~SEQ IDNO:18。采用qRT-PCR的方法对转基因三七细胞系中PnDof1的表达水平以及上述几个关键酶基因的表达量进行定量分析。qRT-PCR反应体系如下:1µLcDNA、10µL 2×Hieff®qPCRSYBR® Green Master Mix、1µLPrimer QF (10 mM)、1 µL Primer QR(10 mM)、7 µLRNase-Free ddH2O;PCR反应条件:94℃,5min,1个循环;94℃、15s,60℃、60s,40个循环;熔解曲线分析:60-95℃。以上每个qRT-PCR反应均做3个重复。为保证实验结果的可靠性,本次实验将cDNA模板稀释至同一水平,并利用2-∆∆Ct法对荧光定量数据进行计算。qRT-PCR结果表明PnDof1在几个转基因细胞系中大量表达(图2),并且PnDof1的过表达显著提高了MVK、ACAT、CAS、HMCAR等皂苷合成关键酶基因的表达水平(图3)。
实施例5:PnDof1基因过表达对三七皂苷合成含量的影响
精确称取1.0g各转基因细胞系和非转基因三七细胞系,加入25mL70%甲醇浸提过夜,超声40min,3000rpm离心10min,取上清液用0.45μm滤膜过滤,得到最终的预处理液。利用Agilent1200系列,色谱柱为Thermo Scientific Hypersil GOLD C18色谱柱(250mm×4.6mm,5.0μm);流动相为水(A)-乙腈(B)进行线性洗脱(V/V),洗脱条件为0min19%B,36min36%B,70min80%B;流速:1mL/min,检测波长:203nm;柱温:30℃,进样量:10μL。所有样品平行测定3次,求其峰面积的平均值,根据保留时间定性,根据峰面积定量,根据标准品的标准曲线计算样品中各单体皂苷的含量,结果表明PnDof1基因的过表达提高了三七中几种重要单体皂苷的含量以及总皂苷的含量(图4)。
序列表
<110> 昆明理工大学
<120> 一种三七DOF转录因子基因PnDof1及其应用
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tgacaacagt gaagcagttc gtaag 25
<210> 6
<211> 25
<212> DNA
<213> 人工序列(Artificial)
<400> 6
caaacataag acctagcata gccca 25
<210> 7
<211> 24
<212> DNA
<213> 人工序列(Artificial)
<400> 7
gacccttatg cgatctccat gact 24
<210> 8
<211> 24
<212> DNA
<213> 人工序列(Artificial)
<400> 8
aattcctccg aggctcagat aatc 24
<210> 9
<211> 24
<212> DNA
<213> 人工序列(Artificial)
<400> 9
acaagcatca gatggctcat ggta 24
<210> 10
<211> 22
<212> DNA
<213> 人工序列(Artificial)
<400> 10
ccaaccacca gaagcaagtt gt 22
<210> 11
<211> 19
<212> DNA
<213> 人工序列(Artificial)
<400> 11
ttccatgcca ccagccaca 19
<210> 12
<211> 21
<212> DNA
<213> 人工序列(Artificial)
<400> 12
ggtcaagcac ctgcaagaca a 21
<210> 13
<211> 25
<212> DNA
<213> 人工序列(Artificial)
<400> 13
tgtaccgaga atccgagctt ataga 25
<210> 14
<211> 21
<212> DNA
<213> 人工序列(Artificial)
<400> 14
atgctctccc cacttcccat c 21
<210> 15
<211> 25
<212> DNA
<213> 人工序列(Artificial)
<400> 15
ttctggaaat tattgctcgg ataag 25
<210> 16
<211> 23
<212> DNA
<213> 人工序列(Artificial)
<400> 16
aggctgccac atttgtcttc aac 23
<210> 17
<211> 22
<212> DNA
<213> 人工序列(Artificial)
<400> 17
ttgaatctcc tgcttcggat ga 22
<210> 18
<211> 20
<212> DNA
<213> 人工序列(Artificial)
<400> 18
cctccaccac cagctcctgt 20
Claims (3)
1.一种三七DOF转录因子基因PnDof1,其核苷酸序列如SEQ IDNO:1所示。
2.权利要求1所述的三七DOF转录因子基因PnDof1在促进三七皂苷合成中的应用。
3.根据权利要求3所述的应用,其特征在于:将三七DOF转录因子基因PnDof1与植物表达载体pCAMBIA2300s连接,构建植物超表达载体;将植物超表达载体通过根癌农杆菌介导转入三七细胞中;以表达载体上具有的抗性标记来筛选转化子,并通过聚合酶链式反应获得阳性转基因三七细胞系,分析阳性转基因三七细胞中皂苷含量,筛选获得三七皂苷含量上升的转基因三七细胞系。
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