CN116478992A - 一种三七诱导型启动子pprp及其应用 - Google Patents
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Abstract
本发明公开了一种三七诱导型启动子PPRP,其核苷酸序列如SEQ ID NO:1所示,本发明通过分子生物学和基因工程相关技术研究证实三七启动子PPRP对几种植物激素、生物胁迫和非生物胁迫的响应,将本发明三七诱导型启动子PPRP与β‑葡萄糖苷酸酶基因构建的表达框转入烟草中表达,通过荧光法定量检测转基因烟草的葡萄糖苷酸酶活性,结果表明转基因烟草在乙烯利、茉莉酸甲酯、尖孢镰刀菌、茄腐镰刀菌、氯化钠、氯化铝、受伤处理后葡萄糖苷酸酶活性明显增强;由此可见三七启动子PPRP受几种激素、生物和非生物胁迫因子的诱导,能用于植物抗逆境基因工程。
Description
技术领域
本发明属于分子生物学以及基因工程技术领域,具体涉及一种三七诱导型启动子PPRP及其应用。
背景技术
启动子是RNA聚合酶识别、结合和开始转录的一段DNA序列,位于结构基因5'端的上游,是基因的重要组成部分,主要调控基因表达的起始时间和表达程度。启动子本身并不控制基因活动,而是通过与转录因子的结合来控制基因活动。不同的植物启动子使植物基因具有不同的表达特性,通过对启动子的结构分析,可预测植物中基因的表达谱及位置(Shelenkov A,Korotkov E.Search of regular sequences in promoters fromeukaryotic genomes.Computational Biology and Chemistry,2009,33(3):196-204)。
高等植物生长发育是不同基因时空上有序表达和协同作用的结果。真核生物基因表达调控过程中,转录是最关键的调控环节,而启动子则是转录水平的重要顺式调控元件。高等植物启动子包含多种类型的顺式作用元件,其中一些抗逆相关的作用元件通过调控基因的表达,使植物在外界胁迫环境下更具有生命力。植物通过增强自身防御机制来调控其生长发育和抵抗不良环境,基因表达变异可能是因为内部顺式调控元件发生改变,对调控元件深入研究可为作物遗传改良提供新策略(Zhang ML,Lv YD,Wang Y,Rose Jocelyn KC,Shen F,Han ZY,Zhang XZ,Xu XF,Wu T,Han ZH.TATA box insertion provides aselection mechanism underpinning adaptations to fe deficiency.Plantphysiology,2017,173(1):715-727)。启动子的研究具有重要实用价值,基因工程中异源蛋白表达取决于导入基因的转录诱导,它能确保目的基因在合适部位以及合适时间下表达(杨鹏芳,段国琴,胡晓炜,缪秀梅,南淑珍,张丽静.高等植物启动子研究概述.分子植物育种,2018,16(05):1482-1493)。
启动子按功能及作用方式可分为诱导型启动子、组织特异型启动子和组成型启动子。组成型启动子,能够调控基因表达,使其基本恒定在一定程度上,从而使基因在不同部位或组织中的表达水平不存在明显差异,一般来源于植物病毒或植物持家基因,如花椰菜花叶病毒35S启动子、水稻EIF5基因启动子、菊花Actin基因启动子CmActin等。组织特异性启动子,其调控作用使得基因往往只在某些特定器官或组织中表达,且表现出发育调节的特性,此类启动子均包含与组织特异性相关的顺式作用元件。例如水稻OsGEX2启动子是花粉特异性启动子,顺式作用元件分析发现其含有许多花粉特异性增强子元件LAT52/56和POLLEN1LE-LAT52(Cook M,Thilmony R.The OsGEX2 gene promoter confers sperm cellexpression in transgenic rice.Plant Molecular Biology Reporter,2012,30(5):1138-1148)。烟草、草莓ToRB7基因启动子序列也有许多共有元件,且元件相对位置相似,两个基因均在根中特异性表达(Vaughan Simon P,James David J,Lindsey Keith,MassiahAndrea J.Characterization of FaRB7,a near root-specific gene from strawberry(Fragaria ananassa Duch.)and promoter activity analysis in homologous andheterologous hosts.Journal of experimental botany,2006,57(14):3901-3910)。特定顺式作用元件对于基因特异性表达至关重要。
诱导型启动子是在植物适应环境和长期进化过程中形成的,能够响应特殊的生物、物理、化学信号,进而提高特定基因转录水平,来适应一定范围内环境变化的一类启动子。一般情况下,诱导型启动子不启动转录或转录活性较低,只有在外界因素诱导刺激下,才可高效启动基因转录。根据诱导信号来源,诱导型启动子可分为光诱导型启动子、热诱导型启动子、创伤诱导型启动子、激素诱导型启动子和真菌诱导型启动子。创伤诱导启动子对生物胁迫响应具有潜在作用,大豆乙烯响应因子基因GmERF3启动子中的创伤诱导元件WIDE,该元件很可能在植物自我修复或养分重吸收过程中被激活(Hernandez-GarciaCarlos M,Finer JJ.A novel cis-acting element in the GmERF3 promotercontributes to inducible gene expression in soybean and tobacco afterwounding.Plant cell reports,2016,35(2):303-316)。生长素、赤霉素和水杨酸等激素会诱导激素响应启动子的表达,一般生长素响应元件与其他激素响应元件共同调节基因表达。例如,水稻中油菜素内酯诱导型BLE3基因启动子含有能够通过油菜素内酯和生长素IAA进行双重调节的生长素响应元件(Yang XG,Hidemitsu Nakamura,Hiroaki Ichikawa,Hidemi Kitano,Setsuko Komatsu.OsBLE3,a brassinolide-enhanced gene,is involvedin the growth of rice.Phytochemistry,2006,67(14):1442-1454)。诱导型启动子不仅能使目的基因的表达产物在特定时期积累,避免植物能量的不必要浪费,而且可以避免基因过表达对植物造成的毒害,以及因此造成的植物减产甚至死亡,已成为近年来植物基因工程的研究热点(杨瑞娟,白建荣,李锐,常利芳.诱导型启动子在植物基因工程中的研究进展.山西农业科学,2018,46(02):292-298)。
发明内容
本发明提供了一种诱导型启动子PPRP,来源于三七,其核苷酸序列如SEQ ID NO:1所示。
本发明另一目的是将该启动子应用在基因工程中,即在激素、生物、非生物胁迫下作为诱导表达启动子调控外源基因在转基因受体植物中的特异高效表达。
本发明涉及分离诱导型启动子片段并鉴定其表达活性,本发明从三七中克隆获得诱导型启动子,该启动子长573bp。生物信息学分析表明该诱导型启动子中包含一系列不同的顺式作用元件。
将本发明分离克隆的诱导型启动子片段置换pBI121载体上的CaMV的35s启动子,由诱导型启动子驱动报告基因GUS的表达框,通过根癌农杆菌(Agrobacteriumtumefaciens)介导将其转入模式植物烟草(Nicotiana tabacum)中表达,并通过进一步实验揭示诱导型启动子的表达特性,为后期利用该启动子调控外源基因在转基因植株中的高效特异表达奠定基础。
将本发明中PPRP启动子驱动GUS的表达框转入烟草中,采用植物激素、生物胁迫和非生物胁迫处理转基因烟草植株,并进行GUS活性的荧光定量分析,检测结果表明,PPRP启动子响应响应几种植物激素、生物胁迫和非生物胁迫的处理。茉莉酸甲酯、乙烯利、氯化铝胁迫、氯化钠胁迫、伤害胁迫、茄腐镰刀菌(Fusarium solani)、尖孢镰刀菌(Fusariumoxysporum)能明显诱导启动子PPRP的活性。
上述启动子PPRP在基因工程中应用于外源基因的诱导表达,具体操作如下:
(1)从三七幼嫩组织中提取基因组DNA,采用扩增PPRP的特异引物,通过聚合酶链式反应(polymerase chain reaction,PCR)扩增出PPRP序列,然后将其连接到pGEM-T载体上,经测序获得具有序列正确的克隆;
(2)用限制性内切酶酶切pGEM-T-PPRP载体,回收启动子片段;同时采用合适的限制性内切酶酶切去除植物表达载体上的组成型表达启动子,通过胶回收得到载体大片段;再将所获得PPRP片段与pBI121-GUS载体片段连接,构建植物诱导表达载体;之后将所构建的植物诱导表达载体通过根癌农杆菌介导转入受体植物中;表达转基因的植株在遭受氯化铝胁迫、氯化钠胁迫、伤害胁迫、茄腐镰刀菌、尖孢镰刀菌侵染时,启动子PPRP驱动的目的基因会被诱导并上调表达水平,此外体内体外的茉莉酸甲酯、乙烯利也会诱导目的基因高水平表达。
基因工程中植物超表达载体常用来自花椰菜花叶病毒的35S启动子,该启动子为组成型表达启动子,目的基因的表达大体恒定在一定水平上,在不同组织、部位表达水平没有明显差异,所以转入植物的外源基因其表达不受控制,导致蛋白大量积累且浪费能量。而诱导型启动子可以在植物受到外界胁迫或化学因素影响时提高目的基因的表达量,在去除胁迫或化学处理后即下调目的基因的表达,可保证在植物受到逆境胁迫时起到保护植物、抵抗外界刺激的效果,反之在适宜的环境中不浪费植物的能量。此外,在基因工程应用中诱导型启动子不但可以避免目的基因的持续表达对植物能量的过度消耗,而且可以消除基因产物积累对植物本身造成的伤害。激素(茉莉酸甲酯、乙烯利)、非生物胁迫(伤害、氯化铝胁迫、氯化钠胁迫)和生物胁迫(茄腐镰刀菌、尖孢镰刀菌)能明显诱导本发明中PPRP启动子的表达活性,因此本发明在抗生物胁迫的基因工程中具有广阔的应用前景。
附图说明
图1是本发明中启动子PPRP扩增产物检测结果;
图2是本发明中部分pBI121-PPRP-GUS转基因烟草基因组DNA的PCR检测结果,其中Marker:DL2501 DNA Marker(上海捷瑞),由2,000bp、1,000bp、750bp、500bp以及250bp五条DNA片段组成;阳性对照:质粒pGEM-T-PPRP为模板的PCR反应;WT:非转基因烟草(野生型)总DNA为模板进行的PCR;
图3是本发明中GUS酶活测定的标准曲线;
图4是本发明中pBI121-PPRP-GUS转基因烟草分别用茉莉酸甲酯(ETH)、乙烯利(MEJA)处理后的GUS活性,图中对照为正常生长的pBI121-PPRP-GUS转基因烟草的GUS活性,P1、P2、P3分别为3个pBI121-PPRP-GUS转基因烟草株系;
图5是本发明中pBI121-PPRP-GUS转基因烟草分别用茄腐镰刀菌、尖孢镰刀菌处理后的GUS活性,图中对照为正常生长的pBI121-PPRP-GUS转基因烟草的GUS活性,P1、P2、P3分别为3个pBI121-PPRP-GUS转基因烟草株系;
图6是本发明中pBI121-PPRP-GUS转基因烟草分别用氯化钠、氯化铝、受伤处理后的GUS活性,图中对照为正常生长的pBI121-PPRP-GUS转基因烟草的GUS活性,P1、P2、P3分别为3个pBI121-PPRP-GUS转基因烟草株系。
具体实施方式
下面通过附图和实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,本实施例中方法如无特殊说明的均按常规方法操作,所用试剂如无特殊说明的采用常规试剂或按常规方法配置的试剂。
实施例1:三七诱导型启动子PPRP的克隆以及序列分析
以提取的三七根基因组DNA为模板,用扩增启动子PPRP的特异引物(上游引物为:5’A TGGCCTATTCAAAGGCTTTC3’,下游引物为:5’TTAGTTACTACATTGGAAGCCAG3’),通过PCR克隆启动子PPRP的序列。反应体系(20μL)为2μL三七基因组DNA、5μL 10×Ex Taq Buffer(含Mg2+20mM)、4μL dNTP Mix(2.5mM each)、1μL上游引物(5μM)、1μL下游引物(5μM)、0.25μLTaKaRa Ex Taq(5U/μL)、36.75μL ddH2O。PCR反应条件:95℃、3min;95℃、30min,59℃、30s,72℃、1min,30个循环;72℃、5min,P CR结束后,取8μL进行琼脂糖凝胶电泳,用以检测扩增产物的特异性以及大小,结果如图1所示。
使用SanPrep柱纯化切胶回收得到PCR扩增产物。T-A连接后,通过热激转化法将连接产物转入大肠杆菌感受态细胞Tran-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。挑选若干个单菌落,摇菌后用扩增PPRP的特异引物检测多克隆位点插入PPRP的克隆。将得到的阳性克隆进行测序,最终获得的启动子PPRP长573bp。
实施例2:PPRP-GUS表达载体构建
pBI121多克隆位点有SmaⅠ和HindⅢ酶切位点,因此在扩增启动子的特异引物分别添加SmaⅠ和HindⅢ的识别位点。采用SanPrep柱式质粒DNA小量抽提试剂盒(上海生工)提取插入PPRP的大肠杆菌质粒pGEM-T-PPRP以及植物表达载体pBI121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SmaI和HindⅢ分别对质粒pGEM-T-PPRP和pBI121进行双酶切(50μL体系),反应体系和操作过程为:分别取20μL pGEM-T-PPRP和pBI121质粒、依次加入3μL 10×T buffer、2μL SmaⅠ、5μL ddH2O,混匀后短时离心,置于37℃反应2h后,再加入2μL HindⅢ、5μL 10×M buffer、13μL ddH2O,混匀后短时离心,置于37℃反应2h。将所有酶切产物进行琼脂糖凝胶电泳,然后使用SanPrep柱式DNA胶回收试剂盒(上海生工)对启动子片段和pBI121载体大片段分别进行胶回收,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。
利用T4 DNA Ligase(TaKaRa),将回收的启动子DNA片段和pBI121载体片段连接起来,反应体系(20μL),操作过程为:取10μL PPRP DNA片段依次加入2μL pBI121载体DNA、2μL10×T4 DNA Ligase Buffer、1μL T4 DNA Ligase、5μL ddH2O,混匀后短暂离心,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Tran-T1中,用含有50mg/L卡那霉素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启动子PPRP的特异引物进行PCR,挑选出PPRP与pBI121成功连接的克隆,加入甘油并置于-80℃保存备用。
提取并纯化上述大肠杆菌Tran-T1中的pBI121-PPRP-GUS质粒,随后用液氮冻融法将上述构建的植物表达载体pBI121-PPRP-GUS转入所制备的根癌农杆菌LBA4404感受态细胞中。操作步骤为:取0.2μg pBI121-PPRP-GUS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,再冰浴2min,之后加入500μL 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卡那霉素的LB固体培养基上,28℃倒置培养。挑选单菌落摇菌,再用扩增PPRP的特异性引物进行PCR反应,检测pBI121-PPRP-GUS是否转入农杆菌中。选择若干阳性克隆,加入甘油后置于-80℃保存备用。
实施例3:农杆菌介导的植物遗传转化以及转基因植物筛选
本实验的转基因受体是烟草,将烟草种子用75%的酒精浸泡30s,无菌水洗涤后用0.1%的HgCl2浸泡8min,然后再用无菌水洗涤若干次,播种于1/2MS培养基上,28℃暗培养5-8d,发芽后转至光照培养箱(25℃,16h/d光照),以后每月用MS培养基继代一次。
将-80℃冰箱中保存的含有pBI121-PPRP-GUS质粒的农杆菌LBA4404菌液取出,取10μL菌液接种于1mL含有20mg/L利福平和50mg/L卡那霉素的LB液体培养基中,28℃、200rpm振荡培养至浑浊。吸取500μL菌液均匀涂布于含有20mg/L利福平和50mg/L卡那霉素的LB固体培养基上,28℃倒置培养至长出菌苔。用接种环刮取3-5环菌苔接种于40mL含25mg/mL乙酰丁香酮的MGL培养基中,28℃、220rpm振荡培养直至OD600约为0.6。将无菌烟草组培苗叶片剪成约1cm2大小的叶盘,浸泡于含有悬浮农杆菌的MGL培养基中,25℃震荡培养15min。用无菌滤纸将叶盘表面的菌液吸干后转入烟草共培养培养基中,22℃暗培养2天。将共培养后的叶盘转接到烟草筛选培养基上,培养于光照培养箱(25℃,16h/d光照)。培养约3周,将分化出来的烟草幼苗切下并继代于含有50mg/L卡那霉素和300mg/L头孢霉素的生根培养基上进行生根培养。
采用CTAB法提取转基因烟草植株叶片的基因组DNA,取1μL基因组DNA进行琼脂糖凝胶电泳检测其完整性和浓度。以转基因植株的基因组DNA为模板用扩增启动子PPRP的特异引物进行PCR反应。PCR结束后,取8μL产物用于琼脂糖凝胶电泳以检测阳性转基因植株。PPRP-GUS转基因烟草共筛选到26株阳性转基因植株,部分转基因烟草的检测结果见图2。
实施例4:转基因烟草的GUS荧光定量检测
对转基因烟草叶片GUS活性的荧光定量分析参考Jefferson等(JeffersonR.Assaying chimeric genes in plants:The GUS gene fusion system.Plant Mol BiolRep.1987,5(4):387–405)的方法,其反应机理是:GUS可与底物4-MUG反应,催化产生4-MU,4-MU在激发波长为365nm、发射波长为455nm条件下产生荧光,产生的荧光值可以通过荧光分光光度计进行定量测定。
将预先处理好的烟草叶片置于装有液氮的研钵中研磨成粉末,加入400μL GUS提取缓冲液,将匀浆转移至1.5mL离心管中,于4℃、12000g离心10min。离心结束后,收集上清于新的离心管中。预先取1mL的4-MUG溶液(1mmol/L)于2.0mL离心管中37℃预热10min。取50μL上清液加入至预热的GUS反应缓冲液中,迅速摇匀,并立即取200μL反应混合液置于1.8mL的终止缓冲液中(操作时间少于30s),作为酶促反应0点样(荧光测定时以此为空白对照),剩余液体继续37℃反应并开始计时。在反应15min、30min、45min时分别取200μL反应混合液,加入至1.8mL终止缓冲液中,用于荧光测定。使用荧光分光光度计在激发波长为365nm,发射波长为455nm的条件下测定各样品的荧光值。制作4-MU标准曲线:将1mM 4-MU母液用反应终止液分别稀释成5nM、10nM、20nM、40nM、60nM,80nM和100nM的不同梯度液,在激发波长为365nm,发射波长为455nm的条件下测定各梯度液的荧光值,以反应终止液为空白对照,用测得的荧光值和4-MU的浓度绘制标准曲线(图3)。取10μL上清液,采用改良的考马斯亮蓝法测定样品的蛋白含量。以一分钟催化4-MUG生成1pmol 4-MU的酶量为一个活力单位,GUS酶活以每μg总蛋白的酶活力计算,即表示为4-MU pmol/min/μg protein。通过标准曲线,计算出转基因烟草的GUS活性。
为了检测三七启动子PPRP对植物激素、生物胁迫和非生物胁迫的响应,分别用几种植物激素、生物胁迫和非生物胁迫因子处理转基因烟草的叶片,并通过上述方法测定处理前后的GUS活性,以正常生长未处理的转基因烟草叶片GUS活性作为对照;如图4,在分别用茉莉酸甲酯、乙烯利处理后,三七启动子PPRP转基因烟草叶片的GUS活性明显上调。其中茉莉酸甲酯的诱导程度明显大于乙烯利;用茄腐镰刀菌、尖孢镰刀菌接种转基因烟草的叶片,均显著上调启动子PPRP的活性(图5)。对转基因烟草叶片进行氯化铝胁迫、氯化钠胁迫、伤胁迫,也能明显诱导GUS基因的表达水平(图6)。上述实验结果表明,三七启动子PPRP响应植物激素、生物和非生物胁迫。显然,三七启动子PPRP是一种植物激素、生物胁迫和非生物胁迫因子诱导型启动子,可应用于植物抗逆基因工程。
Claims (3)
1.一种诱导型启动子PPRP,来源于三七,其核苷酸序列如SEQ ID NO:1所示。
2.权利要求1所述的诱导型启动子PPRP在植物抗逆基因工程中的应用。
3.根据权利要求2所述的应用,其特征在于:在逆境胁迫下调控外源基因在转基因受体植物中的特异高效表达。
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CN117625628A (zh) * | 2024-01-26 | 2024-03-01 | 湖南工程学院 | 一种增强人参抗逆性的ProPgJOX2启动子及其应用 |
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CN117625628A (zh) * | 2024-01-26 | 2024-03-01 | 湖南工程学院 | 一种增强人参抗逆性的ProPgJOX2启动子及其应用 |
CN117625628B (zh) * | 2024-01-26 | 2024-04-12 | 湖南工程学院 | 一种增强人参抗逆性的ProPgJOX2启动子及其应用 |
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