CN116254267A - 橡胶草乳管特异性表达启动子及其应用 - Google Patents
橡胶草乳管特异性表达启动子及其应用 Download PDFInfo
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Abstract
本发明公开了一种橡胶草乳管特异性表达启动子及其应用。该启动子，1)序列表中序列1的DNA序列；2)与序列表中序列1限定的DNA序列具有70％以上同源性，且具有橡胶草乳管特异性表达启动子功能的DNA序列；3)在高严谨条件下可与序列表中序列1限定的DNA序列杂交的核苷酸序列。本发明的启动子可以启动内源或外源基因在橡胶草乳管中特异性表达，适用于绝大部分具有乳管结构的植物。
Description
技术领域
本发明属于生物技术领域，具体涉及一种橡胶草乳管特异性表达启动子及其应用。
背景技术
天然橡胶是重要工业原料之一，由一种特化的“乳管”结构产生并储存。乳管细胞中天然橡胶生物合成由多种基因相互调控，是一个复杂的过程。因此，揭示橡胶合成过程中基因和蛋白的功能，对阐释橡胶合成的分子机制至关重要。
目前，植物转基因技术是验证基因功能或改良农艺产品性状的一种重要手段。而启动子作为基因调控的重要元件，决定下游基因的表达强度和组织特性。RNA聚合酶II识别转录起始位点，与之结合，调控基因的转录。在植物基因工程中，为提高植物某一优质性状，可通过外源基因的导入来实现。因此为使外源基因能在植物体内稳定高效地表达，选择一个合适的启动子至关重要。
启动子主要包括组成型启动子、诱导型启动子和组织特异型启动子。组成型启动子花椰菜花叶病毒(Cauliflower mosaicvirus，CaMV)35S是目前在双子叶植物中应用十分广泛的组成型启动子之一，可以启动基因在植物体内各个组织和细胞表达(Odell J.T.,Nagy F.,and Chua N.H.Identification of DNA sequencesrequired for activity ofthe cauliflower mosaic virus 35S promoter[J].Nature.1985,313(6005):810-812)。而单子叶植物中玉米泛素(Ubiquitin)启动子也较为常见(Alan H.Christensen,PeterH.Quail.Ubiqui tin promoter-based vectors forhigh-level expression ofselectable and/or screenable marker genes inmonocotyledonous plants[J].Pubmed,1996，5(3)：213-218.)。诱导型启动子在植株自然生长状态下，活性很低甚至没有活性。但当受到干旱、虫害、离子浓度等不利条件刺激时，启动子的转录活性显著提高，启动一些抗逆基因的表达，从而应对胁迫。与组成型启动子相比，诱导型启动子的活性会受到外界信号的调控，一般不会影响植物的正常生长。但是，当启动一些具有组织特异性的基因在植株各个组织内均表达，可能会引植株发育畸形、生物量降低甚至植株致死等负面效应(ZhengH.-Q.,Lin S.-Z.,ZhangQ.,et al.Isolation and analysis of aTIR-specificpromoter from poplar[J].Forestry Studies in China.2007,9(2):95-106)。为解决这一问题，研究者利用组织特异性启动子来启动基因的表达。使目的基因只在植物特定的组织或器官中被激活转录。从而减少基因过度表达带来的负面影响。
在天然橡胶合成分子机制的研究中，由于天然橡胶是在植物类异戊二烯/萜类次生代谢途径中产生的，因此在整个植物体内组成型表达橡胶合成途径基因，将预见地会引起醌类、甾醇类和植物激素等其他重要的类异戊二烯生理代谢紊乱，影响植物体的发育(Fray R.,Wallace A.,Fraser P.,et al.Constitutiveexpression of a fruitphytoene synthase gene in transgenic tomatoes causes dwarfismby redirectingmetabolites from the gibberellin pathway[J].The Plant Journal.1995,8:693-701)。为了避免该类问题，乳管组织特异性启动子的研究必不可少。Priya等人克隆了橡胶树REF基因上游启动子序列，并在烟草和拟南芥中验证了启动活性(Priya P.,Venkatachalam P.,and Thulaseedharan A.Molecular cloning andcharacterizationof the rubber elongation factor gene and its promoter sequence fromrubbertree(Hevea brasiliensis):A gene involved in rubber biosynthesis[J].PlantSci.2006,171(4):470-480)。Tata等人在短角蒲公英中证明HbSRPP上游启动子序列在乳管中特异表达(Tata S.,Choi J.,Jung J.-Y.,et al.Laticifer tissue-specificactivation of the Hevea SRPP promoter in Taraxacum brevicorniculatumand itsregulation by light,tapping and cold stress[J].Industrial Crops andProducts.2012,40:219–224)。Fei等人利用5’端缺失法证明Hevein上游-749到-292的区域在C-乳清中特异表达，证明了乳管特异性表达的核心区域(Fei X.W.and Deng X.D.Invitro transient expression system of latex C-serum was used for analysisofhevein promoter in response to abscisic acid in Hevea brasiliensis[J].JIntegr PlantBiol.2008,50(3):338-344)。Ganesh等人以橡胶草为受体证明了橡胶树PEP16上游启动子序列在乳管中特异性启动GUS基因表达(Ganesh I.,Choi S.C.,BaeS.W.,et al.Heterologous activation of the Hevea PEP16 promoter in therubber-producing laticiferous tissues of Taraxacum kok-saghyz[J].Sci Rep.2020,10(1):10844)。但是目前在来源于橡胶草的乳管特异性启动子很少有报道。
发明内容
因此，本专利发明了一种橡胶草乳管特异性启动子，有助于揭示橡胶草乳管结构发育、产胶调控通路的分子机理，更细致地理解植物胚胎发育中形态发生与细胞分化的分子机理，又能广泛应用于植物基因工程研究中，使乳管特异表达在乳管中高效表达，以实现基因功能的探究，进而提高橡胶草的产胶含量，获得优质的橡胶草植株。
本发明的目的是提供一种橡胶草乳管特异性表达启动子及其应用。
本发明所提供的橡胶草乳管特异性表达启动子，来源于橡胶草TkTHFI基因的上游序列：
1)序列表中序列1的DNA序列；
2)与序列表中序列1具有70％以上同源性，且具有橡胶草乳管特异性表达启动子功能的DNA序列；
3)在高严谨条件下可与序列表中序列1限定的DNA序列杂交的核苷酸序列。
其中，序列表中序列1是由2000个脱氧核苷酸组成。上述序列的获得，首先通过橡胶草转录组测序分析筛选出在乳管中特异高表达的基因TkTHFI。然后利用q-PCR验证TkTHFI基因的乳管表达特异性。进而通过PCR方式获得TkTHFI基因上游2000bp序列作为启动子。
含有上述橡胶草乳管特异性表达启动子的表达盒、重组表达载体、转基因细胞系及宿主菌均属于本发明的保护范围。
在所述表达盒中，所述橡胶草乳管特异性表达启动子的下游可连接结构基因，报告基因，调节基因，或者能够干扰内源基因表达的小RNA等。可用来驱动结构基因，报告基因，调节基因，或者天然小RNA或人工合成的小RNA等基因的表达。
所述重组表达载体是由上述表达盒与质粒、病毒或表达载体所构建。所述重组表达载体为重组植物表达载体，含有上述表达盒并且能够将所述的表达盒转入植物宿主细胞、组织或器官等，将所述表达盒转入宿主的基因组中，它包括但不限于双元载体。
上述重组植物表达载体可以利用农杆菌介导遗传转化、发根农杆菌转化、基因枪、原生质体转化法等常规转基因方法转入宿主植物的细胞和组织中，成功转化的植物细胞和组织经过分化诱导获得完整植株。
上述橡胶草乳管特异性启动子可用于启动内源或外源基因在植物乳管内特异表达。
利用橡胶草乳管特异性启动子启动内源或外源基因在植物中表达的方法属于本专利的保护范畴。
所述橡胶草乳管特异性启动子启动内源或外源基因在植物中表达，是将内源或外源基因连入乳管特异性启动子下游，构建植物表达载体，利用农杆菌介导遗传转化法转入植物中，筛乳管中特异表达内源或外源基因的阳性植株。
所述橡胶草乳管特异性启动子还可用来启动基因调控原件的表达。所述基因的调控元件包括能增强外源基因表达或调控基因在植物表达组织的元件，如增强子、沉默子、组成型表达和诱导型表达等调控元件。
所述方法中，所述外源基因为蛋白质编码基因或非编码基因；所述蛋白质编码基因为具有功能的优质基因等；所述非编码基因为正义RNA或反义RNA等。
所述方法中，所述植物为双子叶植物。
所述双子叶植物是橡胶草、莴苣或橡胶树等。
本发明中橡胶草乳管特异性启动子的获得，首先设计启动子引物，以橡胶草基因组DNA为模板，然后通过PCR方式获得TkTHFI上游2000bp序列作为启动子。在乳管特异性启动子下游连入报告基因β-葡糖苷酸酶(β-glucuronidase，GUS)，结果表明GUS基因只在橡胶草乳管中特异表达。说明本发明中的启动子可以用来启动内源或外源基因在橡胶草乳管中特异表达，适用于具有乳管结构的绝大部分植物。
本发明的启动子为后续验证乳管特异表达基因的功能奠定了一定基础。利用本发明的启动子可特异性提高内源或外源基因在乳管中的表达水平，改变乳管结构、提高胶乳含量、或改变天然橡胶分子量等，从而提高产胶植物的橡胶含量及质量。本发明的启动子在产胶植物乳管发育调控、橡胶含量提高以及产胶通路调控机理等方面都具有重要作用。本发明通过PCR的方法获得橡胶草乳管中TkTHFI基因的启动子，并通过转基因方法验证该启动子具有乳管特异性，可以在乳管中特异启动外源基因的表达。本发明中乳管特异表达启动子为揭示产胶植物中乳管结构发育、橡胶含量积累、产胶调控通路解析等方面奠定了基础，具有极大的应用前景。
附图说明
图1为TkTHFI基因橡胶草转录组测序结果示意图
图2为TkTHFI基因组织表达特异性结果示意图
图3为TkTHFI启动子克隆载体pEASY-TkTHFI的结构示意图
图4为pEASY-TkTHFI载体构建结果
图5为TkTHFI启动子序列顺式作用元件分析
图6为pCAMBIA1303-pTkTHFI::GUS表达载体结构示意图
图7为pCAMBIA1303-pTkTHFI::GUS表达载体构建结果
图8为转pCAMBIA1303-pTkTHFI::GUS载体T0代PCR检测DNA水平结果
图9为转pCAMBIA1303-pTkTHFI::GUS载体T0代GUS检测结果
具体实施方式
下述实施例中的方法，如无特别说明，均为常规方法。
实施例1、橡胶草乳管特异性启动子(pTkTHFI)的获得
根据橡胶草转录组测序结果，筛选出在乳管中高表达的基因TkTHFI(图1、图2)，根据基因组测序结果查找TkTHFI基因上游2000bp序列作为启动子序列(pTkTHFI)，并设计引物。利用常规DNA提取试剂盒提取橡胶草叶片中基因组DNA作为模板(全式金
Genomic DNA Kit)，以pTkTHFI-F：GTTTAGGATGTTGTTGTTGACGG为正向引物，pTkTHFI-R：TGTTGGATGATATGATCAGTGAAGC为反向引物。PCR技术扩增pTkTHFI序列。
DNA聚合酶反应体系如表1所示(Takara，
GXL DNAPolymerase)；反应程序为：98℃预变性5min，然后98℃10s，60℃15s，68℃2min30sec，35个循环，最后68℃,5min。PCR成功获得2000bp启动子序列。纯化pTkTHFI目的条带，连接到pEASY-Blunt平末端载体上。选取EcoRI和SpeI进行初步酶切鉴定，大小为4431bp和1525bp。酶切鉴定正确后进行Sanger测序，结果表明，扩增的片段具有序列表中序列1的核苷酸序列，为橡胶草乳管特异性启动子(pTkTHFI)序列。如图3所示，将序列正确已构建好的载体命名为pEASY-pTkTHFI。pEASY-pTkTHFI启动子克隆及载体构建结果如图4所示。
表1.pTkTHFI启动子克隆PCR反应体系
实施例2、橡胶草乳管特异性启动子pTkTHFI顺式作用元件分析
利用在线软件PlantCARE对pTkTHFI启动子序列进行顺式作用元件分析，通过TBtools软件对启动子序列顺式作用元件可视化(图5)。
结果发现，在pTkTHFI启动子中，共预测到17不同的顺式作用元件。例如不同类型的光反应作用元件、干旱低温等非生物胁迫响应元件、参与MeJA响应元件和MYB结合位点等。
实施例3、橡胶草乳管特异性启动子pTkTHFI植物表达载体构建与遗传转化
1、pTkTHFI植物表达载体构建
pCAMBIA1303植物表达载体购自淼灵质粒平台，该载体由35S启动GUS报告基因，植物筛选标记为潮霉素(HPT)。
采用同源重组的方式将启动子序列连入pCAMBIA1303表达载体，并以GUS作为报告基因(pCAMBIA1303-pTkTHFI::GUS)。载体选择BamHI和NcoI进行酶切(下划线所示)，并以限制性内切酶上下游15bp作为同源臂序列(下划线并加粗所示)。
利用常规DNA提取试剂盒提取橡胶草叶片中基因组DNA作为模板，利用PCR技术扩增含有载体同源臂及酶切位点的启动子序列，以pEASY-pTkTHFI为模板，以clonepTkTHFI-F：
正向引物，clonepTkTHFI-R：/>
为反向引物。
用BamHI和NcoI双酶切植物表达载体pCAMBIA1303。切胶纯化回收启动子序列和线性化载体pCAMBIA1303。将上述PCR扩增得到的启动子序列和pCAMBIA1303载体利用诺唯赞同源重组酶(
II One Step CloningKit)进行连接、转化，获得pCAMBIA1303-pTkTHFI::GUS植物表达载体。将重组质粒利用PCR和双酶切进行验证，选取KpnI和SpeI切出11567bp和2008bp大小条带。如图6所示，为pCAMBIA1303-pTkTHFI::GUS表达载体结构示意图。如图7所示，为pCAMBIA1303-pTkTHFI::GUS表达载体构建结果。
2、pCAMBIA1303-pTkTHFI::GUS表达载体遗传转化
利用化学转化法将pCAMBIA1303-pTkTHFI::GUS转入农杆菌EHA105中。以橡胶草的叶片作为外植体，通过农杆菌介导遗传转化法将pCAMBIA1303-pTkTHFI::GUS表达载体转入橡胶草中。
潮霉素作为植物筛选标记，对获得的抗性芽进行筛选。获得4株T0代转pCAMBIA1303-pTkTHFI::GUS阳性植株。提取转基因植株叶片的基因组DNA进行PCR分子检测，引物分别选取pTkTHFI启动子一段序列和GUS基因一段序列，扩增大小为678bp。引物序列如下：THFI-238-F：GCAAGGAAAACGAGGATGAG；GUS-440-R：CAACGAACTGAACTGGCAGA。DNA聚合酶购自康为2×Taq，PCR反应体系如表2所示。反应程序为：95℃预变性10min，然后95℃30sec，56℃30sec，72℃30sec，35个循环，最后72℃,10min。扩增结果条带大小为678bp。如图8所示，共获得4株DNA水平检测阳性转基因植株。
表2.转基因阳性植株PCR检测反应体系
| 试剂 | 10μL |
| Mix | 5μL |
| THFI-238-F | 0.2μL(0.2-0.3μM) |
| GUS-440-R | 0.2μL(0.2-0.3μM) |
| H2O | 4.6μL |
实施例3、橡胶草乳管特异性启动子(pTkTHFI)的功能验证
对pCAMBIA1303-pTkTHFI::GUS转基因阳性植株进行GUS组织化学染色。步骤如下：橡胶草野生型植株作为阴性对照，分别取野生型植株和转基因阳性植株生长状态良好的叶片，置于GUS染色液中(50mM磷酸钠缓冲液，0.5mM K4[Fe(CN)6].3H2O，0.5mM K3[Fe(CN)6]，0.1％Triton×100，10mM EDTA，1mM X-Gluc，ddH2O补至100mL)，37℃放置3-5小时。结束后将叶片取出置于75％乙醇中进行脱水，每隔2h更换新的75％乙醇，直至叶片完全脱水。结果表明：pCAMBIA1303-pTkTHFI::GUS转基因阳性植株只在含有乳管结构的叶脉位置成功染色，而叶片部位均未检测到GUS活性。如图9所示，四株转基因阳性植株均在乳管位置成功显现蓝色，pTkTHFI启动β-葡糖苷酸酶(GUS)报告基因在橡胶草乳管中特异性表达。
Claims (10)
1.一种橡胶草乳管特异性启动子，其序列是下述核苷酸序列之一：
1)序列表中序列1的DNA序列；
2)与序列表中序列1具有70％以上同源性，且具有橡胶草乳管特异性表达启动子功能的DNA序列；
3)在高严谨条件下可与序列表中序列1限定的DNA序列杂交的核苷酸序列。
2.含有权利要求1所述的橡胶草乳管特异性表达启动子的表达盒。
3.含有权利要求1所述的橡胶草乳管特异性表达启动子的重组表达载体、转基因细胞系或宿主菌。
4.权利要求1所述的橡胶草乳管特异性表达启动子在启动目的基因在植物乳管特异性表达中的应用。
5.权利要求1所述的橡胶草乳管特异性表达启动子在培育乳管特异性表达目的基因的转基因植物中的应用。
6.根据权利要求5中所述的应用，其特征在于：所述转基因植株，由橡胶草乳管特异性表达启动子启动内源或外源基因表达；所述橡胶草乳管特异性表达启动子及其启动表达的内源或外源基因通过植物表达载体转入植物中，筛选出在橡胶草乳管中特异表达目的基因的转基因植物。
7.根据权利要求6所述的应用，其特征在于：所述橡胶草乳管特异性表达启动子下游还连接有调控基因表达的调控元件。
8.根据权利要求7所述的应用，其特征在于：所述内源或外源基因为蛋白编码基因和/或非蛋白编码基因。
9.根据权利要求8所述的应用，其特征在于：所述植物为双子叶植物。
10.根据权利要求9所述的应用，其特征在于：所述双子叶植物是橡胶草、莴苣或橡胶树等。
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