CN103243097A - 毛果杨中分离的花器官特异表达启动子及其应用 - Google Patents
毛果杨中分离的花器官特异表达启动子及其应用 Download PDFInfo
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Abstract
本发明公开了从毛果杨(Populus trichocarpa Torr.&Gray)中分离的花器官特异表达启动子及其应用,属于启动子的分离和应用。所述启动子的核苷酸序列为SEQ ID No.1所示。本发明还公开了含有所述花器官特异表达启动子的重组植物表达载体以及重组宿主细胞。功能验证试验发现,本发明启动子驱动的GUS基因仅在烟草萼片和花瓣中特异表达,在烟草的根、茎、叶、雄蕊和心皮等其它组织部位里没有GUS表达活性,说明本发明从毛果杨分离的启动子为花器官特异表达启动子。本发明进一步公开了所述花器官特异表达启动子在改良植物性状、培育植物新品种以及研究植物花器官的发育及调控机制等方面的应用。
Description
技术领域
本发明涉及一种从植物中分离的启动子,尤其涉及从毛果杨(Populustrichocarpa Torr.& Gray)所分离的花器官特异表达启动子,本发明还涉及含有该花器官特异表达启动子的重组植物表达载体以及宿主细胞,本发明进一步涉及它们在研究花器官基因表达模式或转录调控机制、改良植物性状、培育植物新品种等方面的应用,属于植物组织或器官特异性表达启动子的分离及其应用领域。
背景技术
启动子是RNA聚合酶特异性识别和结合的DNA序列,是重要的顺式作用元件,一般位于结构基因5’端上游区。高等植物基因调控主要在转录水平进行,启动子控制着基因表达的起始时间和表达程度,同时对所使用的RNA聚合酶类型也起着决定性作用,所以它是理解基因表达模式和转录调控机制的关键,是植物基因转录调控的中心。
根据启动子的转录模式及功能,可将其分为三类:组成型启动子、组织或器官特异性启动子和诱导型启动子。目前,在植物基因工程中使用的大多数为组成型启动子,使外源目的基因在植物各组织部位高水平表达,但是,在此过程中会出现多种多样的问题,例如不能从时间和空间上有效地调控目的基因的表达,过度消耗细胞内的物质和能量(Gittins JR,Pellny TK,Hiles ER,Rosa C,Biricolti S,et al.(2000)Transgene expressiondriven by heterologous ribulose-1,5-bisphosphate carboxylase/oxygenasesmall-subunit gene promoters in the vegetative tissues of apple(Malus pumilamill.).Planta210:232-240.);大量异源蛋白或代谢产物在植物体内积累,打破植物的代谢平衡,不利于植物生长(Robinson DJ(1996)Environmentalrisk assessment of releases of transgenic plants containing virus-derivedinserts.Transgenic research5:359-362.);引起基因沉默或共抑制现象(Kumpatla SP,Chandrasekharan MB,Iyer LM,Guofu L,Hall TC(1998)Genome intruder scanning and modulation systems and transgene silencing.Trends in Plant Science3:97-104;Mette MF,Aufsatz W,van der Winden J,Matzke MA,Matzke AJ(2000)Transcriptional silencing and promotermethylation triggered by double-stranded RNA.EMBO J19:5194-5201.);此外还存在转基因植物安全性隐忧。因此,科学家们不断寻找更为有效的组织或器官特异性启动子来代替组成型启动子,以期更精确的调控外源基因的表达。
组织或器官特异性启动子调控下的基因转录过程一般只发生在某些特定的组织或者器官中。组织或器官特异性表达启动子可以更加经济有效的调控外源基因的表达,特异地在特定需要的部位发挥作用,这样不仅可以提高外源基因的表达丰度,而且将生物能耗降到最低,从而不影响植株的正常生长。另外,深入研究组织或器官特异性启动子有助于阐明植物形态、发育、代谢途径等基础理论,而且具有广泛地应用价值。对组织或器官特异性表达启动子进行深入地研究已成为当前植物基因工程领域的热点。
目前与植物开花相关的组织或器官特异性表达启动子的研究主要集中在拟南芥(Arabidopsis thaliana)(Nitz I,Berkefeld H,Puzio PS,GrundlerFM(2001)Pyk10,a seedling and root specific gene and promoter fromArabidopsis thaliana.Plant Sci161:337-346;Paul W,Hodge R,Smartt S,Draper J,Scott R(1992)The isolation and characterisation of thetapetum-specific Arabidopsis thaliana A9gene.Plant Mol Biol19:611-622;Bell-Lelong DA,Cusumano JC,Meyer K,Chapple C(1997)Cinnamate-4-hydroxylase expression in Arabidopsis.Regulation in responseto development and the environment.Plant Physiol113:729-738.)、烟草(Nicotiana tabacum)等草本模式植物(Borisjuk NV,Borisjuk LG,LogendraS,Petersen F,Gleba Y,et al.(1999)Production of recombinant proteins inplant root exudates.Nat Biotechnol17:466-469.)、或者马铃薯(Solanumtuberosum)(Trindade LM,Horvath B,Bachem C,Jacobsen E,Visser RG(2003)Isolation and functional characterization of a stolon specific promoterfrom potato(Solanum tuberosum L.).Gene303:77-87.)、玉米(Zea mays)(Taniguchi M,Izawa K,Ku MS,Lin JH,Saito H,et al.(2000)The promoterfor the maize C4pyruvate,orthophosphate dikinase gene directs cell-andtissue-specific transcription in transgenic maize plants.Plant Cell Physiol41:42-48.)、水稻(Oryza sativa)(Takaiwa F,Kikuchi S,Oono K(1986)Thestructure of rice storage protein glutelin precursor deduced from cDNA.FEBSletters206:33-35;Wu HK,Chen T,Chung MC(1996)Analysis of5'region ofglutelin genes from wild rice species.Botanical Bulletin of Academia Sinica37;Yoshihara T,Washida H,Takaiwa F(1996)A45-bp proximal regioncontaining AACA and GCN4motif is sufficient to confer endosperm-specificexpression of the rice storage protein glutelin gene,GluA-3.FEBS Lett383:213-218;Vasconcelos M,Datta K,Oliva N,Khalekuzzaman M,Torrizo L,etal.(2003)Enhanced iron and zinc accumulation in transgenic rice with theferritin gene.Plant Science164:371-378;Datta K,Baisakh N,Oliva N,Torrizo L,Abrigo E,et al.(2003)Bioengineered'golden'indica rice cultivarswith beta-carotene metabolism in the endosperm with hygromycin andmannose selection systems.Plant Biotechnol J1:81-90)、番茄(Solanumlycopersicum)(Sandhu JS,Krasnyanski SF,Domier LL,Korban SS,OsadjanMD,et al.(2000)Oral immunization of mice with transgenic tomato fruitexpressing respiratory syncytial virus-F protein induces a systemic immuneresponse.Transgenic research9:127-135.)、油菜(Brassica napus)(AlbaniD,Robert LS,Donaldson PA,Altosaar I,Arnison PG,et al.(1990)Characterization of a pollen-specific gene family from Brassica napus whichis activated during early microspore development.Plant Mol Biol15:605-622;Ye L,Li C,Song Y(2000)Construction of plant seed-specificexpression vectors pSCB and pSCAB and the obtainment oftransgenicBrassica napus H165expressing poly-3-hydroxybutyrate syntheticgenes.Chinese Science Bulletin45:1206-1211;Zhang J,Li L,Song Y(2002)Identification of seed-specific promoter nap300and its comparison with7Spromoter.Progress in Natural Science12:737-741.)等农作物上,而对于多年生木本植物,尤其对于杨树开花相关的组织或器官特异性表达启动子的研究相对薄弱,鲜有报道。
发明内容
本发明目的之一是提供从毛果杨(Populus trichocarpa Torr.&Gray)中分离的花器官特异表达启动子。
本发明目的之二是提供含有上述花器官特异表达启动子的重组表达载体以及含有该重组表达载体的宿主细胞。
本发明目的之三是将所述的花器官特异表达启动子以及含有该花器官特异表达启动子的重组表达载体应用于研究花器官的基因表达模式或转录调控机制、构建转基因植物、改良植物性状、培育植物新品种等。
为实现上述目的,本发明首先提供了一种从毛果杨(Populustrichocarpa Torr.&Gray)中分离的花器官特异表达启动子PtAP1-2pro,其核苷酸序列为(a)或(b)所示:
(a)、SEQ ID No.1所示的核苷酸序列;
(b)、与SEQ ID NO.1的互补序列在严谨杂交条件能够进行杂交的核苷酸,该核苷酸仍具有花器官特异表达启动子的功能或活性。
优选的,本发明所述的从毛果杨(Populus trichocarpa Torr.&Gray)中所分离的花器官特异表达启动子PtAP1-2pro为SEQ ID No.1所示的核苷酸序列。
为研究PtAP1-2启动子的功能,本发明将PtAP1-2启动子与GUS可操作的连接,构建得到PtAP1-2pro::GUS植物表达载体;此外,将CaMV35S启动子与GUS可操作的连接,构建得到CaMV35Spro::GUS植物表达载体作为阳性对照;以烟草的根、茎、叶及花为受体材料,采用农杆菌介导的瞬时表达法对PtAP1-2启动子进行功能验证,试验结果表明PtAP1-2启动子驱动的GUS基因仅在烟草萼片和花瓣中特异表达,在根、茎、叶、雄蕊和心皮等其它组织部位里没有GUS表达活性;CaMV35S驱动的GUS基因(阳性对照CaMV35Spro::GUS)在根、茎、叶、萼片、花瓣、雄蕊、心皮等各个组织部位均有GUS表达活性。阴性对照(未转化材料)无GUS表达活性。试验结果证实,本发明从毛果杨(Populus trichocarpa Torr.&Gray)中所分离的启动子PtAP1-2pro为花器官特异表达启动子。
本发明进一步提供了含有所述花器官特异表达启动子的重组植物表达载体以及含有该重组植物表达载体的宿主细胞。
将本发明所述花器官特异表达启动子与待转录的异源性DNA序列进行可操作的连接,即得到在植物的花器官的特定部位中(例如:萼片和花瓣)转录或表达所述异源性DNA序列的重组植物表达载体。
所述的重组植物表达载体中还可含有选择标记基因。
另外,可以将本发明的花器官特异表达启动子与标记序列可操作的连接以确定标记序列的活性,所述的标记序列通常包括提供抗生素抗性或除草剂抗性的基因,诸如:四环素抗性基因、潮霉素抗性基因;草苷膦或草丁膦抗性基因等。
可以采用任何植物转化方法将本发明所构建的重组植物表达载体引入到目标植物(所述的目标植物包括被子植物、裸子植物;单子叶植物和双子叶植物;草本植物、木本植物和藤本植物;一年生植物和多年生植物;水生植物和陆生植物等)的细胞、组织或器官中,得到转化体;再由转化体通过植物组织培养方法再生得到完整的植株及其无性系或其后代;所述的转化方法包括:农杆菌介导的转化以及原生质体转化、Ti质粒、Ri质粒、植物病毒载体、显微注射、电穿孔法、微粒轰击等。
本发明花器官特异表达启动子在研究花器官的基因表达模式或转录调控机制、构建转基因植物、改良植物性状、培育植物新品种等方面有广泛的应用。
将本发明的花器官特异表达启动子可操作的与待转录的异源性DNA序列相连接,可以指导或调控待转录的异源基因在植物花器官中的特定部位进行转录或表达,得到具有预期性状的转基因植物或植物新品种。例如,将本发明的花器官特异表达启动子可操作的与待转录的异源DNA序列相连接(其中,该待转录的异源DNA序列还与3′非编码区可操作的连接,所述的3′非编码区可以包含终止子序列、mRNA切割序列等。)得到可以在植物花瓣或萼片中表达该待转录的异源DNA序列的植物表达载体。待转录的异源DNA序列不受限制,可以是调节基因、调节基因的反义基因或者能干扰内源基因表达的小RNA等;所述的待转录的异源DNA序列可以是来自非靶基因物种的核酸分子或基因,或者是起源于或存在于相同的物种中经过人工改造或修饰的核酸分子或基因。通常来说,待转录的异源DNA序列多为提供与下列期望特征有关的DNA序列,包括:植物形态、生理、生长和发育、产量、营养强化、病虫害抗性、环境或化学耐受性等,为植物体提供有益的性状。该待转录的异源DNA序列可以包括具有RNA活性的序列或产生多肽产物的序列等,例如,可以是反义序列、RNAi序列、核酶序列、剪接体、氨基酸编码序列以及它们的片段。
更具体的,本发明所述的花器官特异启动子可以应用于研究相关基因在花器官发育中的功能、调控植物花器官的发育或改良植物花器官的性状(包括改良花色、花香等)。
本发明所涉及到的术语定义
除非另外定义,否则本文所用的所有技术及科学术语都具有与本发明所属领域的普通技术人员通常所了解相同的含义。虽然在本发明的实践或测试中可使用与本文所述者类似或等效的任何方法、装置和材料,但现在描述优选方法、装置和材料。
术语“严谨杂交条件”意指在所属领域中已知的低离子强度和高温的条件。通常,在严谨条件下,探针与其靶序列杂交的可检测程度比与其它序列杂交的可检测程度更高(例如超过本底至少2倍。严谨杂交条件是序列依赖性的,在不同的环境条件下将会不同,较长的序列在较高温度下特异性杂交。通过控制杂交的严谨性或洗涤条件可鉴定与探针100%互补的靶序列。对于核酸杂交的详尽指导可参考有关文献(Tijssen,Techniques inBiochemistry and Molecular Biology-Hybridization with Nucleic Probes,"Overview of principles of hybridization and the strategy of nucleic acidassays.1993)。更具体的,所述严谨条件通常被选择为低于特异序列在规定离子强度pH下的热熔点(Tm)约5-10℃。Tm为在平衡状态下50%与目标互补的探针杂交到目标序列时所处的温度(在指定离子强度、pH和核酸浓度下)(因为目标序列过量存在,所以在Tm下在平衡状态下50%的探针被占据)。严谨条件可为以下条件:其中在pH7.0到8.3下盐浓度低于约1.0M钠离子浓度,通常为约0.01到1.0M钠离子浓度(或其它盐),并且温度对于短探针(包括(但不限于)10到50个核苷酸)而言为至少约30℃,而对于长探针(包括(但不限于)大于50个核苷酸)而言为至少约60℃。严谨条件也可通过加入诸如甲酰胺的去稳定剂来实现。对于选择性或特异性杂交而言,正信号可为至少两倍的背景杂交,视情况为10倍背景杂交。例示性严谨杂交条件可如下:50%甲酰胺,5×SSC和1%SDS,在42℃下培养;或5×SSC,1%SDS,在65℃下培养,在0.2×SSC中洗涤和在65℃下于0.1%SDS中洗涤。所述洗涤可进行5、15、30、60、120分钟或更长时间。
术语“宿主细胞”意指包含本发明多核苷酸的细胞,而不管使用何种方法进行插入以产生重组宿主细胞,例如直接摄取、转导、f配对或所属领域中已知的其它方法。外源性多核苷酸可保持为例如质粒的非整合载体或者可整合入宿主基因组中。
术语“核苷酸”意指单股或双股形式的脱氧核糖核苷酸、脱氧核糖核苷、核糖核苷或核糖核苷酸及其聚合物。除非特定限制,否则所述术语涵盖含有天然核苷酸的已知类似物的核酸,所述类似物具有类似于参考核酸的结合特性并以类似于天然产生的核苷酸的方式进行代谢。除非另外特定限制,否则所述术语也意指寡核苷酸类似物,其包括PNA(肽核酸)、在反义技术中所用的DNA类似物(硫代磷酸酯、磷酰胺酸酯等等)。除非另外指定,否则特定核酸序列也隐含地涵盖其保守修饰的变异体(包括(但不限于)简并密码子取代)和互补序列以及明确指定的序列。特定而言,可通过产生其中一个或一个以上所选(或所有)密码子的第3位经混合碱基和/或脱氧肌苷残基取代的序列来实现简并密码子取代(Batzer等人,Nucleic Acid Res.19:5081(1991);Ohtsuka等人,J.Biol.Chem.260:2605-2608(1985);和Cassol等人,(1992);Rossolini等人,Mol Cell.Probes8:91-98(1994))。
术语“花器官特异表达启动子”:在所述花器官特异表达启动子的调控下,异源基因的表达只限于花器官的特定部位,例如花萼、花瓣、雄蕊、雌蕊或心皮等,并表现发育调控的特性。
术语“异源性DNA序列”指该DNA序列对该特定的宿主细胞而言属于外来的来源,或若来自相同的原始来源但对该原始序列进行了修饰或改造。
术语“内源基因”来自宿主本身的基因,包括DNA或RNA序列。
术语“选择标记基因”:该基因在植物细胞中的表达给予该细胞选择优势,用这些选择性标记基因所转化的这些细胞所具有的选择优势可以是由于它们与非转化细胞的生长相比具有在阴性选择剂(如:抗菌素或除草剂)的存在下生长的能力。选择标记基因还指多种基因的组合,它们在植物细胞中的表达给予该细胞阴性以及阳性的选择优势。
“可操作的连接”指两个或更多个元件之间功能性的连接,可操作的连接的元件可为邻接或非邻接的。
术语“转化”:将异源性DNA序列引入到宿主细胞或有机体的方法。
术语“表达”:内源性基因或转基因在植物细胞中的转录和/或翻译。
术语“编码序列”:转录成RNA的核酸序列。
术语“植物表达载体”:一种或多种用于实现植物转化的DNA载体;本领域中这些载体常被称为二元载体。二元载体连同具有辅助质粒的载体是大多常用于土壤杆菌介导转化的。二元载体通常包括:T-DNA转移所需要的顺式作用序列、经工程化处理以便能够在植物细胞中表达的选择标记物,待转录的异源性DNA序列等。
附图说明
图1pProtest质粒载体图谱。
图2毛果杨PtAP1-2基因启动子克隆及酶切鉴定;(a):PtAP1-2启动子PCR扩增,(b):重组质粒双酶切鉴定;M:1kb DNA ladder,1:PCR产物,2:重组质粒的SacⅠ和KpnⅠ双酶切产物。
图3毛果杨PtAP1-2基因启动子序列分析。
图4毛果杨PtAP1-2pro::GUS植物表达载体构建;
a:PtAP1-2pro::GUS植物表达载体PCR鉴定及双酶切鉴定;
b:PtAP1-2pro::GUS植物表达载体结构示意图;
M:1kb DNA ladder,1:PCR产物,2:植物表达载体SacⅠ和KpnⅠ双酶切产物。
图5烟草根、茎、叶及花组织的GUS组织化学染色;A-E:根、茎、茎横切、叶、花,PtAP1-2pro::GUS:转化PtAP1-2pro::GUS载体的烟草,CaMV35Spro::GUS:转化CaMV35Spro::GUS载体的烟草(阳性对照),WT:野生型烟草(阴性对照)。
具体实施方式
下面结合具体实施例来进一步描述本发明,本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的,并不对本发明的范围构成任何限制。本领域技术人员应该理解的是,在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换,但这些修改和替换均落入本发明的保护范围内。
生物材料与试剂
1.1实验材料
用于提取植物基因组DNA的毛果杨(P.trichocarpa)无性系和遗传转化受体植物野生型烟草W38(N.tabacum cv.W38)均取自林木育种国家工程实验室;大肠杆菌(Escherichia coli)菌株DH5α、根癌农杆菌菌株(Agrobacterium tumefaciens)GV3101由本发明人实验室保存;pProtest质粒载体由美国俄勒冈州立大学Steven H.Strauss教授馈赠,其示意图见图1;克隆载体pMD19-T购自TaKaRa公司。
1.2主要试剂
植物基因组DNA提取试剂盒、琼脂糖凝胶DNA回收试剂盒以及普通质粒小提试剂盒均购自北京天根公司,LA Taq酶、核酸分子量标准(DNAMarker Ladder)、T4-DNA连接酶、限制性内切酶SacⅠ和KpnⅠ等购自TaKaRa公司;GUS(β-葡萄糖苷酶,β-glucuronidase)组织化学染色底物5-溴-4-氯-3-吲哚-β-D葡萄糖苷酸酯(X-Gluc)购自Sigma公司。
实施例1毛果杨PtAP1-2基因启动子的克隆及序列分析
按照北京天根公司植物基因组DNA提取试剂盒的操作流程,以毛果杨无性系叶片为材料提取基因组DNA,用1.2%琼脂糖凝胶电泳进行鉴定。根据毛果杨AP1-2基因5’侧翼序列设计引物:
上游引物5’-AGAGCTCTGCAACACTATTAAT CAATTTATC-3’,
下游引物5’-AGGTACCCT CTCTCTCTCTCTCTAAATG-3’;
在上下游引物的5’端分别加入SacⅠ和KpnⅠ酶切位点。20μL PCR反应体系包括:2μL(100ng)毛果杨基因组DNA,2μL10×LA PCR缓冲液,1.6μL(2.5mmol/L)dNTP,0.4μL10pm/μL上游引物,0.4μL10pm/μL下游引物,0.2μL LA Taq DNA聚合酶(5U/UL),13.4μL ddH2O。PCR反应循环条件为94℃预变性3min,94℃变性30s,55℃退火30s,68℃延伸2.5min,共进行35个循环,最后68℃延伸10min。
PCR产物经1.2%琼脂糖凝胶电泳检测后,切下目的条带,使用凝胶回收试剂盒(天根公司)回收、纯化目的片段。将纯化产物与pMD19-T载体连接,再转化DH5α感受态细胞,接种到含氨苄(100mg/L)的LB平板上37℃培养14h-16h,挑取单菌落进行PCR,并对重组质粒进行限制性内切酶(SacⅠ和KpnⅠ)酶切鉴定,获得阳性克隆,命名为PtAP1-2pro-T。
以毛果杨基因组DNA为模板,通过PCR扩增得到大小约为2.1kb的片段(图2a),与预期基本一致,将此片段回收与pMD19-T载体连接,经菌落PCR和双酶切(SacⅠ和KpnⅠ)(图2b)验证后,证明该启动子片段已克隆到pMD19-T载体上。阳性克隆测序结果表明,该序列长度为2117bp。
使用TSSP-TCM在线软件(Shahmuradov A,Solovyev V V,Gammerman A J(2005)Plant promoter prediction with confidence estimation.Nucleic Acids Res33:1069-1076)对测序结果进行分析,预测转录起始位点;联合运用PLACE(Higo K,Ugawa Y,Iwamoto M,Korenaga T(1999)Plant cis-acting regulatory DNA elements(PLACE)database:1999.NucleicAcids Res27:297-300;Higo K,Ugawa Y,Iwamoto M,Higo H(1998)PLACE:a database of plant cis-acting regulatory DNA elements.NucleicAcids Res26:358-359.)及PlantCARE(Lescot M,Dehais P,Thijs G,Marchal K,Moreau Y,et al.(2002)PlantCARE,a database of plant cis-actingregulatory elements and a portal to tools for in silico analysis of promotersequences.Nucleic Acids Res30:325-327;Rombauts S,Dehais P,VanMontagu M,Rouze P(1999)PlantCARE,a plant cis-acting regulatoryelement database.Nucleic Acids Res27:295-296.)在线软件对其转录调控元件进行分析。
使用TSSP-TCM在线软件对PtAP1-2基因启动子序列进行分析,预测其转录起始位点为G,位于起始密码子上游672bp处。联合使用PLACE和PlantCARE在线软件分析序列,预测其含有的主要启动子基本转录元件(图3)包括:TATA-box位于-50bp处,CAAT-box分别位于-160bp、-206bp、-235bp处,ARE位于-187bp处,ELI-box3位于-414bp处,MRE位于-493bp处,Skn-1_motif位于-509bp处,GT1-motif位于-536bp处,AE-box位于-568bp处,3-AF3binding site位于-704bp处,ATCT-motif位于-826bp处,GCN4_motif位于-767bp和-916bp处,TC-rich repeats位于-1364bp和-1380bp处,Box4位于-519bp、-580bp、-945bp、-1168bp和-1432bp处。
其中,TATA-box为启动子核心元件,保证转录精确开始;CAAT-box可以控制转录的效率和频率;ARE为厌氧诱导反应过程中必不可少的顺式作用元件;ELI-box3为激发子响应元件;MRE为光响应过程中MYB结合位点;Skn-1_motif和GCN4_motif为胚乳表达所需的相关顺式作用元件;GT1-motif和AE-box为光响应元件;3-AF3binding site为保守的DNA模块阵列的一部分;ATCT-motif和Box4为光响应过程中保守DNA模块的一部分;TC-rich repeats为抗病和逆境胁迫响应作用元件。
试验例1PtAP1-2启动子驱动报告基因在烟草花器官中特异表达试验
1.PtAP1-2pro::GUS植物表达载体的构建及工程菌制备
为研究从毛果杨(Populus trichocarpa Torr.&Gray)中所分离的花器官特异表达启动子PtAP1-2(SEQ ID No.1)的功能,将pProtest质粒进行SacⅠ和KpnⅠ双酶切,同时将PtAP1-2pro-T重组质粒进行SacⅠ和KpnⅠ双酶切,回收纯化目的片段,通过T4-DNA连接酶连接,并将连接产物转化大肠杆菌感受态细胞DH5α,新的重组质粒经氨苄霉素(Ampicillin,100mg/L)和PCR筛选后,进行双酶切验证,经菌落PCR和双酶切验证(图4a),获得重组植物表达载体PtAP1-2pro::GUS;同时,按照同样的方法构建CaMV35Spro::GUS植物表达载体作为阳性对照(图4b)。采用液氮冻融法,将表达载体PtAP1-2pro::GUS转入根癌农杆菌GV3101感受态细胞中,经菌落PCR鉴定,获得阳性克隆,完成工程菌制备。
2.PtAP1-2启动子在烟草中的瞬时表达
通过液氮冻融法将PtAP1-2pro::GUS表达载体转化到根癌农杆菌GV3101中。将烟草的根、茎、叶及花分别剪下,形成切口,浸泡于OD600≈0.6的农杆菌菌液中,真空抽滤20min。用无菌滤纸吸除材料表面多余的菌液,平铺于MS固体培养基上,28℃暗培养2d。将共培养后的材料用含头孢霉素(Cefotaxime,250mg/L)的溶液洗涤数次待用。按照Jefferson等(Jefferson RA,Kavanagh TA,Bevan MW(1987)GUS fusions:beta-glucuronidase as a sensitive and versatile gene fusion marker in higherplants.EMBO J6:3901.)的方法对材料进行GUS组织化学染色分析,将材料浸泡于GUS活性检测液,37℃过夜,然后用70%乙醇脱色,观察并拍照,其中GUS活性检测液包含:0.1mol/L K4Fe(CN)6、0.1mol/LK3Fe(CN)6、50mmol/L磷酸钠缓冲液(pH7.0)、10mmol/L Na2EDTA、0.001%(v/v)Triton X-100、0.5mg/ml X-Gluc和20%甲醇。
检测结果表明,PtAP1-2启动子驱动的GUS基因仅在烟草萼片和花瓣中特异表达,在根、茎、叶、雄蕊和心皮等其它组织部位里没有GUS表达活性;阴性对照(未转化材料)无GUS表达活性;阳性对照(CaMV35Spro::GUS)在烟草的根、茎、叶、萼片、花瓣、雄蕊、心皮等各个组织部位均有GUS表达活性(图5)。
Claims (10)
1.从毛果杨(Populus trichocarpa Torr.&Gray)中分离的花器官特异表达启动子,其特征在于,其核苷酸序列为(a)或(b)所示:
(a)、SEQ ID No.1所示的核苷酸序列;
(b)、与SEQ ID NO:1的互补序列在严谨杂交条件下能够进行杂交的核苷酸序列,该核苷酸序列仍具有花器官特异表达启动子活性。
2.一种表达盒,其特征在于,包括:含有权利要求1所述的花器官特异表达启动子和待转录的异源基因序列。
3.含有权利要求1所述的花器官特异表达启动子的重组植物表达载体。
4.按照权利要求3所述的重组植物表达载体,其特征在于,包含:权利要求1所述的花器官特异表达启动子和待转录的异源基因序列;其中,花器官特异表达启动子和待转录的异源基因序列可操作的相连接,待转录的异源基因序列位于所述花器官特异表达启动子的下游。
5.按照权利要求4所述的重组植物表达载体,其特征在于:所述的待转录的异源基因包括:结构基因、结构基因的反义基因、调节基因、调节基因的反义基因或者能干扰内源基因表达的小RNA。
6.含有权利要求3-5任何一项重组植物表达载体的宿主细胞。
7.权利要求1所述的花器官特异表达启动子在构建转基因植物或培育植物新品种中的应用,包括:将权利要求1所述的花器官特异表达启动子和待转录的异源基因序列可操作的连接后转化到植物细胞、组织或器官中得到转化体;将转化体通过组织培养再生得到完整的植株或无性系。
8.权利要求1所述的花器官特异表达启动子在改良植物性状中的应用。
9.权利要求1所述的花器官特异表达启动子在分析花器官的基因表达模式或转录调控机制中的应用。
10.权利要求1所述的花器官特异表达启动子在分析基因功能或构建转基因植物中的应用。
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