CN113717977A - 甘蓝型油菜组织特异型p8启动子及其在制备转基因油菜中的应用 - Google Patents
甘蓝型油菜组织特异型p8启动子及其在制备转基因油菜中的应用 Download PDFInfo
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Abstract
本发明涉及植物基因工程、植物保护和生物技术领域,公开了甘蓝型油菜组织特异型P8启动子及其在制备转基因油菜中的应用。本发明自中双11中克隆了启动子P8,该启动子能够特异性驱动基因在植物中高效表达,而在种子中不表达。利用P8启动子构建了油菜内源抗菌肽BnPRP1的过表达载体,获得了转基因油菜株系,该转基因油菜能够有效提高对菌核病的抗性。本发明提供的启动子驱动目的基因在转基因植物的特定组织或时期表达,避免造成植物自身能源和物质的浪费,提高外源基因在转基因植物中的表达效率,限制其表达部位。可应用于植物的基因工程研究和籽用油菜安全转基因研究。
Description
技术领域
本发明涉及植物基因工程、植物保护和生物技术领域,具体涉及甘蓝型油菜组织特异型P8启动子及其在制备转基因油菜中的应用。
背景技术
植物的生长发育和生命周期是不同的基因在时间和空间上有序表达的结果,植物启动子在基因的表达调控中起着关键作用。启动子是转录水平调控的重要顺式作用元件,也是基因工程表达载体的一个重要组成部分。在某种程度上,启动子决定了基因表达的时空顺序以及表达强度。所以,研究启动子的功能序列对于基因表达调控机制以及日渐成熟的植物基因工程具有非常重要的意义。
启动子在构建能够高水平表达异源表达载体的过程中起到关键作用,它决定了外源基因的转录效率和基因的表达水平。植物启动子按其转录方式可分为组成型、组织特异型及诱导型三类。目前,植物转基因工程中使用的多是组成型启动子。组成型启动子调控的基因表达不受外界环境条件的影响,几乎在所有植物不同组织中都能表达。对许多双子叶植物的转化,通常都使用含花椰菜花叶病毒(CaMV)的35S启动子或者其他两个来源于植物的组成型启动子-拟南芥色氨酸合成酶蛋白β亚基基因(PTSB1)启动子和植物光敏色素B基因(PPHYB)启动子(聂丽娜.植物基因启动子的克隆及其功能研究进展[J].植物遗传资源学报,2008,9(3):385-391.)。而在单子叶植物转化中最常用的是水稻肌动蛋白Actin1启动子和玉米泛素Ubiquitin启动子(张春晓,王文棋,蒋湘宁.植物基因启动子研究进展[J].遗传学报,2004,31(12):1455-1464.)。但是很多时候外源基因在受体植物中的持续高效表达不仅造成生物体内能源的非必要浪费,而且在所有组织中的表达有可能对植株本身具有毒害作用,阻碍植物的正常生长,甚至引起转基因安全问题(Jia S-R(贾士荣).Environmentand food biosafty assessment of transgenic plants.Advanced of Bioengineer,1997,17(6):37-42;Morris S H,Adley C.C.Irish public perceptions and attitudesto modern biotechnology:an overview with a focus on GM foods.Trends inBiotechnology,2001,19(2):43-48.)。因此,组织特异型启动子和诱导型启动子的研究和应用日益受到育种工作者的重视。
目前,在抗逆植物基因工程中应用最广泛的诱导型启动子为rd29A启动子,该启动子能够驱动目的基因在干旱、低温和ABA等非生物胁迫下表达(聂丽娜.植物基因启动子的克隆及其功能研究进展[J].植物遗传资源学报,2008,9(3):385-391.)。但是诱导型启动子的应用具有一定限制,对受体植物进行的外在条件处理,如热激,激素处理等可能引起生物体内一系列的生理生化反应而不利于植株的正常生长。而且化学调控系统中用作诱导物的甲基脱氢皮质醇(Dex,Dexamethasone)、雌二醇(Estradiol)和四环素(Tetracycline)都对生态环境有害,不宜用于生产实践。而使用植物体内自身的组织特异性启动子就可以避免这种问题。显然,外源基因的组织特异性表达必将有效提高转基因作物的生物安全性。近年来在这方面已经取得很大成就。在不同组织中特异性表达的各类启动子已不断得到研究(宋扬等.植物组织特异性启动子研究.生物技术通报,2007,(4):21-24.)。
王劲等克隆到了一个油菜维管束特异性基因BnVSP的启动子,该启动子能够驱动目的基因在目标组织(如导管、筛管)中进行定位表达,从而实现基因的特异性功能(WangJ,ZuoK,et al.OilseedRape Vascular Bundle Specific PromoterBnVSPand UseThereof.WO/2012/083545)。刘胜毅等克隆到了一个油菜花特异性启动子P76247,该启动子能够驱动GUS在拟南芥花中特异性的表达(刘胜毅等.甘蓝型油菜P76247启动子及制备方法和应用,ZL 2014 10058108.6)。
发明内容
本发明的目的在于提供了一种甘蓝型油菜组织特异型P8启动子,所述启动子为SEQ ID NO.1所示。
本发明的另一个目的在于提供了一种甘蓝型油菜组织特异型P8启动子在制备转基因十字花科植物中的应用。
为了达到上述目的,本发明采取以下技术措施:
一种甘蓝型油菜组织特异型P8启动子的获得:
以“中双11”的基因组DNA为模板,设计引物,进行PCR扩增,扩增所用引物P8F:5′-AAAATATTATTCTAATCGTATTA-3'和P8R:5′-CTGGCTAAACGCATCAAAAAG-3′,获得P8启动子,所述启动子P8的核苷酸序列如下所示:
AAAATATTATTCTAATCGTATTAAGTTTTATTAAGAAAACTAAACCTATTAAAATCGAGTAATCAATAATCCCACTAAAAAAAAACTCAAGTCCATAGTTCAACAGAGAATTACCAACTGTAAAATCTTAACGTCTATTTCATAACCGATCCAACGGTGGATAATTTTCTCACACCTCTTCGACATTTCTATGGCTCGGTTATGTTTCCCACACACAGAAAAAAATAATAATAAAAGAACTCCTTTAATTATAAAAAGAGACATAAAATCGGAACAGTGGCGTCTCTCTTTCCTCTTAGCAGTTGTCTTCATTTCCCCAAATCTCTCCCAGAAATCTATCTTCGATATCGCACGCGCCGCCGCAGGCTTCGCCGCCGGCAACTGCTTTTACCCTTACCCTCTTCGTCTCCCCTCTCTTCCTCAATTCATAAAAACCCATAAAGAAGAAATCTTTCTGCTTCGTTTTATCTTCTCCGCATCAGTTGTTTCTCTCTGATTTGCTTTTGTTTCTTTCGTTTCTATCTTGATTGCTCTGTAATAAACGAGAGGGGAGAGAAGAGCACAAAGAAAAAAAAACCCAAAAAAAAAAAGAAAAAAGTTTCAAAAACCAAAAGAGAAAGAAAATGGCTCAAAGTCAAACTCCGAACGGCTCTGTTCTGCCGGTTGGTTTGGGTGCAGCGGGCGCTCAGTTCGGTACGACGTCGCTTTACGTCGGAGATCTGGATGCGAGTGTTACCGATTCGCAGCTTTTTGATGCGTTTAGCCAG。
一种甘蓝型油菜组织特异型P8启动子在制备转基因十字花科植物中的应用,包括利用本发明提供的P8启动子,启动外源基因在十字花科植物中进行表达,获得转基因植物,例如,驱动抗逆基因的表达,获得抗逆植物。
所述的十字花科植物优选为拟南芥,甘蓝型油菜。
包含P8启动子的植物表达载体或包含P8启动子的重组菌株也属于本发明的保护范围。
与现有技术相比,本发明达到了以下技术效果:
1.本发明提供了一个组织特异型启动子,所述启动子能够特异性驱动基因在植物中高效表达,而在种子中不表达,该启动子可以应用到十字花科,例如拟南芥,油菜等植物中。
2.本发明构建的植物表达载体过表达抗菌肽BnPRP1,为油菜抗病育种提供了原材料,具有重要意义,在作物抗菌核病的育种实践、品种改良及品种推广均具有重要的实践指导价值。
3.本发明提供的启动子的时空表达模式可用于研究基因的表达形式,优点在于,来源于植物内源的组织特异性启动子能够精确定位所调控的基因,驱动目的基因在转基因植物的特定组织或时期表达,避免造成植物自身能源和物质的浪费,提高外源基因在转基因植物中的表达效率,限制其表达部位。可应用于植物的基因工程研究和籽用油菜安全转基因研究。
附图说明
图1为P8组织特异性验证的植物表达载体pBI121-P8::GUS图谱。
图2为启动子P8的转基因植株的PCR鉴定;
其中:泳道M:DL2000 DNA marker;1-4:pBI121-P8::GUS转基因阳性苗;5:pBI121-P8::GUS重组质粒阳性对照;6:Col-0野生型拟南芥阴性对照。
图3为pBI121-P8::GUS转基因阳性苗的GUS染色结果示意图;
其中:A:二叶期;B:四叶期;C:拟南芥的茎及成熟叶片;D:花序组织;E:单朵花组织;F:柱头及雄蕊;G:花粉粒;H:角果及种子。
图4为pBI121-P8::SP-PRP植物过表达载体图谱。
图5为pBI121-P8::SP-PRP转基因油菜植株的PCR鉴定;
其中:泳道M为DL 2000DNA marker;泳道1-10:转基因阳性株系;泳道11:野生型Westar,泳道12:P8::SP-PRP重组质粒阳性对照。
图6为pBI121-P8::SP-PRP转基因油菜植株叶片的菌核病抗性分析示意图;
其中:A:转基因油菜立体叶片接种核盘菌后80h的病斑大小;B:离体叶片接种核盘菌后不同时间点的病斑大小统计表;C:转基因株系R5-34体内BnPRP1基因的表达量(**,P<0.01);D:转基因株系叶片粗蛋白对核盘菌生长的影响;E:核盘菌在含有转基因株系R5-34的叶片粗蛋白的平板上培养36h后的菌斑直径(**,P<0.01)。
具体实施方式
为了使本技术领域的人员更好地理解本发明方案,下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分的实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例,都应当属于本发明保护的范围。
下面以以下实施例对本发明的原理和特征进行描述,下述实施例中所用的材料、试剂、载体和农杆菌等,如无特殊说明,均可从公司通过商业途径购买。
本发明以利用P8启动子过表达抗菌肽BnPRP1为例,进行说明,其他的抗逆基因,也能用于制备抗逆转基因油菜。
同时,还可以通过本发明的启动子在油菜中表达其他的外源基因。
实施例1:启动子P8的克隆与序列测定及启动子元件预测
利用CTAB法提取甘蓝型油菜品种“中双11”的基因组DNA(J.萨姆布鲁克.D.W.拉塞尔著,贺福初等译,分子克隆试验指南(第四版)科学出版社.),按照参考基因组序列,设计引物,进行PCR扩增,克隆获得油菜启动子P8(767bp)。扩增所用引物P8F:5′-AAAATATTATTCTAATCGTATTA-3'和P8R:5′-CTGGCTAAACGCATCAAAAAG-3′。
PCR反应体系如下:5×PrimeSTAR Buffer(Mg2+Plus)10μL,dNTP(2.5mM)4μL,5’引物(10μM)1μL,3’引物(10μM)1μL,HS聚合酶0.5μL,DNA模板1μL,无菌水补至50μL,根据具体情况设置循环参数。PCR扩增得到长度为767bp的片段。回收纯化PCR产物,并与测序T载体Simple Cloning Vector进行TA克隆(全式金公司,目录号CT111-01),反应体系为:4μL回收产物,Simple Cloning Vector,25℃恒温孵育器反应5min。连接产物,用热激法转化大肠杆菌DH5α感受态细胞,加400μL新鲜的液体LB培养基,复苏20min,涂于氨苄抗生素的LB平板上,37℃倒置过夜培养。挑选白色单克隆菌落,在含氨苄抗生素的液体LB培养基中扩增培养,测序。获得P8启动子序列,命名为P8,为SEQ IDNO.1所示的多核苷酸。
实施例2:P8启动子的植物表达载体构建及根癌农杆菌转化
用Hind III/BamH I双酶切pBI121质粒。设计两端含有pBI121质粒序列的引物(P8-121F:ACCATGATTACGCCAAGCTTAAAATATTATTCTAATCGTATTA;P8-121R:GACTGACCACCCGGG GATCCCTGGCTAAACGCATCAAAAAG)扩增实施例1中得到的P8的TA克隆质粒,得到两端含有pBI121质粒的P8片段。凝胶回收P8片段及酶切下来pBI121大片段,同源重组连接体系:P8片段回收产物4μL,质粒pBI121大片段回收产物3μL,酶(Exnase II)1μL,5×CE II Buffer 2μL,37℃孵育30min后,转化大肠杆菌(方法前面已述),经过单斑测序后,构建成植物表达载体pBI121-P8::GUS(图1)。采用冻融法将该重组载体转入农杆菌GV3101,具体方法为:取重组载体3μL加入到农杆菌GV3101的感受态中,冰浴15min后,将感受态放入液氮中5min,之后再在37℃孵育5min,加入300μLLB,28℃摇菌1h,将菌液涂布在含有卡那霉素(50mg/L)和利福平(50mg/L)双抗性LB平板上,48h后挑选单克隆,以实施例1中的P8F/P8R引物检测阳性克隆。
实施例3:pBI121-P8::GUS的拟南芥转化及PCR检测
按照文献(Zhang X R.et al.Agrobacterium-mediated transformation ofArabidopsisthaliana using the floral dip method.Nature,2006,1:1-6)中转化方法转化野生型拟南芥Col-0。根据转基因植株所特有的卡那霉素抗性,在含有浓度为50mg/L卡那霉素的MS固体培养基上生长,获得的绿苗初步认为是阳性苗。待绿苗长出两片真叶后,将其移栽到蛭石中,待植株生长到8叶期,取一片真叶用CTAB法提取基因组DNA,采用P8F/P8R引物做阳性苗检测,PCR反应体系如下:1×PCR buffer,MgCl2 1.5mM,dNTPs 0.2mM,引物浓度为0.5μM,ExTaq酶1.5U,模板约100ng,加无菌水至20μL。用1%琼脂糖凝胶电泳检测PCR反应产物,结果表明P8植物表达载体pBI121-P8::GUS已经成功转入拟南芥,如图2所示,泳道M为DL 2000 DNA marker;泳道1-4为pBI121-P8::GUS转基因阳性苗;P8-1,-2,-3,-4为4个转基因株系;泳道5为pBI121-P8::GUS重组质粒阳性对照;泳道6为Col-0野生型拟南芥。
实施例4:甘蓝型油菜P8启动子的GUS染色分析
从实施例3中,拟南芥转化及PCR检测步骤中筛选得到的阳性苗T1代,自交收获种子(即T2代)。取T2代5个株系的不同生长时期组织进行GUS染色。
T2代取染色过程如下:将样品浸泡在GUS染液(GUS染色试剂盒购自北京酷来博科技有限公司,以下相同),37℃过夜。第二天用酒精-乙酸(体积比为1:1)进行脱色,直至叶片变白,之后用蒸馏水漂洗3-5次,体式显微镜(OLYMPUS SZX16)拍照。苗期取不同时间点的整个植株;生殖生长期,叶片取嫩叶和成熟叶片;花取开过的花朵和未开的花蕾的花序;角果取不同时期的角果;种子取开花后10天左右的种子。植株被染成蓝色的部位就是GUS基因表达的部位。
染色结果如图3所示:在拟南芥的整个生长过程中,根、叶片、茎、花组织均被染成蓝色;在角果中,角果皮染成蓝色,但是种子中未出现蓝色。由此可见,该启动子驱动的GUS基因在除种子外的所有组织中表达。这一具有组织特异性表达的启动子在植物基因工程和转基因安全(食用)中具有应用价值。
实施例5:pBI121-P8::SP-PRP植物过表达载体构建
利用实施例1中得到的P8启动子,构建油菜抗菌肽BnPRP1的植物过表达载体pBI121-P8::PRP。采用BamHI/Sac I双酶切实施例2中得到的重组载体pBI121-P8::GUS,采用引物SPF:5’-ATGCGTTTAGCCAGGGATCCATGAAGAACACCAGCAGCTT-3’,PRP-R:5’-GATCGGGGAAATTCGAGCTCTCATGGTGGAGCCATAGGTT-3’,及公司合成的含有SP-BnPRP1序列的质粒puc57-SP-PRP为模板,扩增SP-BnPRP1片段,SP为水稻α-淀粉酶信号肽(αAmy3SP),该信号肽能够指导基因定位于细胞间隙(Chen M H,Huang L F,Li H M.et al.Signal peptide-dependenttargeting of a rice a-Amylase and cargo proteinsto plastids and extracellularcompartments of plantcells[J].Plant Physiology,2004,7(35):1367-1377.)。BnPRP1为本实验室筛选到的油菜体内的抗菌肽(Cao H,Ke T,Liu R,et al.Identification of aNovel Proline-Rich Antimicrobial Peptide from Brassica napus[J].PLOS ONE,2015,10(9):e0137414)。PCR反应体系如实施例1中所述。将得到的基因片段与重组载体pBI121-P8::GUS片段通过同源重组的方法(如实施例2中所述)进行连接,并转化到大肠杆菌DH5α感受态中(方法如实施例1所述),挑取单克隆,PCR鉴定阳性克隆并测序,获得pBI121-P8::SP-PRP植物过表达载体。抗菌肽过表达载体图谱如图4所示。
实施例6:pBI121-P8::SP-PRP植物过表达载体的油菜遗传转化及阳性苗鉴定
将实施例5中得到的抗菌肽过表达载体转化到农杆菌GV3101中,方法如实施例2中所述,鉴定到阳性克隆后,通过下胚轴遗传转化方法,将抗菌肽BnPRP1基因转入油菜品种Westar中。下胚轴遗传转化方法参考文献(王美容.甘蓝型油菜BnWRKY28转录因子功能分析[D].华中农业大学,2015.)中的方法。通过卡那霉素筛选阳性苗,提取阳性苗叶片总DNA,方法同实施例1,采用P8F(实施例1)/PRP-R(实施例5)引物扩增目的片段,结果如图5所示,泳道M为DL2000DNAmarker;泳道1-10为转基因阳性株系;泳道11为野生型Westar,泳道12为P8::SP-PRP重组质粒阳性对照。
实施例7:过表达BnPRP1的转基因油菜的菌核病抗病分析
选择实施例6中的1个转基因株系作为菌核病鉴定材料,实验材料于生长间(16h/光照、8h/黑暗)培养至4-6叶期,采集生长状态相似的叶片离体接种核盘菌(接种的核盘菌菌饼的初始大小是5mm)。记录不同时间的病斑大小,并照相。采集转基因株系的幼嫩叶片,使用TRIzol(Invitrogen公司,货号15596026)法提取总RNA,逆转录试剂盒(Takara公司,货号RR047A)合成第一链cDNA,以油菜Actin基因作为内参基因,采用引物PRPqF/qR检测转BnPRP1基因的阳性转基因植株表达量情况,引物序列如下:
PRPqF:5’-CCTCCGACCCAGAATCCCT-3’;
PRPqR:5’-TCATGGTGGAGCCATAGGTT-3’;
BnActinF:5’-CTGGAATTGCTGACCGTATGAG-3’;
BnActinR:5’-ATCTGTTGGAAAGTGCTGAGGG-3’;
提取转基因株系叶片粗蛋白,参考文献(Wang,Zhuanrong,Wan,Lili,Xin,Qiang,et al.Overexpression of OsPGIP2 confers Sclerotiniasclerotiorum resistance inBrassica napus through increased activation of defense mechanisms[J].Journalof Experimental Botany,2018,69(12):3141-3155)中的方法,将提取的粗蛋白与等体积PDA混合,倒板,然后接种新鲜的核盘菌菌饼,36小时后统计菌斑大小,并用光学显微镜观察核盘菌菌丝的生长状态。
如图6所示,图6中A为接种核盘菌80h后的病斑大小;B为接种核盘菌后不同时间点的病斑扩展情况:24-80hpi:R5-34-1叶片中病斑大小分别是0,0.2,2,3,9.2mm,SD分别为0,0,4472,1.0247,1.5411,3.2711;WT叶片上病斑大小分别是0.1111,3.2222,8.1111,14.1667,24.0556mm,SD分别为0.3333,2.0327,3.3145,6.7175,7.0730;C为转基因株系中目的基因BnPRP1的表达水平:Westar中相对表达量为1,SD=0;R5-34中BnPRP1的表达量为3.5211,SD=0.8767;D为转基因株系叶片粗蛋白对核盘菌的抑制效果以及核盘菌菌丝的生长状态;图E为核盘菌在转基因叶片粗蛋白的平板上的菌斑大小:对照Westar中菌斑直径6.9cm,SD=0.1414;转基因植株R5-34中菌斑直径5.625cm,SD=0.1768。
由图6的结果可以看出,接种核盘菌80h后,转基因株系上的病斑显著小于野生型对照,同时在接种后的各个时间点,转基因株系的病斑一直显著小于野生型对照,说明转基因株系对菌核病的抗性显著增强。同时转基因株系中目的基因BnPRP1的表达量显著高于野生型,说明转基因株系中的目的基因BnPRP1已成功转入,并高量表达。转基因植株叶片的粗蛋白,同样能够显著抑制核盘菌菌丝的生长。在含有转基因植株粗蛋白的平板上,核盘菌的菌丝表现出打结、黏连、顶端分枝畸形等症状,说明转基因植株的粗蛋白能够影响核盘菌的正常生长,从而表现出抗病。
综上所述,通过基因工程技术,通过组织特异性启动子的驱动,在油菜中过量表达抗菌肽BnPRP1能够显著提高油菜的菌核病抗性,证明抗菌肽BnPRP1在植物体内也有显著的抗菌核病效果。本发明为油菜菌核病抗病育种提供了新的靶基因源分子,同时不仅为油菜也为其他植物提供了一个高效表达的启动子,本发明在增强甘蓝型油菜对菌核病抗性方面具有重要的应用前景。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 中国农业科学院油料作物研究所
<120> 甘蓝型油菜组织特异型P8启动子及其在制备转基因油菜中的应用
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 767
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
aaaatattat tctaatcgta ttaagtttta ttaagaaaac taaacctatt aaaatcgagt 60
aatcaataat cccactaaaa aaaaactcaa gtccatagtt caacagagaa ttaccaactg 120
taaaatctta acgtctattt cataaccgat ccaacggtgg ataattttct cacacctctt 180
cgacatttct atggctcggt tatgtttccc acacacagaa aaaaataata ataaaagaac 240
tcctttaatt ataaaaagag acataaaatc ggaacagtgg cgtctctctt tcctcttagc 300
agttgtcttc atttccccaa atctctccca gaaatctatc ttcgatatcg cacgcgccgc 360
cgcaggcttc gccgccggca actgctttta cccttaccct cttcgtctcc cctctcttcc 420
tcaattcata aaaacccata aagaagaaat ctttctgctt cgttttatct tctccgcatc 480
agttgtttct ctctgatttg cttttgtttc tttcgtttct atcttgattg ctctgtaata 540
aacgagaggg gagagaagag cacaaagaaa aaaaaaccca aaaaaaaaaa gaaaaaagtt 600
tcaaaaacca aaagagaaag aaaatggctc aaagtcaaac tccgaacggc tctgttctgc 660
cggttggttt gggtgcagcg ggcgctcagt tcggtacgac gtcgctttac gtcggagatc 720
tggatgcgag tgttaccgat tcgcagcttt ttgatgcgtt tagccag 767
<210> 2
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
aaaatattat tctaatcgta tta 23
<210> 3
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ctggctaaac gcatcaaaaa g 21
<210> 4
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
accatgatta cgccaagctt aaaatattat tctaatcgta tta 43
<210> 5
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gactgaccac ccggggatcc ctggctaaac gcatcaaaaa g 41
<210> 6
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atgcgtttag ccagggatcc atgaagaaca ccagcagctt 40
<210> 7
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gatcggggaa attcgagctc tcatggtgga gccataggtt 40
<210> 8
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
cctccgaccc agaatccct 19
<210> 9
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
tcatggtgga gccataggtt 20
<210> 10
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ctggaattgc tgaccgtatg ag 22
<210> 11
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
atctgttgga aagtgctgag gg 22
Claims (7)
1.一种分离自甘蓝型油菜的启动子,其为SEQ ID NO.1所示。
2.包含权利要求1所述的启动子的植物表达载体。
3.包含权利要求1所述的启动子的重组菌株。
4.权利要求1所述的启动子在制备十字花科转基因植物中的应用。
5.权利要求1所述的启动子在制备十字花科抗逆转基因植物中的应用。
6.根据权利要求4或5所述的应用,所述的十字花科植物为拟南芥或甘蓝型油菜。
7.根据权利要求5所述的应用,所述的抗逆为抗菌。
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WO2002097103A2 (en) * | 2001-05-31 | 2002-12-05 | Syngenta Limited | Constitutive plant promotor |
CN102220327A (zh) * | 2011-05-27 | 2011-10-19 | 中国农业科学院油料作物研究所 | 甘蓝型油菜BnPABP8启动子及制备方法和应用 |
CN102220325A (zh) * | 2011-05-12 | 2011-10-19 | 中国农业科学院油料作物研究所 | 甘蓝型油菜BnPABP3启动子的制备方法及其应用 |
CN104530204A (zh) * | 2014-12-19 | 2015-04-22 | 中国农业科学院油料作物研究所 | 一种油菜抗菌肽BnPRP1及其应用 |
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Publication number | Priority date | Publication date | Assignee | Title |
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WO2002097103A2 (en) * | 2001-05-31 | 2002-12-05 | Syngenta Limited | Constitutive plant promotor |
CN102220325A (zh) * | 2011-05-12 | 2011-10-19 | 中国农业科学院油料作物研究所 | 甘蓝型油菜BnPABP3启动子的制备方法及其应用 |
CN102220327A (zh) * | 2011-05-27 | 2011-10-19 | 中国农业科学院油料作物研究所 | 甘蓝型油菜BnPABP8启动子及制备方法和应用 |
CN104530204A (zh) * | 2014-12-19 | 2015-04-22 | 中国农业科学院油料作物研究所 | 一种油菜抗菌肽BnPRP1及其应用 |
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