CN102286486B - 花粉特异性启动子的分离、鉴定及其应用 - Google Patents
花粉特异性启动子的分离、鉴定及其应用 Download PDFInfo
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Abstract
本发明提供了一种花粉特异性启动子及其应用。该启动子来源于白菜型油菜(Brassica rapa),是一个Δ8鞘脂脱氢酶基因的上游序列。本发明的启动子是一个稳定的特异性高的花粉特异性启动子。利用本发明的启动子可以在花粉中启动表达各种基因,可用于培育雄性不育作物品种和培育消除或大幅度减少花粉的园林绿化植物等。
Description
技术领域
本发明涉及一种植物花粉特异性启动子及其应用。具体地,涉及一个Δ8鞘脂脱氢酶基因启动子的获得和植物表达载体的构建,以及其在培育植物雄性不育系、花粉消除或大幅度减少的园林绿化植物中的应用。
背景技术
在高等植物的发育过程中,雄配子的发育是个非常复杂的过程,其调控机制十分复杂。它包括一系列器官分化及严格控制的细胞及生化变化,同时伴随着大量基因的协同表达。在花粉发育过程中,大约涉及20000种基因的表达,其中大约10%为花粉特异表达[1],而花粉特异表达的启动子对于这些基因在花粉中的特异表达起到了十分重要的作用。
启动子是RNA聚合酶能够识别并与之结合,从而起始基因转录的一段DNA序列。启动子通常位于基因上游,是基因表达调控的重要元件,它基本决定一个基因是否表达、何时表达和何处表达。组织特异性基因的启动子对该基因的组织特异性表达起重要作用。
利用杂种优势可以显著的提高作用的产量,改善其品质。近十几年来,杂种优势育种已成为许多作物的主要育种方法。杂交繁育是传统的将两种植物的所需遗传性状导入一种植物以培育植物新品种的方法。无论对于自花授粉作物或异花授粉作物杂交,为了获得可靠的性状一致的杂种,应确保亲本系之一为雄性不育。产生雄性不育目前有多种技术,一种方法是人工地或机械化地除去雌性亲本植物的花药或穗状雄花,该植物只有通过雄性亲本的花粉才具有可育性,然而该方法费时费力而且不完全可靠。另外一种方法是利用化学去雄制种,即选用某种化学药剂,在植物生长发育的某一时期喷洒母本,直接杀伤或抑制雄性器官及花粉,造成生理雄性不育,以达到去雄的目的。但这种方法因去雄不彻底,易受环境影响,效果不稳定,或价格太昂贵等,在应用上也受到限制。基因工程提供了另一种替代方法,来获得植物雄性不育系。例如在花药或花粉特异表达损害细胞的基因,导致花粉败育,从而获得雄性不育系。
转基因雄性不育及其恢复的一个关键是花药或花粉特异表达的启动子,花药或花粉特异表达启动子可以直接驱动人工雄性不育基因在花粉或花药中特异表达而对雌蕊和其它器官没有影响。对于花粉特异性启动子的研究不仅有助于在理论上研究花粉发育过程中基因表达的调控机制,而且为创造植物转基因雄性不育奠定了基础。目前仅从番茄[2,3]、矮牵牛[4]、玉米[5,6,7]、烟草[8,9]、金鱼草[10]、拟南芥[11]、小麦[12]和大小麦[13]、水稻[14]等少数植物中分离鉴定了花粉特异表达的启动子。
利用花药或花粉特异的启动子通过基因工程创造雄性不育的方法主要有以下几种:1,借助编码细胞毒素基因的特异表达导致植物的雄性不育。例如,Zhan等利用水稻花粉特异表达的启动子与核糖核苷酸酶基因Barnase连接构建嵌合基因转化烟草,得到了雄性不育的转基因植株[14]。2,利用反义RNA技术获得雄性不育植株。Van der Meer将chsA基因的反义RNA基因与花药特异表达的启动子串联,转化矮牵牛得到了雄性不育的矮牵牛植株[15]。3,通过提早降解胼胝质获得雄性不育植株。Worrall等将经过修饰的β-1,3-葡聚糖酶基因与拟南芥绒毡层特异表达的启动子A3和A9重组,使转基因植株在减数分裂时期表达过量的β-1,3-葡聚糖酶,从而出现了不同程度的雄性不育现象[16]。在这些方法中,第一种方法的应用最为广泛,而其中用的几乎都是细胞毒素基因Barnase基因,该基因已在水稻[14,20]、烟草[17]、小麦[18]、玉米[19]和油菜[20-22]等植物上得到了应用。
油菜(芸苔属,十字花科)是世界上最重要的油料作物之一,通过分离油菜花药或花粉特异性启动子来创建雄性不育系进行杂种优势利用是提高油菜产量和品质的有效方法之一,该方面研究不仅有助于深入理解启动子调控油菜中基因转录的调控机制及花药或花粉特异表达方式的本质,同时更重要的是其潜在的巨大农业生产价值。对于芸苔属花粉特异性启动子的研究鲜有报道,目前仅有Dzelzkalns等研究发现芸苔属SLG基因上游序列的两个区域(-415到-291和-117到-8)具有启动花粉特异性表达的活性[23]。
白菜型油菜Δ8-鞘脂脱氢酶基因BrD8B是一种花器官特异性表达的基因,我们进一步证实BrD8B基因的启动子为花粉特异性表达的启动子,且特异性强,它的获得为通过基因工程的方法获得植物雄性不育创造了有利条件,为外源基因(对花粉或花粉生成有影响有基因)在花粉中特异性表达提供基础。理论上,将此启动子与细胞毒素基因如Barnase基因或花粉发育的重要基因如chsA(查耳酮合成酶)基因的反义基因相连构建载体转化植物都可得到雄性不育转基因植物。另外,利用此方法获得雄性不育在培育无花粉或花粉显著减少的园林绿化植物上也具有潜在的应用价值,这可能对花粉过敏症有一定的缓解作用。因此,对于花粉特异性表达启动子的研究具有很大的应用价值。白菜型油菜Δ8-鞘脂脱氢酶基因BrD8B启动子的序列和功能尚未见报道。
发明内容
本发明提供了一个花粉特异性表达启动子proBrD8B,其核苷酸序列如SEQ ID NO:1所示,这个启动子来源于白菜型油菜(Brassica rapa)的DNA片段,该启动子大小1292bp。
本发明还提供了一种花粉特异性表达的载体,其特性在于转入了前述的白菜型油菜花粉特异性表达的启动子。利用前述的白菜型油菜特异性表达的启动子置换植物表达载体pBI121上的CaMV35S启动子,构建成在启动子下游含有GUS基因的新的植物表达载体,命名为pBI-ProBrD8B。通过真空渗透法转化模式植物拟南芥,成功获得转基因植株。GUS组织化学染色鉴定BrD8B基因的启动子为花粉特异性表达的启动子,pBI-ProBrD8B为一种花粉特异性表达的载体。
本发明的表达载体可通过使用Ti质粒,Ri质粒,植物病毒载体,直接的DNA转化,微注射,电穿孔等方式导入植物细胞。
可使用本发明的方法转化的植物宿主选自由烟草、小麦、水稻、玉米、棉花、油菜、向日葵、大豆、番茄、蓖麻、芝麻和花生等植物组成的组。
本发明提供的花粉特异性表达的启动子及其植物表达载体,可应用于利用基因工程的方法获得植物尤其是油菜雄性不育系以及培育无花粉或花粉大量减少的园林绿化植物等方面。
具体地,一方面,本发明提供花粉特异性启动子,其特征在于:它的核苷酸序列是:
(a)序列表中序列1所述的核苷酸序列;或
(b)与(a)所述的核苷酸序列互补的核苷酸序列。
另一方面,本发明提供含有权利要求1所述的启动子的表达盒。优选地,所述的表达盒特征在于:所述花粉特异性启动子的下游连接结构基因、调节基因、结构基因的反义基因、调节基因的反义基因或者能够干扰内源基因表达的小RNA。优选地,所述花粉特异性启动子的下游连接Barnase基因、花粉发育的重要基因如chsA基因的反义基因等。
另一方面,本发明提供含有上述启动子的重组表达载体,其特征在于:转入了上述的花粉特异性表达的启动子。
另一方面,本发明提供含有上述表达盒的重组表达载体。优选地,所述的重组表达载体特征在于是由权利要求2或3所述的表达盒与质粒、病毒或转运载体所构建的重组载体。更优选地,所述的重组表达载体为双元载体或共合载体。
另一方面,本发明提供宿主细胞,其包含上述任一方面的重组表达载体。
另一方面,本发明提供所述的花粉特异性启动子在培育植物雄性不育系、花粉消除或大幅度减少的植物中的应用。优选地,所述植物是油菜、拟南芥、烟草、玉米、水稻、向日葵、大豆、番茄、蓖麻、棉花、芝麻和花生。
另一方面,本发明提供获得所述花粉特异性启动子的方法,其特征在于是以白菜型油菜(Brassica rapa)的基因组DNA为模板,用序列表中序列2和序列3的核苷酸序列作为一对引物进行PCR扩增得到。
附图说明
图1.含Δ8鞘脂脱氢酶基因BrD8B启动子的植物表达载体的构建。
图2.BrD8B启动子驱动GUS基因在花药特异表达的染色图(表达GUS基因的细胞经染色呈蓝色,如箭头所示)。A:播种7天的拟南芥幼苗;B:成熟拟南芥叶片;C:成熟花朵和未成熟花蕾;D:未成熟果荚和种子。在植株的根、茎、叶和果荚中都检测不到GUS基因的表达;在开放花朵中显示GUS基因在花药中表达。
图3.BrD8B启动子驱动GUS基因在花粉特异表达的染色图(表达GUS基因的细胞经染色呈蓝色,如箭头所示)。A:单个花药(箭头示花粉的位置);B:单个花粉。
具体实施方式
下面参考实施例和附图详细描述本发明。本领域的普通技术人员可以理解的是,下述实施例是举例说明的目的,其不应以任何方式解释为对本发明的限制。本发明的保护范围由后附的权利要求所限定。
实施例1.Δ8鞘脂脱氢酶基因BrD8B启动子的获得
1.白菜型油菜基因组DNA的制备
以本组种植的白菜型油菜活体植株为材料,取其幼嫩叶片(约100mg),置于研钵中加入液氮充分研磨至材料完全破碎。然后将研磨充分的材料转入1.5ml离心管中,加入600μl预热的CTAB提提液(参见“精编分子生物学实验指南”2001,科学出版社,颜子颖,王海林译),混匀。65℃水浴30min。加入等体积氯仿,轻柔抽提约5min。于12000rpm室温离心10min。取上清,加入1/2体积异丙醇混匀,室温放置10min沉淀DNA。用枪头将沉淀出的DNA挑出,加入70%乙醇中洗涤两次。除去70%乙醇,空气中吹干。溶于适量灭菌ddH2O,-20℃保存。
2.Δ8鞘脂脱氢酶基因BrD8B启动子的克隆
设计引物从所提取的白菜型油菜DNA中克隆BrD8B启动子的DNA片段。所用反应体系如下:
PCR反应体系(50μl):
DNA模板:1μl(50ng)
Pfu聚合酶(购自全式金公司):0.5μl
10×buffer:5μl
正向引物:2.5μl
反向引物:2.5μl
dNTP:2μl
H2O:36.5μl
PCR程序:预变性95℃4min,变性94℃30s,退火50℃40s,延伸72℃2min 30s,35个循环,延伸72℃10min。
PCR产物电泳(1%琼脂糖凝胶浓度)后切胶回收目的片段(BrD8B启动子,约1.3kb),连入pEASY-Blunt载体(购自Transgen公司),进行测序。测序结果为该启动子长1292bp,序列参照序列表SEQ ID NO:1中。通过软件分析,该启动子具有高等植物启动子所应具有的调控元件,如TATA盒、CAAT盒等。BrD8B启动子DNA片段的克隆所用引物见序列表SEQ ID NO:2和SEQ ID NO:3。
实施例2.Δ8鞘脂脱氢酶基因BrD8B启动子植物表达载体的构建
从pEASY-Blunt载体上使用限制性内切酶Hind III和BamH I(购自Takara公司)进行双酶切,与同样双酶切的pBI121(购自Clontech公司)载体连接,构建将pBI121上的CaMV35S启动子置换为BrD8B启动子的植物表达载体,测序验证,并将其命名为pBI-proBrD8B,其载体图见图2。pBI121载体含有GUS基因,经染色其呈现蓝色,用于检测启动子的特异性表达。
实施例3.转pBI-proBrD8B的拟南芥的获得
1.植物表达载体转入农杆菌
挑取农杆菌GV3101(Biovector,Inc)单菌落接种在5ml YEP液体培养基(10g/L酵母提取物,10g/L蛋白胨,5g/L NaCl)中,28℃,200rpm,振荡过夜培养。将2ml过夜培养的菌液加到含有50ml YEP培养基中,28℃,220rpm,振荡培养至OD600在0.5-1.0之间。5000rpm离心菌液,弃上清,沉淀悬浮于10ml 0.5M NaCl,4℃,5000rpm离心5min,弃上清,用1ml预冷的20mM CaCl2重悬细胞。取0.2ml加入约0.5-1μgpBI-proBrD8B,轻轻混匀,液氮速冻1min,37℃热激5min,加入1ml YEP溶液,28℃振荡培养2-4h。5000rpm离心菌液,弃上清,将细胞重悬于0.2ml YEP培养基中,均匀涂布在含卡那霉素(Kan,50μg/mL)(鼎国昌盛公司)的YEP平板上,28℃培养48h。随机挑取单菌落进行PCR和酶切验证,获得含有目标基因GUS植物表达载体pBI-proBrD8B的农杆菌。
2.真空渗透法转化拟南芥
将成熟拟南芥(生态型Col-1)种子(购自美国拟南芥生物资源中心ABRC)于4℃浸泡1天打破休眠,在20%的Blench消毒水中浸泡10-15min后用无菌水漂洗3遍,播种于装有MS培养基的培养瓶或培养皿中。16h光照/8h黑暗条件下22℃下培养至其长出2-3片真叶后,移栽至装有蛭石(与营养土按2∶1混匀)的培养钵中。约4周后顶端出现花蕾,打顶后约5~7日侧枝产生花蕾。去除已开的花,准备侵染。
用10ml新鲜的添加Kan的YEP培养基28℃过夜培养含有目标基因植物表达载体的农杆菌,再取5ml转入100ml新鲜的YEP+Kan的培养基,过夜培养22h。2500rpm离心农杆菌菌液,弃上清后,用100ml转化Buffer(MS基本培养基+蔗糖5%+Silwet-77,pH5.8)重悬至OD600=1.0后用真空浸透法[24]浸渍拟南芥花序。
3.转基因拟南芥的筛选和鉴定
将收获的T0代种子于4℃浸泡3天打破休眠,在20%的Blench消毒水中浸泡10~15min后用无菌水漂洗3遍,播种于含Kan(50μg/mL)抗生素的MS培养基上。16h光照/8h黑暗下22℃培养10天左右可见子叶伸出,此时可见抗性植株呈深绿色,子叶健康。而未转化植株呈黄色或白色,子叶萎蔫。此时将阳性植株移栽至装有蛭石和营养土的培养钵中自然培养至收获。重复筛选获得T2代植株及种子。将T2代种子继续筛选,后代都为抗性植株的则为纯合株系。
实施例4.转基因植株的GUS检测
将T2代转基因纯合植株的组织浸泡于染色缓冲液中(含10mmol/LEDTA二钠盐,100mmol/L NaPO4pH 7.0,0.5mM K4Fe(CN)6,0.5mMK3Fe(CN)6,0.1%Triton X-100,500μg/mL X-Gluc(5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid,cyclo-hexylammoniumsalt),200mbar抽真空15min,使染液能浸入到组织的内部,37℃浸泡过夜。移去染液,用0.1mol/L的磷酸缓冲液冲洗2-3次。再用70%的乙醇脱色,更换乙醇2-3次。染色的组织或器官4℃下保存于0.1mol/L磷酸缓冲液(pH7.0)的溶液中。至少对三个以上株系进行GUS染色鉴定,图2和图3示具有代表性的结果。
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Claims (11)
1.花粉特异性启动子,其特征在于:它的核苷酸序列是:
(a)序列表中序列1所述的核苷酸序列;或
(b)与(a)所述的核苷酸序列互补的核苷酸序列。
2.含有权利要求1所述的启动子的表达盒。
3.根据权利要求2所述的表达盒,其特征在于:所述花粉特异性启动子的下游连接结构基因、调节基因、结构基因的反义基因、调节基因的反义基因或者能够干扰内源基因表达的小RNA。
4.含有权利要求1所述启动子的重组表达载体,其特征在于:转入了权利要求1所述的花粉特异性表达的启动子。
5.含有权利要求2或3所述的表达盒的重组表达载体。
6.根据权利要求5所述的重组表达载体,其特征在于:所述重组表达载体是由权利要求2或3所述的表达盒与质粒、病毒所构建的重组载体。
7.根据权利要求6所述的重组表达载体,其特征在于:所述重组表达载体为双元载体。
8.宿主细胞,其包含权利要求4-7任一所述的重组表达载体。
9.权利要求1所述的花粉特异性启动子在培育植物雄性不育系、花粉消除或大幅度减少的植物中的应用。
10.权利要求9的应用,其中所述植物是油菜、拟南芥、玉米、水稻、烟草、向日葵、大豆、番茄、蓖麻、棉花、芝麻和花生。
11.一种权利要求1所述的花粉特异性启动子的获得方法,其特征在于是以白菜型油菜(Brassica rapa)的基因组DNA为模板,用序列表中序列2和序列3的核苷酸序列作为一对引物进行PCR扩增得到。
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石东乔等.甘蓝型油菜BcNA1基因启动子在转基因烟草中对GUS基因表达的调控.《植物生理学报》.2001,(第04期), * |
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