CN108795953A - 一种悬铃木开花调控基因及其在控制植物开花中的应用 - Google Patents
一种悬铃木开花调控基因及其在控制植物开花中的应用 Download PDFInfo
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Abstract
本发明公开了一种悬铃木开花调控基因及其在控制植物开花中的应用。其中悬铃木开花调控基因(PlacTFL1)具有如由SEQ ID NO.1所示的核苷酸序列。本发明通过对PlacTFL1基因的开发,不仅揭示悬铃木开花调控机理,丰富基本真双子叶植物的开花理论,同时为培养无花无果悬铃木提供可能。
Description
技术领域
本发明涉及植物基因工程和生物技术领域,尤其涉及一种悬铃木开花调控基因(PlacTFL1)以及该基因在控制植物开花中的应用。
背景技术
TERMINAL FLOWER 1(TFL1)和FLOWERING LOCUS T(FT)是参与开花诱导的两个重要基因,它们是编码与phosphatidylethanolamine-binding proteins(PEBPs)同源的开花调控因子,PEBPs参与细菌、动物和植物生长与分化的多个信号途径(Ahn et al 2006;Hanzawa et al 2005;Karlgren et al 2011)。TFL1和FT氨基酸序列的一致性大约在60%,但是功能相反(Ahn et al 2006;Hanzawa et al 2005)。FT促进营养生长向生殖发育的转换,而TFL1抑制成花转换。
TFL1是拟南芥的茎端分生组织决定基因(shoot meristem identity gene),它在营养生长和花序生长阶段的顶端分生组织表达,以维持茎端分生组织和花序分生组织的无限性,从而调控营养生长和生殖生长的进程(Bradley et al 1997)。在tfl1突变体中,本应形成侧生花序的位置通常发育成单朵花,且茎端也发育成单朵花;而超量表达TFL1的转基因植株,其营养生长阶段和生殖生长阶段均显著延长,说明TFL1对于植物营养生长和花序发育的维持均起着重要作用(Ratcliffe et al 1998)。通过构建LFY、TFL1、AP1超量表达转基因植株以及结合各种突变体,揭示了TFL1与LFY、AP1在成花过程中的作用关系:LFY、AP1在花分生组织顶端抑制TFL1的转录,促成并维持花分生组织的特性;TFL1则在茎端抑制LFY、AP1的活性,保证正常的花序结构形成(Parcy et al 2002)。进一步研究显示,TFL1蛋白是一个均匀分布在分生组织处的移动信号,但它不能进入分生组织侧面的细胞,从而保证原基细胞建立自己的属性(Conti and Bradley 2007)。
TFL1的功能在许多核心真双子植物中得到了验证,如番茄的TFL1同源基因SELF-PRUNING(SP)影响茎的生长(Jiang et al 2013);豌豆的TFL1同源基因PsTFL1a维持无限生长(Foucher et al 2003);玫瑰和草莓的TFL1同源基因(RoKSN和FvKSN)功能缺失型突变体表现为持续开花(Iwata et al 2012) 等。
悬铃木(Platanus spp.)是山龙眼目悬铃木科悬铃木属雌雄同株异花的落叶乔木树种,属于基本真双子叶植物,是被子植物进化地位较为原始的种类。二球悬铃木(英国梧桐,P.acerifolia Willd)是一球悬铃木(美国梧桐,P. occidentalis Linn.)和三球悬铃木(法国梧桐,P.orientalis Linn.)的杂交种,由于其良好的遮阴效果、极强的抗逆性等优点,成为城市道路和庭院的极佳景观植物。然而,每年春天开花期飘散的大量花粉以及果实干裂后散落的大量果毛不仅污染环境,人体吸入后还可能导致严重的花粉病或呼吸困难(Iglesias et al 2007;Pourkhabbaz et al 2010)。为此,园林科研工作者针对悬铃木“落果飞毛”问题做了大量的探索和尝试,包括化学药剂落果、物理修剪控果、选育少果品系及辐射诱变育种等,但问题没有得到根本的解决。所以,随着植物基因工程的快速发展,开发不开花基因在植物中的应用,为无球果悬铃木的培育带来了新的途径。
发明内容
本发明要解决的技术问题是提供一种悬铃木开花调控基因(以下简称为PlacTFL1)。
本发明要解决的另外一个技术问题是提供一种制备PlacTFL1基因的方法。
本发明还要解决的一个技术问题是提供制备PlacTFL1基因所使用的引物。
本发明还要解决的一个技术问题是PlacTFL1基因在控制植物开花中的应用。
对于PlacTFL1基因,本发明采用的技术方案是,具有如SEQ ID NO.1所示的核苷酸序列。
对于PlacTFL1基因编码的蛋白,具有如由SEQ ID NO.2所示的氨基酸序列。
对于制备方法,本发明采用的技术方案是,用改良后的CTAB法从二球悬铃木样品中提取总RNA,根据同源克隆和悬铃木的转录组数据,结合5’Tail 和3’RACE延伸技术,由此获得悬铃木开花调控基因(PlacTFL1)。
对于引物,本发明采用的技术方案是,引物为5’Tail引物,其中:
PlacTFL1-tR1引物,其具有如SEQ ID NO.3所示的核苷酸序列;
PlacTFL1-tR2引物,其具有如SEQ ID NO.4所示的核苷酸序列;
PlacTFL1-tR3引物,其具有如SEQ ID NO.5所示的核苷酸序列。
引物为命名为PlacTFL1-rF的3’RACE引物,其具有如SEQ ID NO.6所示的核苷酸序列。
引物为全长扩增引物,其中:
上游引物为PlacTFL1-F,其具有如SEQ ID NO.7所示的核苷酸序列。
下游引物为PlacTFL1-R,其具有如SEQ ID NO.8所示的核苷酸序列。
另外,本发明的PlacTFL1基因在控制植物开花中的应用。
本发明的有益效果是:
通过对PlacTFL1基因的开发,不仅揭示悬铃木开花调控机理,丰富基本真双子叶植物的开花理论,同时为培养无花无果悬铃木提供可能。
附图说明
下面结合附图和具体实施方式对本发明作进一步详细的说明。
图1是本发明实施例的PlacTFL1基因在拟南芥中超量表达的表型,其中:
a野生型拟南芥(左)以及35S:PlacTFL1转基因中等表型(中)和强表型(右)。b35S:PlacTFL1转基因植株莲座叶增多。c 35S:PlacTFL1转基因植株营养生长超半年。d、e分别为35S:PlacTFL1转基因植株维持顶端分生组织特性和进入花的分化。f 35S:PlacTFL1转基因植株茎木质化程度高,粗壮。标尺:10mm(a-c),1mm(d-f)。
图2是本发明实施例的PlacTFL1基因在短牵牛中超量表达的表型分析,其中:aPlacTFL1转基因株系表达量检测。b转基因株系开花前分枝数和主枝叶片数统计。c W115(左)以及35S:PlacTFL1转基因23号株系的中等表型(中) 和6号株系的强表型(右)。d 35S:PlacTFL1转基因23号株系(左)和6号株系(右)生长数月后的表型。标尺:10cm。
具体实施方式
实施例1:PlacTFL1基因的分离克隆
用改良后的CTAB法从二球悬铃木样品中提取总RNA,根据同源克隆和悬铃木的转录组数据,结合5’Tail和3’RACE延伸技术,获得了一种新的TFL1 同源基因,将其命名为PlacTFL1。该基因编码172个氨基酸,由4个外显子和3个内含子组成,gDNA长度1632bp。
PlacTFL1基因的核苷酸序列如SEQ ID NO.1所示,PlacTFL1基因编码蛋白的氨基酸序列如SEQ ID NO.2所示。其所使用的引物如表1。
表1
PCR反应条件为94℃3min;94℃30s,55℃30s,72℃30s(35cycles);72℃10min。
实施例2:载体构建
载有PlacTFL1全长编码序列的pMD18-T载体用SalI和KpnI限制性内切酶进行酶切,目的片段连接至P2300载体的相应位点,该载体含有CaMV 35S 启动子和Nos 3’转录终止子,构建成35S:PlacTFL1。所有构建的质粒都通过 PCR以及酶切进行验证。质粒最终通过分别电转转化到GV3101和AGL0农杆菌中。
实施例3:PlacTFL1基因在拟南芥中的超量表达
通过浸花法将悬铃木TFL1基因的超表载体转化哥伦比亚野生型拟南芥 (Col-0),转化后的T1代种子在含有50μg ml-1卡那霉素和50μg ml-1头孢霉素的MS琼脂培养基中进行筛选,获得T1代35S:PlacTFL1转基因株系38株,并观察阳性植株的表型。通过卡方检测,选择符合3:1分离比的T2代转基因株系,种植在22℃、长日照条件(16/8h,白/昼)的人工气候箱,每个株系统计16株转化体的开花时间和开花形态特征。
表2:PlacTFL1基因在拟南芥中异位表达T2代株系表型统计
如图1、表2所示,PlacTFL1在拟南芥中的超量表达导致拟南芥严重晚花,而且在T1代筛选出5株平均莲座叶50片的呈多年生晚花株系,种植超过半年直至死亡仍未开花。T1代为中等表型株系的T2代晚花表型增强,如35S:PlacTFL1的T2-20、T2-24,它们虽然在20-40片莲座叶时能抽薹,产生很多的花序,却不能开花,顶端维持花序分生组织特性。而其他一些表型稍弱的株系则在花序分支到一定数量时进入花的分化。另外,悬铃木的TFL1-like基因转化拟南芥对其营养生长的促进还体现在植物高大、叶片壮硕以及茎粗壮。
通过卡方检测找出符合3:1分离比的T1代株系,分别种植T2代进行开花时间及其形态变化统计(16株)。数值为平均值±标准差,斜杠代表不开花株系数据无法统计,星号代表显著性差异(*P<0.05)。
实施例4:PlacTFL1基因在矮牵牛中的超量表达
将PlacTFL1超表载体转化W115野生型矮牵牛。通过采集植株顶端嫩叶、升汞消毒、农杆菌菌液侵染、外植体共培养、选择培养以及切芽、生根、炼苗、移栽等步骤,获得矮牵牛转化植株。通过PCR检测确定转基因植株。采用Trizol 试剂盒提取W115及T1代转基因植株的总RNA,再反转录。以PhEF1α为内参基因,鉴定PlacTFL1在矮牵牛转基因株系中的表达水平。
我们共获得了25个35S:PlacTFL1转化矮牵牛T0代阳性株系,通过半定量检测发现有表达的株系仅有3株。如图2所示,表型观测和数据统计发现, 6号和23号株系有明显的晚花,尤其是6号株系维持花序分生组织一年多至今未开花,高达近两米。由于6号株系不开花也没有后代种子,所以也无法统计数据。而23号株系则属于中等晚花表型,生长到一定时间便能开花,但开了一朵花的23号株系又回复到营养生长状态,继续形成叶片。另外,35S:TFL1 转化矮牵牛的表型和表达量之间正相关。
以上所述的本发明实施方式,并不构成对本发明保护范围的限定。任何在本发明的精神和原则之内所作的修改、等同替换和改进等,均应包含在本发明的权利要求保护范围之内。
序列表
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Claims (8)
1.一种悬铃木开花调控基因,其特征在于,具有如由SEQ ID NO.1所示的核苷酸序列。
2.如权利要求1所述悬铃木开花调控基因编码的蛋白,其特征在于,具有如SEQ IDNO.2所示的氨基酸序列。
3.一种制备权利要求1所述的悬铃木开花调控基因的方法,其特征在于,用改良后的CTAB法从二球悬铃木样品中提取总RNA,根据同源克隆和悬铃木的转录组数据,结合5’Tail和3’RACE延伸技术,由此获得所述悬铃木开花调控基因。
4.制备如权利要求1所述的悬铃木开花调控基因所使用的引物,其特征在于,所述引物为5’Tail引物,其中:
PlacTFL1-tR1,其具有如SEQ ID NO.3所示的核苷酸序列;
PlacTFL1-tR2,其具有如SEQ ID NO.4所示的核苷酸序列;
PlacTFL1-tR3,其具有如SEQ ID NO.5所示的核苷酸序列。
5.制备如权利要求1所述的悬铃木开花调控基因所使用的引物,其特征在于,所述引物是命名为PlacTFL1-rF的3’RACE引物,其具有如SEQ ID NO.6所示的核苷酸序列。
6.制备如权利要求1所述的悬铃木开花调控基因所使用的引物,其特征在于,所述引物为全长扩增引物,其中:
上游引物为PlacTFL1-F,其具有如SEQ ID NO.7所示的核苷酸序列。
下游引物为PlacTFL1-R,其具有如SEQ ID NO.8所示的核苷酸序列。
7.如权利要求1所述的悬铃木开花调控基因在控制植物开花中的应用。
8.如权利要求7所述的应用,其特征在于,所述植物包括拟南芥、矮牵牛和悬铃木。
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