CN114957425B - 一种矮牵牛PhSPL9-like转录因子及其应用 - Google Patents
一种矮牵牛PhSPL9-like转录因子及其应用 Download PDFInfo
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- CN114957425B CN114957425B CN202210761750.5A CN202210761750A CN114957425B CN 114957425 B CN114957425 B CN 114957425B CN 202210761750 A CN202210761750 A CN 202210761750A CN 114957425 B CN114957425 B CN 114957425B
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Abstract
本发明提出了一种矮牵牛PhSPL9‑like转录因子及其应用，属于基因工程技术领域。矮牵牛PhSPL9‑like转录因子，其核苷酸序列如SEQ ID NO.1所示，其编码蛋白的氨基酸序列如SEQ ID NO.2所示。本发明通过对PhSPL9‑like转录因子的开发，不仅揭示矮牵牛开花和花器官大小的调控机理，丰富植物的开花和花器官大小理论，同时为培养花期和花器官大小适宜的优良花卉品种提供可能。
Description
技术领域
本发明涉及基因工程技术领域，具体涉及一种矮牵牛PhSPL9-like转录因子及其应用。
背景技术
矮牵牛(Petunia hybrida)为茄科矮牵牛属草本花卉，花色丰富，株型多样，花期长，素有“花坛之王”的美誉，大规模应用于园林景观和家庭园艺。矮牵牛生长周期较短，遗传转化体系成熟且易转化，已成为分子生物学研究的模式植物(Vandenbussche et al.,2016)，矮牵牛基因组数据的公布和组学分析等技术的成熟，为矮牵牛重要功能基因的分子机理研究提供了极大便利(Bombarely et al.,2016)。
开花和花器官大小是花发育至关重要的过程，同时开花和花器官大小也是影响植物交配系统进化和保障繁殖的关键因子，具有重要的进化意义。通常情况下，开花是保障植物成功繁殖后代的前提，而花器官大小则是影响植物交配概率的重要影响因子。一般来说，自主性自花授粉植物的花器官体积比依赖传粉者的自花授粉和异花授粉植物更小。这是因为花器官的大小往往与传粉者造访传粉的概率密切相关，在不同植物以及同一植物的不同花型中，传粉者更倾向于造访尺寸更大、更醒目的花器官，这种偏好给异花传粉植物的花器官进化设置了选择方向(张栩佳等,2014；Muhammad et al.,2022)；这虽然会在一定程度上减少遗传多样性，但是却更好地确保了繁殖成功率，丰富了自然界中植物交配系统的特征多样性。
此外，调控植物花器官变大对提升花卉的观赏和经济价值具有重要的意义。一方面，花器官丰盈硕大的观赏植物在市场上更受消费者青睐；另一方面，对于花、果为主要产品的经济植物而言，花器官变大即意味着经济价值的提升，在生产实践中具有广泛的应用前景。同时，花器官的大小还是自然环境对对植物生存能力(基因型)的选择。在相对干旱的环境下，大花基因型植株更易受到环境胁迫影响而难于生存，因此逆境环境更倾向于选择小花型品种(张栩佳等,2014)。因此，调控植物的开花和花器官大小，不仅对植物观赏性状、适应性和繁殖能力的改良具有重要的指导意义，同时拓展花器官大小的转录调控网络，并为器官大小调控网络增添新的依据，具有十分重要的理论意义。
调控植物开花和花器官大小的措施有很多，如，传统栽培措施调控，通过调节温度、施加植物生长调节剂(如生长素、赤霉素等)，改变植物生长环境等。然而上面这些措施的应用，在生产中需要投入大力的人力、物力和财力，且对植物的影响多数并不是从根本上改变的。因此，通过基因工程从根本上改良植物的基因型，从而培育出更优的、具有更高观赏价值的花期和花器官大小适宜的植物。目前控制植物开花和调控花器官大小的基因已相继被克隆，其功能也被不断深入解析，通过转基因技术改良植物花期和花器官大小已成为一种快捷有效的措施(Ahmed et al.,2020；David et al.,2021；Wang et al.,2021；Zhaoet al.,2021)。
发明内容
本发明的目的在于克服现有技术的不足，提供了一种矮牵牛PhSPL9-like转录因子及其在控制植物开花和花器官大小中的应用；该应用是PhSPL9-like基因的核苷酸编码序列(即为PhSPL9-like转录因子)的克隆，表达载体的构建，将PhSPL9-like基因的完整翻译区(PhSPL9-like转录因子)与花椰菜花叶病毒启动子结合后转入植物体(宿主拟南芥和矮牵牛)内，得到转基因植株PhSPL9-like，对转基因植株进行分子鉴定和表型观察，以评估该基因在调控植物开花和花器官大小的应用前景：转基因植物的开花时间和花器官大小发生显著性的变化。
本发明的技术方案是这样实现的：
本发明提供一种矮牵牛PhSPL9-like转录因子，其核苷酸序列如SEQ ID NO.1所示。
本发明进一步保护如权利要求1所述矮牵牛PhSPL9-like转录因子的编码蛋白，其特征在于，其氨基酸序列如SEQ ID NO.2所示。
本发明进一步保护制备上述的矮牵牛PhSPL9-like转录因子所使用的引物对，所述引物对为部分扩增、全长扩增引物及amicroRNA-PhSPL9-like(amiRNA-PhSPL9-like)茎环结构扩增引物，所述部分扩增引物由上游引物PhSPL9-like-F1、下游引物PhSPL9-like-R1和上游引物PhSPL9-like-F2、下游引物PhSPL9-like-R2组成；所述全长扩增引物由上游引物PhSPL9-like-F1和下游引物PhSPL9-like-R2组成；所述amicroRNA-PhSPL9-like(amiRNA-PhSPL9-like)茎环结构扩增引物由上游引物amiRNA-PhSPL9-like-F和下游引物amiRNA-PhSPL9-like-R组成，其中：
上游引物PhSPL9-like-F1的核苷酸序列如SEQ ID NO.3所示；
下游引物PhSPL9-like-R1的核苷酸序列如SEQ ID NO.4所示；
上游引物PhSPL9-like-F2的核苷酸序列如SEQ ID NO.5所示；
下游引物PhSPL9-like-R2的核苷酸序列如SEQ ID NO.6所示；
上游引物amiRNA-PhSPL9-like-F的核苷酸序列如SEQ ID NO.7所示；
下游引物为amiRNA-PhSPL9-like-R的核苷酸序列如SEQ ID NO.8所示。
本发明进一步保护一种利用上述引物对获得PhSPL9-like转录因子的方法，包括以下步骤：
以矮牵牛不同组织cDNA为模板和上述PhSPL9-like转录因子的引物对，进行PCR扩增，纯化得到PhSPL9-like转录因子；其中，
PCR体系：2×Taq DNA polymerase 10μl，上游引物1μl，下游引物1μl，模板1μl，ddH2O 7μl，总共20μl。
PCR的条件：98℃预变性，2min；98℃，10s，55℃，15s，72℃，1min，35个循环；72℃，1min，72℃延伸，10min。
本发明进一步保护一种重组表达载体，其特征在于，所述重组表达载体含有上述PhSPL9-like转录因子的植物表达载体。
作为本发明的进一步改进，所述植物表达载体为pCAMBIA2300。
本发明进一步保护一种上述重组表达载体的构建方法，包括以下步骤：将目的片段导入克隆载体18T中，然后进行将含目的基因的克隆载体PhSPL9-like-/>18T和pCAMBIA2300用Sal I和Kpn I双酶切进行酶切，再将得到的目的片段连接到酶切过的pCAMBIA2300载体上即得到重组表达载体pCAMBIA2300-PhSPL9-like；
本发明进一步保护一种上述重组表达载体的构建方法，包括以下步骤：利用microRNA抑制技术，以PhSPL9-like编码区序列为模板，获得将要干涉的片段，并合成amiRNA-PhSPL9-like茎环结构序列扩增的引物，将载有amiRNA-PhSPL9-like茎环结构序列的B/C中间载体用KpnI和SalI限制性内切酶进行酶切，目的片段连接至pCAMBIA2300载体的相应位点即得到重组表达载体pCAMBIA2300-amiRNA-PhSPL9-like。
本发明进一步保护一种含有上述重组表达载体的宿主细胞，所述宿主细胞为农杆菌GV3101或AGL0。
本发明进一步保护下述各项之一在促进植物开花和花器官大小的应用，其特征在于，包括：
(1)上述矮的牵牛PhSPL9-like转录因子；
(2)上述的重组表达载体；
(3)上述的宿主细胞。
作为本发明的进一步改进，所述植物为拟南芥或矮牵牛。
本发明具有如下有益效果：本发明通过对PhSPL9-like转录因子的开发，不仅揭示矮牵牛开花和花器官大小的调控机理，丰富植物的开花和花器官大小理论，同时为培养花期和花器官大小适宜的优良花卉品种提供可能。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。
图1为实施例的PhSPL9-like转录因子在拟南芥中超量表达的表型分析图；
其中：a W115(左)以及35S:PhSPL9-like转基因株系(右)早花表型，b-c35S:PhSPL9-like转基因株系表达量检测，d-e转基因株系开花时间和莲座叶数目统计分析，标尺：10cm。
图2为实施例的PhSPL9-like转录因子在矮牵牛中超量表达的表型分析图
其中：a W115(左)以及35S:PhSPL9-like转基因株系(右)早花表型，b-c35S:PhSPL9-like转基因株系表达量检测，d-m转基因株系花器官变大表型，n-p转基因株系开花时间、叶片长宽比和花朵直径数据统计分析，标尺：10cm。
图3为实施例的PhSPL9-like转录因子在矮牵牛中利用microRNA抑制技术进行干涉后的表型分析图；
其中：a W115(左)以及35S:amiRNA-PhSPL9-like转基因株系(右)晚花表型。b-f转基因株系花器官变小表型，g-i 35S:amiRNA-PhSPL9-like转基因株系表达量检测，j-m转基因株系开花时间、叶片长宽比、节间距和花朵直径统计分析，标尺：10cm。
具体实施方式
下面将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前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1：PhSPL9-like基因的分离克隆
用trizol法从矮牵牛不同组织样品中提取总RNA，根据矮牵牛基因组和转录组数据，获得了一个新的SPL9/15同源基因，将其命名为PhSPL9-like。该基因编码374个氨基酸，由3个外显子和2个内含子组成，编码区长度1125bp。
PhSPL9-like基因的核苷酸序列如SEQ ID NO.1所示，PhSPL9-like基因编码蛋白的氨基酸序列如SEQ ID NO.2所示。
PCR反应条件为98℃2min；98℃10s，55℃15s，72℃1min(35cycles)；72℃10min。
其所使用的引物如表1。
表1
| 名称 | 序列 |
| PhSPL9-like-F1 | CTACACATAAACAGGCAAATGG |
| PhSPL9-like-R1 | GGAAAGCAGTGATAGCGCTCGTCCCTCATTTACTGGTAAGT |
| PhSPL9-like-F2 | CGAGCGCTATCACTGCTTTCCTCCAATAGTGATTCATCGAT |
| PhSPL9-like-R2 | GGAAGCAAATTATCGACAAGAG |
实施例2：载体构建
将载有PhSPL9-like全长编码序列的18-T载体用Sal I和BamH I限制性内切酶进行酶切，目的片段连接至pCAMBIA2300载体的相应位点，该载体含有CaMV35S启动子和Nos 3’转录终止子，构建成35S:PhSPL9-like。
利用microRNA抑制技术，以PhSPL9-like编码区序列为模板，在http://psams.carringtonlab.org/amirna/designer#start(Carbonell et al 2014)上获得将要干涉的片段，并合成amiRNA-PhSPL9-like茎环结构序列扩增的引物，其所使用的引物如表2。将载有amiRNA-PhSPL9-like茎环结构序列的B/C中间载体用KpnI和SalI限制性内切酶进行酶切，目的片段连接至pCAMBIA2300载体的相应位点，构建成35S:amiRNA-PhSPL9-like。所有构建的质粒都通过PCR及双酶切进行验证，且质粒最终分别转化到GV3101和AGL0农杆菌中。
表2
实施例3：PhSPL9-like基因在拟南芥中的超量表达
通过花序侵染法将矮牵牛PhSPL9-like的超量表达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。
本发明共获得了42个35S:PhSPL9-like转化拟南芥T1代阳性株系，选取3个有表型的株系，通过半定量和定量检测PhSPL9-like的表达水平，发现3个有表型的株系其表达量均高于野生型拟南芥。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35株转基因阳性植株的开花时间、莲座叶和茎生叶的形态特征。如图1、表3所示，PhSPL9-like在拟南芥中超量表达后，表型观测和数据统计发现，5号、18号和24号株系有明显的早花表型，尤其是18号株系，开花时间比野生型提前11d左右，同时莲座叶数目显著性减少，但茎生叶数目没有显著性的差异。此外，35S:PhSPL9-like转化拟南芥的表型和表达量之间呈正相关。数值为平均值±标准差，星号代表显著性差异(*P＜0.05，**P＜0.01)。
表3 PhSPL9-like超量表达后在拟南芥中的表型数据分析
实施例4：PhSPL9-like基因在矮牵牛中的超量表达
将PhSPL9-like超量表达载体转化W115野生型矮牵牛。通过采集植株顶端嫩叶、升汞对外植体进行消毒、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检测确定转基因阳性株系。采用Trizol法提取W115及T1代转基因株系的总RNA，再反转录。以PhEF1α为内参基因，鉴定PhSPL9-like在矮牵牛转基因株系中的表达水平。
本发明共获得了34个35S:PhSPL9-like转化矮牵牛T1代阳性株系，选取3个有表型的株系，通过半定量和定量检测PhSPL9-like的表达水平，发现3个有表型的株系其表达量均高于野生型矮牵牛。对3个有表型的株系进行T2代分析，各选取25株T2代进行表型观测和数据统计。如图2和表3所示，发现6号、13号和24号株系有明显的早花表型，尤其是13号株系，开花时间比野生型提前14d左右，同时花朵直径和叶片长宽比与野生型相比较大，而株高、节间距、和分枝数与野生型相比没有显著性的差异。此外，35S:PhSPL9-like转化矮牵牛的表型和表达量之间呈正相关。数值为平均值±标准差，星号代表显著性差异(*P＜0.05，**P＜0.01)。
表4 PhSPL9-like超量表达后在矮牵牛中的表型数据分析
实施例5：PhSPL9-like基因在矮牵牛中的干涉表达
将35S:amiRNA-PhSPL9-like载体转化W115野生型矮牵牛。方法同上，获得矮牵牛转化植株、阳性株系检测及表达量检测。
本发明共获得了27个35S:amiRNA-PhSPL9-like转化矮牵牛T1代阳性株系，选取3个有表型的株系，通过半定量和定量检测amiRNA-PhSPL9的表达水平，发现3个有表型的株系其表达量均高于野生型矮牵牛。对3个有表型的株系进行T2代分析，各选取25株T2代进行表型观测和数据统计，如图3和表5所示，发现7号、17号和26号株系有明显的晚花表型，尤其是17号株系，开花时间比野生型延迟了16d左右。同时转基因株系的节间距变短、叶片长宽比和花朵直径变小，而株高和分枝数与野生型相比没有显著性的差异。研究结果表明35S:amiRNA-PhSPL9-like转化矮牵牛的表型和表达量之间呈正相关。数值为平均值±标准差，星号代表显著性差异(*P＜0.05，**P＜0.01)。
表5 PhSPL9-like干涉表达后在矮牵牛中的表型数据分析
以上所述仅为本发明的较佳实施例而已，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> 广州市林业和园林科学研究院
浙江省园林植物与花卉研究所（浙江省萧山棉麻研究所）
<120> 一种矮牵牛PhSPL9-like转录因子及其应用
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1125
<212> DNA
<213> 矮牵牛(Petunia hybrida)
<400> 1
atggaaaacg gtggttggaa ctttttcagc aataacaata ttgaaagttg cagcagcaac 60
atcaatggtg gaagtactac tactgtggga ggacaaatta gtaattctac tcatcatgca 120
tgggacttct ggcaacttgg tagtagtagt actaatactt cattaagcca tgaccttgag 180
tggaatgcta acaatatttc acttagccat gacccaacta ttaggcagcc caaaacattc 240
aatttatata atcctaattt gaatcttaac ttaggaaata agaagcatta tattgaaaat 300
tatgagggtg gtagagaggt agaggggttt gctacgagta agagagggaa accctacttt 360
tgtggtggcg gtggtggtga tggaggggcg gcaatgccgg cggcgttggt ggtgccgagg 420
tgtcaggtgg agggatgcca ggtggtgctg gtgaatgcca agacatatca tagaagacat 480
aaagtctgtg aaatgcatgc taaagctcct aaagttgtgc ttcttggcct tgaacaacgg 540
ttttgtcaac agtgtagcag gtttcattca gtgagtgagt ttgatgagtc aaagaggagc 600
tgcagaagga gattagcagg acacaatgag agaagaagaa agagctctca agaacatttt 660
gcaaacacca gaaacaactt tcaaggtaag tttcgggact taccagtaaa tgagggacga 720
gcgctatcac tgctttcctc caatagtgat tcatcgatac caaaatgcaa tctccctgca 780
agatcatata gttccagtat tgatgagctg attgctggaa gtagagcaac tagtctagct 840
cgacaacttc tccttgagaa agactggcat aggcatcaac atcgtcagaa tctgagcaac 900
caaccaaaaa atacgagctt tactcatgac tgcagccatg tagccctcga atcacatggt 960
tggggacgta tcgatgatgt tgctgaacat ttgacattga atcttatgca tgttcgaaat 1020
tctgaatttg gattcttgcc cggacaaaag cagcctaagg aagaaggaaa agaagaatgg 1080
tgtccaggac attggaactc ctttgcagga ggtcatgttt cctga 1125
<210> 2
<211> 374
<212> PRT
<213> 矮牵牛(Petunia hybrida)
<400> 2
Met Glu Asn Gly Gly Trp Asn Phe Phe Ser Asn Asn Asn Ile Glu Ser
1 5 10 15
Cys Ser Ser Asn Ile Asn Gly Gly Ser Thr Thr Thr Val Gly Gly Gln
20 25 30
Ile Ser Asn Ser Thr His His Ala Trp Asp Phe Trp Gln Leu Gly Ser
35 40 45
Ser Ser Thr Asn Thr Ser Leu Ser His Asp Leu Glu Trp Asn Ala Asn
50 55 60
Asn Ile Ser Leu Ser His Asp Pro Thr Ile Arg Gln Pro Lys Thr Phe
65 70 75 80
Asn Leu Tyr Asn Pro Asn Leu Asn Leu Asn Leu Gly Asn Lys Lys His
85 90 95
Tyr Ile Glu Asn Tyr Glu Gly Gly Arg Glu Val Glu Gly Phe Ala Thr
100 105 110
Ser Lys Arg Gly Lys Pro Tyr Phe Cys Gly Gly Gly Gly Gly Asp Gly
115 120 125
Gly Ala Ala Met Pro Ala Ala Leu Val Val Pro Arg Cys Gln Val Glu
130 135 140
Gly Cys Gln Val Val Leu Val Asn Ala Lys Thr Tyr His Arg Arg His
145 150 155 160
Lys Val Cys Glu Met His Ala Lys Ala Pro Lys Val Val Leu Leu Gly
165 170 175
Leu Glu Gln Arg Phe Cys Gln Gln Cys Ser Arg Phe His Ser Val Ser
180 185 190
Glu Phe Asp Glu Ser Lys Arg Ser Cys Arg Arg Arg Leu Ala Gly His
195 200 205
Asn Glu Arg Arg Arg Lys Ser Ser Gln Glu His Phe Ala Asn Thr Arg
210 215 220
Asn Asn Phe Gln Gly Lys Phe Arg Asp Leu Pro Val Asn Glu Gly Arg
225 230 235 240
Ala Leu Ser Leu Leu Ser Ser Asn Ser Asp Ser Ser Ile Pro Lys Cys
245 250 255
Asn Leu Pro Ala Arg Ser Tyr Ser Ser Ser Ile Asp Glu Leu Ile Ala
260 265 270
Gly Ser Arg Ala Thr Ser Leu Ala Arg Gln Leu Leu Leu Glu Lys Asp
275 280 285
Trp His Arg His Gln His Arg Gln Asn Leu Ser Asn Gln Pro Lys Asn
290 295 300
Thr Ser Phe Thr His Asp Cys Ser His Val Ala Leu Glu Ser His Gly
305 310 315 320
Trp Gly Arg Ile Asp Asp Val Ala Glu His Leu Thr Leu Asn Leu Met
325 330 335
His Val Arg Asn Ser Glu Phe Gly Phe Leu Pro Gly Gln Lys Gln Pro
340 345 350
Lys Glu Glu Gly Lys Glu Glu Trp Cys Pro Gly His Trp Asn Ser Phe
355 360 365
Ala Gly Gly His Val Ser
370
<210> 3
<211> 22
<212> DNA
<213> 人工序列(无)
<400> 3
ctacacataa acaggcaaat gg 22
<210> 4
<211> 41
<212> DNA
<213> 人工序列(无)
<400> 4
ggaaagcagt gatagcgctc gtccctcatt tactggtaag t 41
<210> 5
<211> 41
<212> DNA
<213> 人工序列(无)
<400> 5
cgagcgctat cactgctttc ctccaatagt gattcatcga t 41
<210> 6
<211> 22
<212> DNA
<213> 人工序列(无)
<400> 6
ggaagcaaat tatcgacaag ag 22
<210> 7
<211> 75
<212> DNA
<213> 人工序列(无)
<400> 7
tgtataagtg aaatattgtt agcaaatgat gatcacattc gttatctatt ttttttgcta 60
acaagatttc actta 75
<210> 8
<211> 75
<212> DNA
<213> 人工序列(无)
<400> 8
aatgtaagtg aaatcttgtt agcaaaaaaa atagataacg aatgtgatca tcatttgcta 60
acaatatttc actta 75
Claims (10)
1.一种矮牵牛PhSPL9-like转录因子，其特征在于，其核苷酸序列如SEQ ID NO.1所示。
2.如权利要求1所述矮牵牛PhSPL9-like转录因子的编码蛋白，其特征在于，其氨基酸序列如SEQ ID NO.2所示。
3.一种制备如权利要求1所述的矮牵牛PhSPL9-like转录因子的方法，其特征在于，包括以下步骤：
以矮牵牛不同组织cDNA为模板和PhSPL9-like转录因子的引物对，进行PCR扩增，纯化得到PhSPL9-like转录因子；其中，
PCR体系：2×Taq DNA polymerase 10μl，上游引物1μl，下游引物1μl，模板1μl，ddH2O 7μl，总共20μl；
PCR的条件：98℃预变性，2min；98℃，10s，55℃，15s，72℃，1min，35个循环；72℃，1min，72℃延伸，10min；
所述PhSPL9-like转录因子的引物对为部分扩增、全长扩增引物及amicroRNA-PhSPL9-like（amiRNA-PhSPL9-like）茎环结构扩增引物，所述部分扩增引物由上游引物PhSPL9- like-F1、下游引物PhSPL9-like-R1和上游引物PhSPL9-like-F2、下游引物PhSPL9-like-R2组成；所述全长扩增引物由上游引物PhSPL9-like-F1和下游引物PhSPL9-like-R2组成；所述amicroRNA-PhSPL9-like（amiRNA-PhSPL9-like）茎环结构扩增引物由上游引物amiRNA- PhSPL9-like-F和下游引物amiRNA-PhSPL9-like-R组成，其中：
上游引物PhSPL9-like-F1的核苷酸序列如SEQ ID NO.3所示；
下游引物PhSPL9-like-R1的核苷酸序列如SEQ ID NO.4所示；
上游引物PhSPL9-like-F2的核苷酸序列如SEQ ID NO.5所示；
下游引物PhSPL9-like-R2的核苷酸序列如SEQ ID NO.6所示；
上游引物amiRNA-PhSPL9-like-F的核苷酸序列如SEQ ID NO.7所示；
下游引物为amiRNA-PhSPL9-like-R的核苷酸序列如SEQ ID NO.8所示。
4.一种重组表达载体，其特征在于，所述重组表达载体含有权利要求1所述PhSPL9-like转录因子的植物表达载体。
5.根据权利要求4所述重组表达载体，其特征在于，所述植物表达载体为pCAMBIA2300。
6.一种权利要求4或5所述重组表达载体的构建方法，其特征在于，包括以下步骤：将目的片段导入克隆载体pMD®18T中，然后进行将含目的基因的克隆载体PhSPL9-like-pMD®18T和pCAMBIA2300用Sal I和Kpn I双酶切进行酶切，再将得到的目的片段连接到酶切过的pCAMBIA2300载体上即得到重组表达载体pCAMBIA2300-PhSPL9-like。
7.一种权利要求4或5所述重组表达载体的构建方法，其特征在于，包括以下步骤：利用microRNA抑制技术，以PhSPL9-like编码区序列为模板，获得将要干涉的片段，并合成amiRNA-PhSPL9-like茎环结构序列扩增的引物，将载有amiRNA-PhSPL9-like茎环结构序列的B/C中间载体用KpnI和SalI限制性内切酶进行酶切，目的片段连接至pCAMBIA2300载体的相应位点即得到重组表达载体pCAMBIA2300-amiRNA-PhSPL9-like。
8.一种含有权利要求4或5所述重组表达载体的宿主细胞，其特征在于，所述宿主细胞为农杆菌GV3101或AGL0。
9.下述各项之一在矮牵牛开花时间减少、花朵直径增大、叶片长宽比提高中的应用，其特征在于，包括：
（1）权利要求1所述矮的牵牛PhSPL9-like转录因子；
（2）权利要求5或6所述的重组表达载体；
（3）权利要求8所述的宿主细胞。
10.下述各项之一在拟南芥开花时间减少、莲座叶数目降低中的应用，其特征在于，包括：
（1）权利要求1所述矮的牵牛PhSPL9-like转录因子；
（2）权利要求5或6所述的重组表达载体；
（3）权利要求8所述的宿主细胞。
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| CN1244585A (zh) * | 1998-08-07 | 2000-02-16 | 农林水产省农业生物资源研究所长 | 通过导入矮牵牛属转录因子PetSPL2的基因以缩短花序节间的方法 |
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