CN115058434A - 一种调控月季花瓣颜色的基因RcNHX2及其应用 - Google Patents
一种调控月季花瓣颜色的基因RcNHX2及其应用 Download PDFInfo
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Abstract
本发明提供一种调控月季花瓣颜色的基因RcNHX2及其应用。本发明第一方面提供一种调控月季花瓣颜色的基因RcNHX2,所述基因具有以下核苷酸序列中的一种:1)SEQ ID NO.1所示的核苷酸序列;2)SEQ ID NO.1所示的核苷酸序列通过一个或几个核苷酸的取代、缺失、添加后衍生产生的核苷酸序列;3)与SEQ ID NO.1具有至少80%同源性的核苷酸序列。本发明研究发现月季中液泡pH相关基因RcNHX2影响月季花瓣的颜色,在调控月季花色中具有重要的作用,为月季育种提供了重要的基础。
Description
技术领域
本发明涉及基因工程技术领域,具体涉及一种调控月季花瓣颜色的基因RcNHX2及其应用。
背景技术
花色是花卉品质的一个重要特性,广义的花色是指显花植物花器官中一切花瓣状结构的颜色,狭义仅指花瓣的颜色,自然界中花卉颜色种类繁多,鲜艳的花色在植物性杂交中发挥重要作用,对人类也具有很大的吸引力,通过杂交育种的手段可以部分实现花色的多样化。
月季普遍缺少蓝色系的花瓣,蓝色花花色素的形成机理以及使花显蓝色的基因工程研究一直是近年来的研究热点,因此,如何通过基因工程的手段,使月季花色转蓝,得到蓝色系花瓣的月季成为了本领域技术人员亟待解决的技术问题。
发明内容
本发明提供一种调控月季花瓣颜色的基因RcNHX2及其应用,通过基因工程的手段,改变月季的花瓣颜色,为月季花色转蓝育种提供基础。
本发明第一方面提供一种调控月季花瓣颜色的基因RcNHX2,所述基因具有以下核苷酸序列中的一种:
1)SEQ ID NO.1所示的核苷酸序列;
2)SEQ ID NO.1所示的核苷酸序列通过一个或几个核苷酸的取代、缺失、添加后衍生产生的核苷酸序列;
3)与SEQ ID NO.1具有至少80%同源性的核苷酸序列。
本发明第二方面提供一种调控月季花瓣颜色的蛋白,所述蛋白由上述任一所述的基因RcNHX2编码得到。
本发明第三方面提供一种重组载体,所述重组载体包含上述任一所述的核苷酸序列。
进一步地,所述重组载体为包含所述核苷酸序列的TRV2病毒或pCAMBIA2300-GFP载体。
本发明第四方面提供一种重组转化体,所述重组转化体包含上述任一所述的重组载体。
进一步地,所述重组转化体为包含所述重组载体的农杆菌GV3101。
本发明第五方面提供上述所述的基因RcNHX2在调控月季花瓣颜色中的应用。
本发明第六方面提供一种调控月季花瓣颜色的方法,具体包括如下步骤:
将上述任一所述的基因RcNHX2连接到载体上,构建得到重组载体;
将所述重组载体转入农杆菌GV3101,构建得到重组转化体;
将所述重组转化体侵染所述月季。
进一步地,所述重组转化体侵染的方法为摩擦接种法或真空渗透法。
进一步地,所述月季为蓝色狂想曲月季、月月粉月季、仙境月季中的一种。
本发明研究发现月季中液泡pH相关基因RcNHX2影响月季花瓣的颜色,在蓝色狂想曲与月月粉月季的VIGS植株中花瓣颜色变浅,并且,RcNHX2基因在蓝色狂想曲与月月粉月季的沉默突变株系中表达量显著降低;在仙境月季的瞬时过表达植株中花瓣颜色由粉红转向淡蓝紫,并且,RcNHX2基因在仙境月季瞬时过表达突变株系中的表达量显著升高,因此,本发明提供的RcNHX2基因在调控月季花色转蓝中具有重要的作用,为月季花色转蓝育种提供了重要的基础。
附图说明
图1为不同花色的蓝色狂想曲月季的图片;
图2为RcNHX2在不同花色的蓝色狂想曲月季中的表达量;
图3为蓝色狂想曲月季在摩擦接种TRV1+TRV2-RcNHX2,开花后1天的沉默突变株系表型;
图4为蓝色狂想曲月季在摩擦接种TRV1+TRV2-RcNHX2,开花后2天的对照和沉默突变株系表型;
图5为月月粉月季通过花苞抽真空法侵染TRV1+TRV2-RcNHX2,开花后2天的对照和沉默突变株系表型;
图6为仙境月季通过抽真空瞬时过表达pCAMBIA2300-RcNHX2,开花后2天的对照和过表达突变株系表型;
图7为蓝色狂想曲月季在摩擦接种TRV1+TRV2-RcNHX2,开花后2天的对照和沉默突变株系花瓣中RcNHX2基因的表达量;
图8为月月粉月季通过花苞抽真空法侵染TRV1+TRV2-RcNHX2,开花后2天的对照和沉默突变株系花瓣中RcNHX2基因的表达量;
图9为仙境月季通过抽真空瞬时过表达pCAMBIA2300-RcNHX2,开花后2天的对照和过表达突变株系花瓣中RcNHX2基因的表达量。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将结合本发明的实施例,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。
下列实施例中未注明具体条件的实验方法,通常按照常规条件,例如Sambrook等分子克隆:实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件,或按照制造厂商所建议的条件。所采用的试剂、载体,若无特殊说明,均为市售或公开渠道可以获得的。
在本发明中,可选用本领域已知的各种载体,如市售的载体,包括质粒等。
实施例1月季RcNHX2基因的克隆
提取野生型月季品种月月粉花瓣总RNA,提取试剂盒为RNAplant(市售),利用反转录试剂盒(市售)将总RNA反转录成cDNA。根据转录组测序结果设计引物,引物序列如SEQ IDNO.3和SEQ ID NO.4所示,采用PCR方法从蝴蝶兰cDNA中扩增出一条1632bp的条带。PCR产物回收,获取RcNHX2基因,其核苷酸序列如SEQ ID NO.1所示,该核苷酸序列编码的氨基酸序列如SEQ ID NO.2所示,由543个氨基酸残基组成,分子量为60.2千道尔顿。
实施例2不同花色的蓝色狂想曲月季的表达谱验证
1、提取如图1所示,不同花色的蓝色狂想曲月季的RNA,花色分别为白、粉、粉间、间、蓝,其中,粉间与间均为介于粉色与蓝色之间的颜色,粉间偏向于粉,间偏向于蓝紫,提取试剂盒为RNAplant(市售),利用反转录试剂盒(市售)将总RNA反转录成cDNA;
2、根据同源序列Blast数据比对结果设计引物,引物序列如SEQ ID NO.5和SEQ IDNO.6所示;
3、以不同花色的蓝色狂想曲月季反转录得到的cDNA为模板,对RcNHX2进行不同发育时期的表达谱验证。
验证结果如图2所示,RcNHX2的表达量随着花瓣颜色的变化而变化,且在蓝色花瓣中表达受到显著诱导,说明RcNHX2可能参与了蓝色狂想曲月季花瓣颜色的形成。
实施例3TRV载体介导的病毒诱导蓝色狂想曲月季的RcNHX2基因沉默
将RcNHX2基因的开放阅读框可操作地连接于TRV2病毒载体,形成含有该基因片段的TRV2-RcNHX2重组载体,然后将该重组载体转入农杆菌GV3101,通过农杆菌介导的方法,侵染蓝色狂想曲月季花苞下方4-5个叶片,低温处理2-3天,保湿避光培养至其开花,观察蓝色狂想曲月季的表型,具体步骤如下:
1、通过同源重组法将目的基因RcNHX2靠近3′端/5′端的特异片段插入TRV2载体,构建其VIGS沉默载体TRV2-RcNHX2,同时构建对照组载体TRV2-GFP,由于TRV2病毒包括衣壳蛋白(cp)基因、多克隆位点(mcs)等,用于插入目的基因,TRV1病毒包括编码RNA依赖的RNA聚合酶、运动蛋白和16kd蛋白的基因,是VIGS体系的辅助病毒载体,需搭配TRV1病毒共同侵染月季,具体地,将TRV2-RcNHX2载体、TRV1病毒、TRV2-GFP载体分别转入农杆菌感受态GV3101中;
2、准备菌液:
取于-80℃保存的TRV2-RcNHX2、TRV2-GFP、TRV1农杆菌菌液分别小摇(5mLkan+Rif抗性液体LB+10μL菌液)16小时后以1:100的比例小摇转大摇(20mL LB+200μL菌液);
3、过夜培养菌体:
①集菌,离心(9min,3700rpm,4℃)弃上清;
②加MgCl2(10mmol/L)5mL,混匀吹打,离心(9min,3700rpm,4℃)弃上清。加MgCl2(10mmol/L)5mL,混匀吹打,离心(9min,3700rpm,4℃)弃上清。加MgCl2(10mmol/L)适量1-2mL;
③测OD值(单注射0.6/共注射1.2先加少量MgCl2之后调整浓度)
OD1×V1=OD2×V2
④加促表达AS和MES,AS的体积(μL)为1.5*菌液终体积,MES的体积(μL)为20*菌液终体积;
⑤将TRV2-RcNHX2和TRV1农杆菌菌液混合,将TRV2-GFP和TRV1农杆菌菌液混合,混合结束后再室温下放置3-4h备用。
4、取材:选取还未开放,花苞还未呈色,长势良好且一致的花苞;
5、将真空泵内压强抽至0.08-0.09Mpa,以0.08-0.09Mpa压强维持5分钟,然后缓慢释放至压强为0Mpa,重复3次,与金刚砂共同摩擦接种至蓝色狂想曲月季花苞下方4-5个叶片;
6、将月季置于10℃保存3天,利于TRV病毒的向上传导,之后进行土培或水培,待开花后,观察花瓣表型。
图3为蓝色狂想曲月季在摩擦接种TRV1+TRV2-RcNHX2,开花后1天的沉默突变株系表型,图4为蓝色狂想曲月季在摩擦接种TRV1+TRV2-RcNHX2,开花后2天的对照和沉默突变株系表型,其中,a为对照株系表型,b为沉默突变株系表型,如图4所示,蓝色狂想曲月季沉默突变株系的箭头所指区域花瓣颜色明显变浅。
实施例4TRV载体介导的病毒诱导月月粉月季的RcNHX2基因沉默
参考实施例3提供的方法,诱导月月粉月季的RcNHX2基因沉默,观察月月粉月季花瓣表型。
图5为月月粉月季通过花苞抽真空法侵染TRV1+TRV2-RcNHX2,开花后2天的对照和沉默突变株系表型,其中,a为对照株系表型,b为沉默突变株系表型,如图5所示,月月粉月季沉默突变株系的花瓣颜色明显变浅。
实施例5pCAMBIA2300载体介导的仙境月季RcNHX2瞬时过表达
将RcNHX2基因的全长CDS序列可操作地连接于pCAMBIA2300-GFP载体,形成含有该基因片段的pCAMBIA2300-RcNHX2载体,GFP用于检验目标基因是否转入载体中,将该载体转入农杆菌GV3101,通过农杆菌介导的方法,通过真空渗透法将其渗入仙境月季的花苞中,保湿避光培养至其开花,观察仙境月季的表型。
图6为仙境月季通过抽真空瞬时过表达pCAMBIA2300-RcNHX2,开花后2天的对照和过表达突变株系表型,其中,a为对照株系表型,b为瞬时过表达株系表型,如图6所示,仙境月季瞬时过表达突变株系的花瓣颜色明显发生变化,由粉红转向淡蓝紫。
实施例6TRV病毒诱导蓝色狂想曲、月月粉及pCAMBIA2300载体介导的突变株系中,RcNHX2基因的表达验证
1、选取如图4-6所示的对照和沉默/瞬时过表达突变株系的花瓣,提取其总RNA,提取试剂盒为RNAplant(市售),利用反转录试剂盒(市售)将总RNA反转录成cDNA;
2、根据转录组测序数据设计引物,引物序列如SEQ ID NO.5和SEQ ID NO.6所示;
3、对照和沉默/瞬时过表达突变株系花瓣的反转录得到cDNA为模板,对RcNHX2进行基因沉默/瞬时过表达效率验证。
验证结果如图7-9所示,RcNHX2基因的表达在蓝色狂想曲月季及月月粉月季突变株系花瓣中均显著降低,证明RcNHX2沉默效率较高;在仙境月季过表达突变株系中显著升高,证明RcNHX2过表达效率较高,也进一步说明了RcNHX2参与了不同月季品种花瓣颜色的形成。
综上,本发明研究发现月季中液泡pH相关基因RcNHX2影响月季花瓣的颜色,在蓝色狂想曲与月月粉月季的VIGS植株中花瓣颜色变浅,并且,RcNHX2基因在蓝色狂想曲与月月粉月季的沉默突变株系中表达量显著降低;在仙境月季的瞬时过表达植株中花瓣颜色由粉红转向淡蓝紫,并且,RcNHX2基因在仙境月季瞬时过表达突变株系中的表达量显著升高,因此,本发明提供的RcNHX2基因在调控月季花色转蓝中具有重要的作用,为月季花色转蓝育种提供了重要的基础。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
序列表
<110> 上海师范大学
<120> 一种调控月季花瓣颜色的基因RcNHX2及其应用
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<170> SIPOSequenceListing 1.0
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<212> DNA
<213> Rosa chinensis
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ttagtattca gtgaagatct tttctttatt taccttctac cacctattat ttttaatgcc 300
gggtttcagg tgaaaaagaa gcagtttttc cgtaatttca ttactattgt aatgttcggt 360
gctattggta cattagtatc ctgcaccatt atagcatttg gtgctacaca aatctttaag 420
aaattggaca ttggttcgct ggacataggg gattatctcg caattggtgc aatatttgct 480
gcaacggatt ctgtatgcac gttgcaggtg ctccatcagg atgagactcc tttactgtac 540
agtcttgtat tcggcgaggg agttgttaac gatgctacat ctgtggtgct tttcaatgct 600
atccagagct ttgatctaac acaccttgat tccagaatcg ccttgaagtt tatgggcaac 660
tttttgtatt tgttttttgc aagcaccatg ctaggagtga ttacagggct gctaagtgct 720
ttcattatca aaaagcttta ttttgcaagg cactcaacag atcgtgaggt cgctcttatg 780
atgctcatgg catacctttc atatatactg gctgaactat tctatttgag tggcattctc 840
actgtattct tttgtgggat tgtgatgtcc cattacacct ggcacaatgt gacagagagt 900
tcaagagtca cgaccaagca tgcttttgca accttgtcat ttgtttgcga gacttttatc 960
ttcctctatg ttggtatgga tgccttggac attgaaaagt ggagatttgt aagtgatagt 1020
cctggaacgt cagtggcagt gagttcaata ctgctaagtc ttgttatgct tggaagagca 1080
gcttttgttt ttcctttatc ctttttctca aacttattta agaaaaatca aagcgagaaa 1140
attagcctcc agcagcaagt ggtaatatgg tgggctggtc ttatgagagg tgctgtgtct 1200
atagcgcttg cttataatca gtttacaaga tctggtcaca ctcaattgcg agcaaatgca 1260
atcatgatca ctagcacaat aagtgttgtt cttgtcagca cagtggtgtt tggtttgatg 1320
acgaaacctc ttattagatt attgctgcct cataaacaat tgaccagcac aaacagcatt 1380
atgtcagacc caccctctcc aaaatcagtc attgttccac tacttgggca ggattctgaa 1440
gctgatctga gcggtcatga ggtgcgtcgt ccagccagca tacgtgatct tctgacgact 1500
ccaacacaca ctgtacatcg ctactggcgt aagtttgata atgctttcat gcgtccagta 1560
tttggtggtc ggggttttgt tccctttgtt cccggctcac caactgaacg gaacaacact 1620
caatggcaat ga 1632
<210> 2
<211> 543
<212> PRT
<213> Rosa chinensis
<400> 2
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Asn Leu Ser Ala Ser Asp His Ser Ser Val Val Ser Leu Asn Leu Phe
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Val Ala Leu Leu Cys Ala Cys Ile Val Ile Gly His Leu Leu Glu Glu
35 40 45
Asn Arg Trp Val Asn Glu Ser Ile Thr Ala Leu Leu Ile Gly Val Ser
50 55 60
Thr Gly Val Leu Ile Leu Leu Ile Ser Gly Gly Lys Ser Ser His Leu
65 70 75 80
Leu Val Phe Ser Glu Asp Leu Phe Phe Ile Tyr Leu Leu Pro Pro Ile
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Ile Phe Asn Ala Gly Phe Gln Val Lys Lys Lys Gln Phe Phe Arg Asn
100 105 110
Phe Ile Thr Ile Val Met Phe Gly Ala Ile Gly Thr Leu Val Ser Cys
115 120 125
Thr Ile Ile Ala Phe Gly Ala Thr Gln Ile Phe Lys Lys Leu Asp Ile
130 135 140
Gly Ser Leu Asp Ile Gly Asp Tyr Leu Ala Ile Gly Ala Ile Phe Ala
145 150 155 160
Ala Thr Asp Ser Val Cys Thr Leu Gln Val Leu His Gln Asp Glu Thr
165 170 175
Pro Leu Leu Tyr Ser Leu Val Phe Gly Glu Gly Val Val Asn Asp Ala
180 185 190
Thr Ser Val Val Leu Phe Asn Ala Ile Gln Ser Phe Asp Leu Thr His
195 200 205
Leu Asp Ser Arg Ile Ala Leu Lys Phe Met Gly Asn Phe Leu Tyr Leu
210 215 220
Phe Phe Ala Ser Thr Met Leu Gly Val Ile Thr Gly Leu Leu Ser Ala
225 230 235 240
Phe Ile Ile Lys Lys Leu Tyr Phe Ala Arg His Ser Thr Asp Arg Glu
245 250 255
Val Ala Leu Met Met Leu Met Ala Tyr Leu Ser Tyr Ile Leu Ala Glu
260 265 270
Leu Phe Tyr Leu Ser Gly Ile Leu Thr Val Phe Phe Cys Gly Ile Val
275 280 285
Met Ser His Tyr Thr Trp His Asn Val Thr Glu Ser Ser Arg Val Thr
290 295 300
Thr Lys His Ala Phe Ala Thr Leu Ser Phe Val Cys Glu Thr Phe Ile
305 310 315 320
Phe Leu Tyr Val Gly Met Asp Ala Leu Asp Ile Glu Lys Trp Arg Phe
325 330 335
Val Ser Asp Ser Pro Gly Thr Ser Val Ala Val Ser Ser Ile Leu Leu
340 345 350
Ser Leu Val Met Leu Gly Arg Ala Ala Phe Val Phe Pro Leu Ser Phe
355 360 365
Phe Ser Asn Leu Phe Lys Lys Asn Gln Ser Glu Lys Ile Ser Leu Gln
370 375 380
Gln Gln Val Val Ile Trp Trp Ala Gly Leu Met Arg Gly Ala Val Ser
385 390 395 400
Ile Ala Leu Ala Tyr Asn Gln Phe Thr Arg Ser Gly His Thr Gln Leu
405 410 415
Arg Ala Asn Ala Ile Met Ile Thr Ser Thr Ile Ser Val Val Leu Val
420 425 430
Ser Thr Val Val Phe Gly Leu Met Thr Lys Pro Leu Ile Arg Leu Leu
435 440 445
Leu Pro His Lys Gln Leu Thr Ser Thr Asn Ser Ile Met Ser Asp Pro
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Ala Asp Leu Ser Gly His Glu Val Arg Arg Pro Ala Ser Ile Arg Asp
485 490 495
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Asp Asn Ala Phe Met Arg Pro Val Phe Gly Gly Arg Gly Phe Val Pro
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<210> 3
<211> 24
<212> DNA
<213> 人工序列(Artificial sequences)
<400> 3
atggcttctc atttggccat gttg 24
<210> 4
<211> 23
<212> DNA
<213> 人工序列(Artificial sequences)
<400> 4
tcattgccat tgagtgttgt tcc 23
<210> 5
<211> 20
<212> DNA
<213> 人工序列(Artificial sequences)
<400> 5
tggcgttcgg attgtcttct 20
<210> 6
<211> 20
<212> DNA
<213> 人工序列(Artificial sequences)
<400> 6
cgcaaagcaa cgcaacaaag 20
Claims (10)
1.一种调控月季花瓣颜色的基因RcNHX2,其特征在于,所述基因具有以下核苷酸序列中的一种:
1)SEQ ID NO.1所示的核苷酸序列;
2)SEQ ID NO.1所示的核苷酸序列通过一个或几个核苷酸的取代、缺失、添加后衍生产生的核苷酸序列;
3)与SEQ ID NO.1具有至少80%同源性的核苷酸序列。
2.一种调控月季花瓣颜色的蛋白,其特征在于,所述蛋白由权利要求1所述的基因RcNHX2编码得到。
3.一种重组载体,其特征在于,所述重组载体包含权利要求1所述的核苷酸序列。
4.根据权利要求3所述的重组载体,其特征在于,所述重组载体为包含所述核苷酸序列的TRV2病毒或pCAMBIA2300-GFP载体。
5.一种重组转化体,其特征在于,所述重组转化体包含权利要求3或4所述的重组载体。
6.根据权利要求5所述的重组转化体,其特征在于,所述重组转化体为包含所述重组载体的农杆菌GV3101。
7.权利要求1所述的基因在调控月季花瓣颜色中的应用。
8.一种调控月季花瓣颜色的方法,其特征在于,具体包括如下步骤:
将权利要求1所述的基因RcNHX2连接到载体上,构建得到重组载体;
将所述重组载体转入农杆菌GV3101,构建得到重组转化体;
将所述重组转化体侵染所述月季。
9.根据权利要求8所述的方法,其特征在于,所述重组转化体侵染的方法为摩擦接种法或真空渗透法。
10.根据权利要求8所述的方法,其特征在于,所述月季为蓝色狂想曲月季、月月粉月季、仙境月季中的一种。
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CN114807160A (zh) * | 2022-03-10 | 2022-07-29 | 上海师范大学 | 调控月季花瓣颜色的基因RcGT、蛋白、重组载体、重组转化体、应用和方法 |
CN115094066A (zh) * | 2022-05-06 | 2022-09-23 | 上海师范大学 | 一种调控月季花瓣颜色的基因RcTTG1及其应用 |
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