CN108017696B - 菊花CmTFL1c基因及其应用 - Google Patents
菊花CmTFL1c基因及其应用 Download PDFInfo
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- CN108017696B CN108017696B CN201711407496.4A CN201711407496A CN108017696B CN 108017696 B CN108017696 B CN 108017696B CN 201711407496 A CN201711407496 A CN 201711407496A CN 108017696 B CN108017696 B CN 108017696B
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Abstract
本发明提供了菊花CmTFL1c基因及其应用,属于植物基因工程领域。所述菊花CmTFL1c基因序列如SEQ ID No.1所示,其编码蛋白序列如SEQ ID No.2所示。通过农杆菌介导法侵染菊花叶盘,将该基因转入菊花中,得到转CmTFL1c基因菊花株系,结果发现转CmTFL1c基因菊花株系侧枝数量增加,地表覆盖率增加,进而花量也增加,并且花期推迟15‑20天。可见本发明的菊花CmTFL1c基因具有推迟花期、促进菊花侧枝生长、增加花量的功能,将该基因应用于植物性状改良,具有良好的应用前景。
Description
技术领域
本发明属于植物基因工程领域,具体地说,涉及菊花CmTFL1c基因、其编码蛋白及其在促进植物侧枝生长,增加开花量中的应用。
背景技术
菊花是原产我国的十大名花之一,由长期人工培育和天然种间杂交而来。菊花在叶、花型、瓣型上变化很大,色、香、姿、韵俱佳,与梅兰竹共称为“花卉四君子”,不仅为我国人民所喜爱,而且现已为世界各国广泛栽培。菊花品种繁多,我国多达3000多种,全世界7000多种,但大多数品种的自然花期较集中且短,大多在10月-11月,其自然花期在一定程度上制约着菊花的周年生产和应用范围,随着人们的观赏需求不断增长,培育不同花色、花径、株高及连续开花等观赏性状的菊花品种已经成为菊花育种的一个重要目标。
TFL1基因属植物FT/TFLl(FLOWERING LOCUS T/TERMlNAL FLOWER l)基因家族,TFL1基因抑制花序分生组织向花分生组织的转变,从而抑制开花使植物花期推后。TFL1基因在植物花序分生组织类型的维持及花期的调控方面起着极其重要的作用。对拟南芥中TFL1基因研究发现,拟南芥TFL1基因控制花序分生组织和花分生组织发育,通过抑制LFY基因在花序分生组织中的活性而维持花序分生组织的无限生长模式。
在蔷薇属植物中,TFL1同源基因是植物花序分生组织特异性表达基因,主要表达于茎顶端的分生组织中,抑制花分生组织特异基因LFY和AP1的表达,延迟植株开花。
近年来,TFL1在植物开花响应机制被广泛研究。大量的研究表明TFL1及其同源基因在不同植物的表达模式及其功能不尽相同。目前菊花CmTFL1c基因在菊花花期以及分枝调控中的相关功能未有报道。
发明内容
本发明的目的是提供菊花CmTFL1c基因及其应用。
本发明首先提供菊花CmTFL1c蛋白,其具有:
1)如SEQ ID No.2所示的氨基酸序列;或
2)SEQ ID No.2所示的氨基酸序列经取代、缺失和/或增加一个或多个氨基酸且具有同等活性的由1)衍生的蛋白质。
本发明提供了编码菊花CmTFL1c蛋白的基因,其具有:
1)SEQ ID No.1所示的核苷酸序列;或
2)SEQ ID No.1所示核苷酸序列经取代、缺失和/或增加一个或几个核苷酸;或
3)在严格条件下与1)限定的DNA序列杂交的核苷酸序列。
本发明提供了含有上述编码菊花CmTFL1c蛋白的基因的生物材料,所述生物材料为载体、宿主细胞或表达盒。
本发明提供了上述菊花CmTFL1c蛋白或编码其的基因或含有该基因的生物材料在促进植物侧枝生长中的应用。
本发明提供了上述菊花CmTFL1c蛋白或编码其的基因或含有该基因的生物材料在增加植物开花量中的应用。
本发明提供了上述菊花CmTFL1c蛋白或编码其的基因或含有该基因的生物材料在促进植物腋芽发生中的应用。
本发明提供了上述菊花CmTFL1c蛋白或编码其的基因或含有该基因的生物材料在促使匍匐性植株增加地面覆盖率中的应用。
本发明提供了上述菊花CmTFL1c蛋白或编码其的基因或含有该基因的生物材料在推迟植物花期中的应用。
本发明提供了上述菊花CmTFL1c蛋白或编码其的基因或含有该基因的生物材料在制备转基因植物中的应用。
所述转基因植物腋芽生长旺盛、侧枝数量多或花量多。
本发明提供了上述菊花CmTFL1c蛋白或编码其的基因或含有该基因的生物材料在植物种质资源改良中的应用。
本发明提供的菊花CmTFL1c基因序列如SEQ ID No.1所示,其编码蛋白序列如SEQID No.2所示。通过农杆菌介导法侵染菊花叶盘,将该基因转入菊花中,得到转CmTFL1c基因菊花株系,结果发现转CmTFL1c基因菊花株系侧枝数量增加,地表覆盖率增加,进而花量也增加,并且花期推迟15-20天。可见本发明的菊花CmTFL1c基因具有推迟植物花期、促进植物侧枝生长、增加花量的功能,将该基因可作为匍匐性菊花改良性状的候选基因,应用于植物性状改良,具有良好的应用前景。
附图说明
图1为图1CmTFL1c基因的PCR扩增电泳图,其中M.DL 2000;CK.ddH2O;1~5.CmTFL1c基因。
图2为不同植物TFL1的系统发育树。
图3为pCAMBIA1301-pmi-CmTFL1c载体构建菌液PCR电泳图,图中M,DL2000Maker;1~7为载体构建菌液PCR结果。
图4为pCAMBIA1301-pmi-CmTFL1c载体转化农杆菌感受态菌液PCR电泳图。
图5A-图5D为转CmTFL1c基因菊花再生过程,其中图5A表示筛选培养15d,图5B表示获得的抗性愈伤组织,图5C表示分化得到的抗性芽,图5D为获得的抗性植株。
图6为转基因菊花抗性苗PMI基因的PCR鉴定结果图,M,DL2000Maker;1,阳性质粒对照;2为阴性对照,3-8为为检测的抗性植株,但是仅有5,7,8为阳性,说明这3个株系为转基因植株,其余的3,4,6为假阳性。
图7转CmTFL1c基因25#株系与野生种花期的比较,相同培育时间下,左为野生种,右为转CmTFL1c基因25#株系。
图8为野生型菊花(右)与转CmTFL1c基因菊花(左)腋芽生长对比图。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。
植物双元表达载体pCAMBIA1301-pmi,pEASY-Blunt-CmTFL1c质粒为实验室保存,其中pCAMBIA1301-pmi双元表达载体参考王叶(透明颤菌血红蛋白基因表达载体构建及转化地被菊研究,2012)实验所用,pEASY-Blunt购自北京全式金生物技术有限公司,按照使用说明书连接CmTFL1c基因。KOD高保真Taq酶购自北京擎科新业生物技术有限公司,其它限制性内切酶购自北京拜尔迪生物技术有限公司,农杆菌感受态购自北京华岳洋生物科技有限公司。
实施例1菊花CmTFL1c基因的获得
1.试验材料
以菊花‘金不凋’茎尖组织为材料。
2.质粒、菌种及试验试剂
RNA反转录试剂盒(TAKARA)购于北京六合通经贸有限公司;Trans1-T1DH5α感受态细胞、DNAmarker、克隆载体pEASY-Blunt购于北京全式金生物技术有限公司。DNA凝胶回收试剂盒(AXYGEN)、LB肉汤(Coolaber),氨苄(Amp)、卡那霉素(Kan)、异丙基硫代半乳糖苷(IPTG),X-Gal、琼脂(Agar)、琼脂糖购自拜尔迪生物技术有限公司;超快型植物RNA提取试剂盒、GelRed核酸染料购自北京华越洋生物科技有限公司;2xPCR Mix购自北京擎科新业生物技术有限公司、引物合成及测序服务由北京睿博兴科生物技术有限公司提供。
3.试验方法
(1)菊花‘金不凋’茎尖组织RNA提取
使用北京华越洋超快型植物RNA提取试剂盒提取菊花‘金不凋’茎尖组织总RNA,具体方法见使用说明书。
(2)cDNA合成
使用TAKARA的RNA反转录试剂盒PrimeScriptTM RT reagent Kit with gDNAEraser,按说明书进行反转录操作。
(3)菊花CmTFL1c基因全长克隆
以菊花cDNA为模版,根据菊花转录组数据(本课题组前期测序,未发表)设计特异性引物TcF(TCGTCGTCTTCATCATGTC)和TcR(TCATCTTCTACGGGCTGCAT)进行PCR扩增。反应体系为cDNA模板1.0μl,TcF 0.4μl,TcR 0.4μl,2×PCRMix 12.5μl,ddH2O补足至25μl。PCR扩增程序:95℃预变性5min;95℃变性30s,54℃退火30s,72℃延伸1min,30个循环;72℃再延伸10min;4℃保存。
(4)PCR产物的回收、连接和转化
将PCR产物经1.0%琼脂糖凝胶电泳分析,使用AXYGEN胶回收试剂盒对PCR产物进行回收。回收的DNA片段与pEASY-Blunt载体连接,转化Trans1-T1DH5α感受态细胞。将转化质粒后的大肠杆菌均匀涂布在含X-gal和IPTG的固体培养基上,经过蓝白斑筛选,挑取白色阳性单克隆菌落进行PCR鉴定,将阳性克隆送往北京睿博兴科生物技术有限公司进行序列测定。
4.结果与分析
利用菊花特异性引物,在菊花中共克隆得到1条长度约为530bp左右的目的条带(图1)。测序结果经过BLAST比对,为TFL1同源基因,命名为CmTFL1c。CmTFL1c的ORF为522bp,编码173个氨基酸。根据预测的蛋白质与其它植物的TFL1蛋白进行同源比对显示,CmTFL1c蛋白(ALL28249.1)与芝麻CEN-like蛋白(XP_011076204.1)相似性高达82%,此外与荷花CEN-like蛋白(XP_010278585.1)、苹果CEN-like蛋白(NP_001280770.1)相似性均为80%。系统进化树进一步表明CmTFL1c为TFL1蛋白家族(图2)。
实施例2含有菊花CmTFL1c基因的植物表达载体的构建及菊花的遗传转化表型分析
1、构建以甘露糖(PMI基因)为筛选标记的安全表达载体(pCAMBIA1301-pmi-CmTFL1c),需要将Hyg位点替换为目的基因,达到无抗生素标记的安全性。Hyg位点上下游均为XhoI的酶切位点,为了保证目的基因连接方向正确,选择PCR融合的方法构建植物表达载体,在目的基因片段的上下游分别引入pCAMBIA1301-pmi表达载体连接位置的序列15-20bp,具体的方法为:
(1)目的基因的扩增
利用实验室前期工作保存的pEASY-Blunt-CmTFL1c质粒为模板,设计基因特异性引物进行PCR扩增,引物为CmTFL1c-pmi-F(AGATGTTTAGATAGAGACTCGAGATGTCAAGAATGAATGAGCCACTTGC)和CmTFL1a-pmi-R(TGTAATAATACCTCTTTCTCGAGTCATCTTCTACGGGCTGCATTTTCTCT)。反应体系为:1μl cDNA模板,1μl上游和下游引物(10μmolL-1),12.5μl PCR MIX(含500μMdNTP,20mMTris-HCl,100mMKCl,3mM MgCl2,0.1U·μl-1Taq Polymerase),ddH2O补齐至25μl.反应程序为94℃30s,58℃1min,72℃30s,35个循环,72℃10min,最后10℃保存。1%琼脂糖凝胶电泳检测扩增条带。
(2)以CmTFL1c基因片段为引物分别扩增质粒模板,反应体系为如下:
反应程序为98℃2min,98℃30s,68℃6min 30s,35循环,4℃保存。
(3)上述PCR产物中加入2μL DpnI,37℃消化2h。
(4)上述酶切产物经琼脂糖电泳后切胶纯化,取10μL转化大肠杆菌DH5α,37℃过夜培养。
(5)挑取单菌落进行PCR检测。
(6)将上述扩增出目的条带的菌液接菌于LB培养基中,扩大培养,并提质粒测序。测序正确的质粒保存于-20℃冰箱中。
本发明采用PCR融合技术进行pCAMBIA1301-pmi表达载体与目的基因CmTFL1c连接,将表达载体上面的hptII筛选标记基因由CmTFL1c替代,上游同样是CaMV35S启动子驱动,下游为CaMV35S ployA终止。在植物体内表达时的筛选标记为Pmi基因,即甘露糖异构酶基因,具有甘露糖抗性。接后的重组载体经菌落PCR、测序鉴定,表明pCAMBIA1301-pmi-CmTFL1c,载体均构建成功(图3)。
2、转化农杆菌感受态
将测序正确的pCAMBIA1301-pmi-CmTFL1c质粒通过冻融法转化农杆菌感受态。
(1)将-80℃保存的农杆菌感受态置于冰上融化,待完全融化后立刻加入质粒,轻轻吹打混匀,冰浴30min。
(2)液氮速冻1min,37℃金属浴热激5min,然后再冰浴2min。
(3)无菌条件下,加入700μl液体LB培养基(无抗生素),28℃,180rpm震荡培养4-5h。
(4)室温5000rpm离心1min,去掉上清液,保留200μl,用枪头轻轻吹打混匀后涂布于5LB固体培养基(附加50mg/L Rif和50mg/mL Kan)上,28℃倒置暗培养2-3天,直至长出单克隆菌斑。
(5)挑取单菌斑利用菌液PCR进行阳性鉴定,PCR检测为阳性的菌斑摇菌后,用于后续转基因试验,或1:1加入灭菌的30%甘油,于-80℃保存备用。
利用冻融法转化农杆菌感受态EHA105,菌落PCR鉴定表明载体质粒已成功转入到农杆菌中(图4)。
3、CmTFL1c基因在菊花中的遗传转化
3.1叶盘的转化及筛选
地被菊‘粉地毯’的遗传转化体系参考王叶(2013)建立的以pmi为安全标记的菊花遗传转化体系,并根据实验做适当的调整。地被菊‘粉地毯’叶盘转化的基本培养基配方如下:
M1培养基:MS培养基+0.5g/L 6-BA+0.1g/L NAA+30g/L蔗糖+7g/L琼脂,PH5.8-6.0
M2培养基:M1培养基+400mg/L Car,PH5.8-6.0
M3培养基:M2培养基+8mg/L mannose,PH5.8-6.0
M4培养基:M1培养基+300mg/L Car+10mg/L mannose,PH5.8-6.0
M5培养基:MS培养基+7mg/L mannose,PH5.8-6.0
具体方法为:
(1)取经测序鉴定为阳性农杆菌pCAMBIA1301-pmi-CmTFL1c,若是在-80℃中保存,需在LB固体培养基(50mg/L Kan+50mg/L Rif)上划线活化。待长起单菌落后,接种于3mL同样含双抗的LB液体培养基中小摇过夜。将浑浊的菌液按照1:100的比例转入含两抗的LB培养液中继续扩大培养,28℃,180rmp避光震荡培养至OD600=0.4-0.6之间。
(2)将菌液放入50ml的离心管中,5500r/min,常温离心15min,收集菌体,弃去上清液,用1/2MS(1/2MS+30g/L蔗糖,PH5.8)培养基重悬至OD600=0.4-0.6之间。
(3)以无菌的苗龄为30d左右的‘粉地毯’幼苗为试材,选取中上部分厚实健壮的叶片,避开主叶脉切成边长为0.5cm的方块,并在方块中间划切几道。叶片近轴面朝下,平铺在M1培养基上,于正常的光照培养下培养20h,不超过24h。
(4)经过预培养的‘粉地毯’叶盘完全浸入在农杆菌重悬液中,轻轻晃动10min。取出叶盘并用无菌滤纸将叶盘表面的菌液吸干。然后将叶盘平铺在M1培养基上,暗培养2d。
(5)共培养后的叶盘周围可以看到星点状的菌落,将叶盘放于含有400mg/L Car的无菌水中,冲洗3次,并且用无菌滤纸吸取水份,将叶盘转移至M2培养基上,正常培养4d。
(6)经过脱菌的叶盘转移到M3较低筛选压的培养基上,进行筛选培养15d后可以看到叶盘膨大,边缘形成少量愈伤组织。将叶盘转接至较高筛选压d的M4培养基上,每15d转接到新鲜的培养基上。
(7)筛选培养约45-60d,叶片边缘的愈伤组织分化出抗性芽,待抗性芽继续生长到1cm的时候,切下来放至M5生根培养基上。约20d,抗性芽生根,30d后长成完整的植株。
菊花‘粉地毯’经过预培养、共培养、延迟培养后转接在含8mg/L甘露糖浓度的培养基上15d(图5A),大部分叶盘开始逐渐褐化,部分叶盘边缘出现少量的愈伤组织(图5B)。继续筛选培养约35d,在抗性愈伤组织中分化出抗性芽(图5C),而大部分愈伤组织在不断增大或者褐化,并不分化。待抗性芽长至1cm,将丛生芽分开单株转移到生根培养基中(含7mg/L甘露糖),约20d后逐渐生根,茎尖也不断有新叶长出,35d左右根系健壮(图5D)。一共侵染叶盘共1000个,仅27个叶盘分化出抗性芽,分化率为2.7%。
3.2转基因菊花的筛选
抗性苗PCR检测:取生根的抗性苗少量叶片,利用Edward方法粗提菊花叶片DNA,利用筛选标记PMI基因引物进行PCR扩增,检测筛选阳性苗。检测引物为PMI-F(ACTCATTAACTCAGTGCAAAACTATGCCTGGG)和PMI-R(CGGCCGTGGCCTTTGACAGTCAC)。结果显示共获得57棵抗性苗,提取抗性苗叶片DNA,以筛选标记基因特异引物进行PCR检测,结果显示共检测出3株抗性苗具有1100bp的目的条带(图6),而野生型和阴性对照无条带。
3.3转基因菊花的表型分析
将上述鉴定为阳性的转基因苗扩繁至7-10株,并且生根后转移到营养土:珍珠岩=2:1的栽培基质中。放于温室中培养,直至开花。栽培条件为在长日照(16h光照/8h黑暗)下生长4个月,之后转移到短日照条件(12h光照/12h黑暗)下促进开花。温度在23-25℃。观察记录野生型和转基因菊花的花期(统计自移植至现蕾及第一朵花开需要的天数)。
转CmTFL1c基因菊花25#株系花期明显推迟,如图7,野生型菊花的花序已经完全开放,但是转CmTFL1c基因25#株系花蕾刚露色。统计从移植到温室至第一朵花完全展瓣时需要的天数,野生菊花需要177d开花,25#转基因株系需要194d。
3.4转CmTFL1c基因促进菊花侧枝生长
菊花茎尖由营养生长向生殖生长转变时,腋芽会逐渐发育,促进分枝。与野生菊花相比,转CmTFL1c基因菊花同样在茎尖可以肉眼看到花蕾的形成,呈半圆形,转CmTFL1c基因26#株系的腋芽明显多于野生型,几乎每个叶腋处均有腋芽产生(图8)。而且腋芽的生长并不影响茎尖花蕾的发育及开花,在顶端花序逐渐开放后,叶腋处的花序陆续开放。这说明CmTFL1c基因促进腋芽生长,促进侧枝的生长。对于匍匐性菊花而言,该性状不仅增大了覆盖面积,并且增加了花量。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
序列表
<110> 北京林业大学
<120> 菊花CmTFL1c基因及其应用
<130> KHP171118850.2
<160> 8
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atgtcaagaa tgaatgagcc acttgcagta ggaagagtta taggagaggt ggtggacgca 60
ttcacaccaa gtgtgaagct aagtgtaaca tataatctca ataagatggt ctgtaatgga 120
catgagctca tgcctaatgt cattacttct aaacctcgtg ttgatatcgg tggtgaagac 180
atgagatctg cttatactct tatcatgacc gatccagacg ttccaggccc aagtgatcct 240
tacctaagag aacatcttca ctggattgtt acagacattc ctggtaccac tgatgcttct 300
tttggaaaag agattgtgag ctatgaaata ccaaagccgg tgatagggat tcaccgatat 360
gtgttcttat tgttcaagca gaaaacaaga aaatcggtga ctccaccggc ttccagggac 420
catttcaaca ctcggagctt ctgtcacgaa catggattag ggttaccggt tgcagctgta 480
tatttcaatg ctcaaagaga aaatgcagcc cgtagaagat ga 522
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<211> 173
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<213> 人工序列(Artificial Sequence)
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Tyr Thr Leu Ile Met Thr Asp Pro Asp Val Pro Gly Pro Ser Asp Pro
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Tyr Leu Arg Glu His Leu His Trp Ile Val Thr Asp Ile Pro Gly Thr
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<213> 人工序列(Artificial Sequence)
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cggccgtggc ctttgacagt cac 23
Claims (6)
1.菊花CmTFL1c蛋白或其编码基因或含有所述编码基因的生物材料在促进菊花侧枝生长中的应用;所述菊花CmTFL1c蛋白的氨基酸序列如SEQ ID No.2所示;所述编码基因的核苷酸序列如SEQ ID No.1所示;所述生物材料为载体或表达盒。
2.菊花CmTFL1c蛋白或其编码基因或含有所述编码基因的生物材料在增加菊花开花量中的应用;所述菊花CmTFL1c蛋白的氨基酸序列如SEQ ID No.2所示;所述编码基因的核苷酸序列如SEQ ID No.1所示;所述生物材料为载体或表达盒。
3.菊花CmTFL1c蛋白或其编码基因或含有所述编码基因的生物材料在促进菊花腋芽发生中的应用;所述菊花CmTFL1c蛋白的氨基酸序列如SEQ ID No.2所示;所述编码基因的核苷酸序列如SEQ ID No.1所示;所述生物材料为载体或表达盒。
4.菊花CmTFL1c蛋白或其编码基因或含有所述编码基因的生物材料在促使菊花植株增加地面覆盖率中的应用;所述菊花CmTFL1c蛋白的氨基酸序列如SEQ ID No.2所示;所述编码基因的核苷酸序列如SEQ ID No.1所示;所述生物材料为载体或表达盒。
5.菊花CmTFL1c蛋白或其编码基因或含有所述编码基因的生物材料在制备开花量增多、分枝增多或腋芽增多的转基因菊花中的应用;所述菊花CmTFL1c蛋白的氨基酸序列如SEQ ID No.2所示;所述编码基因的核苷酸序列如SEQ ID No.1所示;所述生物材料为载体或表达盒。
6.菊花CmTFL1c蛋白或其编码基因或含有所述编码基因的生物材料在菊花种质资源改良中的应用;所述改良为菊花侧枝数量增加、菊花地表覆盖率增加、或菊花花量增加;所述菊花CmTFL1c蛋白的氨基酸序列如SEQ ID No.2所示;所述编码基因的核苷酸序列如SEQ IDNo.1所示;所述生物材料为载体或表达盒。
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