CN116855531A - 牡丹PsASMT基因或其载体在改变植物花色中的应用 - Google Patents
牡丹PsASMT基因或其载体在改变植物花色中的应用 Download PDFInfo
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Abstract
本发明公开了牡丹PsASMT基因或其载体在改变植物花色中的应用。本发明通过将构建的PsASMT基因过量表达载体转化到烟草中进行表达，降低了植物，尤其是烟草花瓣中花色苷含量，抑制了花色苷合成相关基因表达，创制了淡粉色烟草新种质。
Description
技术领域
本发明涉及牡丹PsASMT基因或其载体在改变植物花色中的应用，属于基因新用途领域。
背景技术
N-乙酰基-5-羟色胺甲基转移酶(N-acetylserotonin methyltransferas，ASMT)位于褪黑素合成途径中，催化N-乙酰-5-羟色胺(N-Acetyl-5-hydroxytryptamine)进行O-甲基转移反应生成褪黑素。
ASMT基因首次于2011年在水稻中得到克隆(Kang K,Kong K,Park S,etal.Molecular cloning of a plant N-acetylserotonin methyltransferase and itsexpression characteristics in rice.Journal ofPineal Research,2011,50:304-309)，随后它在苹果(Zuo BX,Zheng XD,He PL,et al.Overexpression ofMzASMTimprovesmelatoninproduction and enhances drought tolerance in transgenic Arabidopsisthaliana plants.Journal of Pineal Research,2014,57:408-417)、核桃(Ma K,Xu RQ,Zhao Y,et al.WalnutN-Acetylserotonin methyltransferase gene family genome-wide identification and diverse functions characterization during flower buddevelopment.Frontiers in Plant Science,2022,13)等植物中也被相继分离。目前，ASMT基因在植物中的应用较多，如Xu等将长春花ASMT基因在番茄中进行超表达，发现转基因植株对高温胁迫的耐受性显著增强(XuW,Cai SY,Zhang Y,et al.Melatonin enhancesthermotolerance by promoting cellular protein protection in tomatoplants.Journal ofPineal Research,2016,4:457-469)；Byeon等敲除水稻ASMT基因后，发现植株褪黑素含量显著降低，叶片衰老，产量减少(ByeonY,Back K.Low melatoninproduction by suppression of either serotonin N-acetyltransferase or N-acetylserotonin methyltransferase in rice causes seedling growth retardationwith yield penalty,abiotic stress susceptibility,and enhanced coleoptilegrowth under anoxic conditions.Journal ofPineal Research,2016,3:348-359)；Wei等发现木薯ASMT基因通过被上游转录因子激活，从而提高木薯对枯萎病的抗性(Wei YX,Liu GY,Bai YJ,et al.Two transcriptional activators of N-acetylserotonin O-methyltransferase 2and melatonin biosynthesis in cassava.JournalofExperimental Botany,2017,17:4997-5006)；发明专利CN110184247A公布了紫花苜蓿ASMT基因核酸序列，过量表达ASMT能够降低萜类和多酚类物质含量，提高生物碱类物质含量。这些研究结果表明，ASMT基因在调控次生代谢产物含量、增强植株抗逆性等方面具有重要作用。而有关ASMT基因在调控植物花色方面的应用至今未见报道。
发明内容
发明目的：本发明所要解决的技术问题是提供了牡丹PsASMT基因或其载体在改变植物花色中的应用。
技术方案：为解决上述技术问题，本发明提供的牡丹PsASMT基因或其载体在改变植物花色中的应用，所述牡丹为任一品种的牡丹。
其中，所述应用包括改变植物花瓣色泽参数a*值。
其中，所述应用包括影响植物花色苷合成。
其中，所述应用包括改变植物花色苷合成相关基因的表达情况，所述相关基因包括CHS1、CHS2、CHI、DFR1、DFR2、ANS1或ANS2中的一种或几种。
其中，所述植物包括烟草。
本发明还提供了一种利用牡丹PsASMT基因改变烟草花色的方法，包括以下步骤：构建牡丹PsASMT基因过量表达载体，转染至烟草(Nicotiana tabacum L.)中，即得改变花色后的烟草。
其中，所述构建牡丹PsASMT基因过量表达载体包括以下步骤：
(1)以牡丹叶片DNA为模板进行PCR扩增，将扩增产物进行凝胶电泳分析，回收含酶切位点的PsASMT大片段；
(2)分别用BamHI和Kpn I对pCAMBIA1301质粒进行双酶切，进行凝胶电泳分析，回收纯化质粒pCAMBIA1301大片段；
(3)用连接酶连接步骤(1)中的PsASMT大片段和步骤(2)中的pCAMBIA1301大片段，得到连接产物；
(4)将步骤(3)中的连接产物转化至感受态细胞，培养，挑取阳性单克隆扩大培养，获得牡丹PsASMT基因过量表达载体pCAMBIA1301-PsASMT。
其中，步骤(1)中所述PCR扩增的引物对序列如SEQ ID NO.5和SEQ ID NO.6所示。
其中，步骤(1)中所述扩增产物的序列如SEQ ID NO.23所示。
其中，步骤(2)中所述双酶切的体系为：CutSmart Buffer、pCAMBIA1301质粒、BamHI、Kpn I和ddH2O。
有益效果：与现有技术相比，本发明具有如下显著优点：本发明通过将构建的PsASMT基因过量表达载体转化到烟草中进行表达，降低了植物，尤其是烟草花瓣中花色苷含量，抑制了花色苷合成相关基因表达，创制了淡粉色烟草新种质。
附图说明
图1为牡丹PsASMT氨基酸序列；
图2为牡丹PsASMT基因和其他植物ASMT基因构建的进化树；
图3为牡丹PsASMT基因cDNA全长扩增结果检测；
图4为牡丹与其他植物ASMT氨基酸序列比对；
图5为野生型和转PsASMT基因的烟草植株花色表型以及花色苷提取液比较；其中，离心管中的液体为花色苷提取液，野生型烟草植株花色为红色；转PsASMT基因的烟草植株花色为淡粉色；
图6为野生型和转PsASMT基因的烟草花瓣色泽参数a*值比较；其中，不同小写字母表示差异显著(p<0.05)；
图7为野生型和转PsASMT基因的烟草花瓣中花色苷含量比较；其中，不同小写字母表示差异显著(p<0.05)；
图8为野生型和转PsASMT基因的烟草花瓣中花色苷合成相关基因的表达水平；其中，CHS为查耳酮合酶基因；CHI为查耳酮异构酶基因；DFR为二氢黄酮醇-4-还原酶基因；ANS为花色素合成酶基因；不同小写字母表示差异显著(p<0.05)。
具体实施方式
下面结合附图对本发明的技术方案作进一步说明。
实施例1牡丹PsASMT基因cDNA全长序列的克隆
PsASMT基因cDNA全长序列的获得：在扬州大学国家芍药种质资源库中选用牡丹‘花王’的花瓣为材料，采用MiniBEST P1ant RNAExtraction Kit(TaKaRa)试剂盒提取总RNA。采用PrimeScriptTMII1st Strand cDNA Synthesis Kit(TaKaRa)反转录生产cDNA的第一条链，然后进行第二轮反转录、PCR扩增。其中，第一轮反转录体系为：1.0μL RNA、1.0μLOligo dT Primer(50μM)、1.0μL dNTP Mixture(10mM each)、7.0μL RNase Free ddH2O；反应条件为：65℃反应5min，于冰上迅速冷却。第二轮反转录体系为：10.0μL第一轮反应液、4.0μL 5×PrimeScript II Buffer、0.5μLRNase Inhibitor(40U/μL)、1.0μLPrimeScriptII RTase(200U/μL)、4.5μLRNase Free ddH2O；反应条件为：42℃反应60min，95℃反应5min，于冰上冷却。PCR扩增体系为：12.5μL 2×PhantaFlash Master Mix(Vazyme)、1.0μLForward Primer(5'-GAGATGGGAGACATAAAG-3'(SEQ ID NO.3)、1.0μLReverse Primer(5'-TGGAAACGCCTCAATTAC-3'(SEQ ID NO.4)、2.0μL第二轮反转录产物、8.5μL ddH2O。其中，引物由擎科生物(Tsingke，北京)合成，浓度为10μM。反应程序：98℃预变性30s；98℃变性10s，60℃退火5s，72℃延伸5s，共35个循环；72℃延伸1min。将PCR产物进行1％琼脂糖凝胶电泳检测，结果见图3(其中，M：DL2000 marker；1：全长扩增产物)，牡丹PsASMT基因的cDNA全长序列为1507bp，具有起始密码子ATG、完整开放阅读框1068bp、终止密码子TAG、5'非编码区67bp、3'非编码区372bp，共编码355个氨基酸。其中，牡丹PsASMT基因的核苷酸序列如SEQID NO.1所示：ATAAATTTGAAGCTACACCTGCTATTATAAACAAGTTAAAGATTCATTTTGAGAGAAAAAGAAAGAGATGGGAGACATAAAGAGAGAAGCAAAGAGTGAAGAAGAAGAAGAAGAAGCAGCAGCAGCCGAAGTTGATATGTGGAGATACATATTCGGGTTTGTGGAAATGGCGGTGGTGAAATGTGGCATTGAGCTTGGGATAGCTGATGCCATTGAAAGCCATGGAAACCCCATGACACTCTCCGACCTATCATCTGCTCTTGGCTGCGTTCCATCTCAACTCCACCGCATCATGAGGTTTCTGGTCCACCGCCGAATATTTAAAGAGGAATCCACAAGCCAAGGTTCCATTTGCTATGTCCAAACGCCACTTTCCAACCGTTTGATGCGACACGGAGAAAAGGGCATGGCTGCTTTTGTGTTGCTAGAGAGCAGCCCAGTGATGCTGGCACCATGGCATGATCTAAGTGCTCGTGTACTAGGTAATGGAGCTTCACCATTTGAGGCTGCTCATGGTAAGGATGTATGGAGCTATGCAGGAGAACATTCTGGGCACAGCAAGCTCTTCAATGACGCAATGGCTTGCGATGCTAGAGTTGTGGTGCCCGCGATAATTAACGGTTGTCCAGAGGTATTAAATGGATTGGGCAGTTTGGTGGATGTGGGTGGGGGTGATGGGACTGCTCTAAGCATCTTGGTCAAGGACTGTCCTTGGATTCGAGGCATCAACTTTGATCTTCCTCATGTTGTTTCCGTTGCACCTAAGTGTGTTCGTGTTGAGCACGTTGGGGGCGACATGTTCGAAACTGTGCCAAAGGCCGATGCGGCTTATCTCATGTGGGTTCTGCATGATTGGGACGATGATGAGTGCATCCAAATCTTGAGAAAATGTAGGGAAGCTATTCCAGAGGACAAAGGGAAAGTGATAATTGTAGAAGCTGTGATCGAA GAAAAGGACAATGATAAGCTTAAGGATGTGAGGCTAATGTTAGACATGGTGATGATGGCTCATACTAACAAAGGCAAGGAGAGGACCTTGAAGGAATGGGCATATGTTCTTGAGGAGGCTGGGTTTAGTCGACACACAGTCAAACCCATTCGCGCGGTGCAATCGGTAATTGAGGCGTTTCCATGATCTATAGACGTTCCCGAATGCAGATTTGTAGCCCAAAGTTTGTTTTCCACAACATGGATCATTCTCTCAATGGTTTGAATAACAGTTTCTATGATAAATTGTACTATATTTGAATAATCGCCTATGATGTAAGGTTATTCCTGCGACCTGGAACCACGAAAACTGTCTCTTCTCATTAGCTAAAGCTGCGTAAAAGTTGTATTAAAACAAAATATAAGGTTAATCCTGTCTTTGCTTTCTCTTTAGTATATGTGTATATTATATATATGATTATGTAAATCATTGTAATATTATTATATTATATTATACTTTGTTGCCTTAAGTGACTATGTGTAGTGTATGTATAAGCTATGCACAGTTTTAAGGACATTA。
实施例2牡丹PsASMT基因推测得到的氨基酸序列与其他植物的序列比对
将牡丹与其他植物中ASMT基因推测得到的氨基酸序列分别保存为TXT文件，再载入DNAMAN5.2.2软件中进行同源性比对。如图1所示，PsASMT基因开放阅读框为1068bp，共编码355个氨基酸；对其他物种ASMT蛋白进行进化树分析，发现牡丹PsASMT与拟南芥AtASMT(AT4G35160)同源性最高(图2)。如图4所示，牡丹和其他植物均具有典型的ASMT保守结构域、S-腺苷-1-蛋氨酸和质子受体。其中，牡丹PsASMT基因编码的蛋白质的氨基酸序列如SEQID NO.2所示：MGDIKREAKSEEEEEEAAAAEVDMWRYIFGFVEMAVVKCGIELGIADAIESHGNPMTLSDLSSALGCVPSQLHRIMRFLVHRRIFKEESTSQGSICYVQTPLSNRLMRHGEKGMAAFVLLESSPVMLAPWHDLSARVLGNGASPFEAAHGKDVWSYAGEHSGHSKLFNDAMACDARVVVPAIINGCPEVLNGLGSLVDVGGGDGTALSILVKDCPWIRGINFDLPHVVSVAPKCVRVEHVGGDMFETVPKADAAYLMWVLHDWDDDECIQILRKCREAIPEDKGKVIIVEAVIEEKDNDKLKDVRLMLDMVMMAHTNKGKERTLKEWAYVLEEAGFSRHTVKPIRAVQSVIEAFP*。
实施例3牡丹PsASMT基因过量表达载体在烟草中的表达
牡丹PsASMT基因过量表达载体构建：设计含有酶切位点BamHI和Kpn I用于扩增PsASMT序列的引物(上游引物：5'-gagaacacgggggacgagctcATGGGAGACATAAAGAGAGAAGCAA-3'(SEQ ID NO.5)，下游引物：5'-gctcaccatgtcgactctagaTGGAAACGCCTCAATTACCG-3'(SEQID NO.6))，引物由擎科生物(Tsingke，北京)合成，浓度为10μM。PCR扩增体系：12.5μL 2×Phanta Flash Master Mix(Vazyme)、1μL Forward Primer、1μLReverse Primer、2μL DNA模板(浓度为500ng/μL，采用MiniBEST Plant Genomic DNA Extraction Kit(TakaRa)试剂盒提取牡丹叶片DNA作为模板)、8.5μL ddH2O。反应程序：98℃预变性30s；98℃变性10s，52℃退火10s，72℃延伸10s，共35个循环；72℃延伸1min。其中，PCR产物的序列如SEQ IDNO.23所示：ATGGGAGACATAAAGAGAGAAGCAAAGAGTGAAGAAGAAGAAGAAGAAGCAGCAGCAGCCGAAGTTGATATGTGGAGATACATATTCGGGTTTGTGGAAATGGCGGTGGTGAAATGTGGCATTGAGCTTGGGATAGCTGATGCCATTGAAAGCCATGGAAACCCCATGACACTCTCCGACCTATCATCTGCTCTTGGCTGCGTTCCATCTCAACTCCACCGCATCATGAGGTTTCTGGTCCACCGCCGAATATTTAAAGAGGAATCCACAAGCCAAGGTTCCATTTGCTATGTCCAAACGCCACTTTCCAACCGTTTGATGCGACACGGAGAAAAGGGCATGGCTGCTTTTGTGTTGCTAGAGAGCAGCCCAGTGATGCTGGCACCATGGCATGATCTAAGTGCTCGTGTACTAGGTAATGGAGCTTCACCATTTGAGGCTGCTCATGGTAAGGATGTATGGAGCTATGCAGGAGAACATTCTGGGCACAGCAAGCTCTTCAATGACGCAATGGCTTGCGATGCTAGAGTTGTGGTGCCCGCGATAATTAACGGTTGTCCAGAGGTATTAAATGGATTGGGCAGTTTGGTGGATGTGGGTGGGGGTGATGGGACTGCTCTAAGCATCTTGGTCAAGGACTGTCCTTGGATTCGAGGCATCAACTTTGATCTTCCTCATGTTGTTTCCGTTGCACCTAAGTGTGTTCGTGTTGAGCACGTTGGGGGCGACATGTTCGAAACTGTGCCAAAGGCCGATGCGGCTTATCTCATGTGGGTTCTGCATGATTGGGACGATGATGAGTGCATCCAAATCTTGAGAAAATGTAGGGAAGCTATTCCAGAGGACAAAGGGAAAGTGATAATTGTAGAAGCTGTGATCGAAGAAAAGGACAATGATAAGCTTAAGGATGTGAGGCTAATGTTAGACATGGTGATGATGGCTCATACTAACAAAGGCAAGGAGAGGACCTTGAAGGAATGGGCATATGTTCTTGAGGAGGCTGGGTTTAGTCGACACACAGTCAAACCCATTCGCGCGGTGCAATCGGTAATTGAGGCGTTTCCATGA。
反应结束后将PCR产物进行1％琼脂糖凝胶电泳分析，并采用DNAGel ExtractionKit(Tsingke)凝胶回收试剂盒回收含酶切位点的PsASMT大片段。取双元表达载体pCAMBIA1301质粒用BamH I和Kpn I(NEB)进行双酶切，反应体系为：2.0μL10×CutSmartBuffer、7μLpCAMBIA1301质粒、0.4μLBamHI(20000U/mL)、0.4μL Kpn I(20000U/mL)、10.2μLddH2O；37℃反应1h。双酶切产物经过琼脂糖凝胶电泳分析，采用DNA Gel Extraction Kit(Tsingke)凝胶回收试剂盒回收纯化质粒pCAMBIA1301大片段。用plus One stepPCR Cloning Kit(Novoprotein)试剂盒采用同源重组的方法连接两个回收的产物，反应体系为：2μL 5×反应缓冲液、0.5μL/>plus重组酶、4μLpCAMBIA1301大片段、1μLPsASMT大片段；在50℃金属浴连接15min后置于冰上冷却，连接产物转化TreliefTM5α感受态细胞(Tsingke)，而后在LB平板上(含有Kan 50mg/L)37℃过夜培养，挑取阳性单克隆扩大培养，进行测序验证，直至pCAMBIA1301-PsASMT过量表达载体构建成功。
牡丹PsASMT基因过量表达载体pCAMBIA1301-PsASMT转化烟草：取5μLpCAMBIA1301-PsASMT过量表达载体质粒转化GV3101(pSoup-p19)感受态细胞(TOLOBIO)，而后在YEB平板上(含有利福平Rif50 mg/L和卡那霉素Kan 50mg/L)28℃培养2d。挑取阳性单克隆于YEB液体培养基中(含有Rif50 mg/L和Kan 50mg/L)，28℃，200rpm培养过夜。取所摇菌液2mL，加到50mL含有相同抗生素的YEB液体培养基(含有Rif50 mg/L和Kan 50mg/L)中，在相同条件下培养至OD600＝0.4。将摇好的菌倒入50mL离心管中，室温离心5000rpm、10min，弃上清备用。向离心管中加入5mLMS0(MS0液体基本培养基，无琼脂和蔗糖；Solarbio，M8521，北京)将溶解菌体，用枪打匀，倒入加有400μL乙酰丁香酮AS(20mg/mL)的小三角瓶中，然后加MS0至50mL。取武汉伯远生物科技有限公司提供的‘K326’烟草(Nicotianatabacum L.)的无菌苗叶片，切成小块(约1cm×1cm)，共切100-150片叶片，放入加有50mLMS0(MS0液体基本培养基，无琼脂和蔗糖)的小三角瓶中，将叶片倒入覆有纱布的烧杯，过滤掉MS0液体培养基，取经过滤后的叶片加入50mLMS0+乙酰丁香酮AS的小三瓶中，侵染8min，侵染时不断轻轻摇动；侵染完成后，滤去菌液，取出叶片，用无菌滤纸吸干叶片表面多余的菌液，将叶片接种于共培养培养基(MS0+3.0mg/L 6-BA+0.1mg/LNAA+30g/L蔗糖+6.66％琼脂，其中6-BA为6-苄氨基嘌呤，NAA为萘乙酸)中，暗培养3d；共培养结束后，转入抗性芽筛选分化培养基(MS0+3.0mg/L 6-BA+0.1mg/LNAA+30g/L蔗糖+6.66％琼脂+100mg/L羧苄青霉素Cb+25mg/L潮霉素Hyg)中进行选择培养，两周继代一次，直到分化出芽；当分生不定芽达2cm以上时，切下不定芽，转入生根筛选培养基(1/2MS培养基(Solarbio，M8526)+0.3mg/L IBA+30g/L蔗糖+6.66％琼脂+50mg/L Cb+8mg/L Hyg)进行生根筛选。经过4-6个月的培养，可获得转PsASMT基因烟草。
转PsASMT基因烟草植株花色表型鉴定：对转PsASMT基因烟草进行培养，培养条件为：28℃16h/23℃8h。结果见图5(1和2为2个重复实验)，在烟草植株花朵开放后，观察到野生型烟草花瓣为红色，而转PsASMT基因烟草花瓣为淡粉色，表明过量表达PsASMT基因具有改变植物花色的功能。
实施例4烟草植株花瓣色泽相关性指标测定
烟草植株花瓣色泽参数a*值测定：采用便携式成像分光色差仪(RM 200QC，美国爱色丽公司)分别测定野生型烟草和转PsASMT基因烟草的花瓣色泽参数a*值。与野生型烟草相比，转PsASMT基因烟草花瓣具有显著较低的a*值，表明转PsASMT基因烟草花瓣色泽较浅(图6)。
烟草植株花瓣中花色苷含量测定：分别称取1.0g野生型烟草和转PsASMT基因烟草的新鲜花瓣，用液氮将花瓣研磨成粉状，用6mL的提取液(CH3OH、ddH2O和HCl的体积比为70:29.9:0.1)溶解，遮光条件下在4℃恒温振荡24h进行花色苷提取，然后用离心机以12,000rpm的转速离心10min，吸取上清液，通过0.22μm微孔滤膜过滤，采用分光光度计测定520nm下的吸光度。从图7可以看出，与野生型烟草相比，转PsASMT基因烟草花瓣中花色苷含量显著较低，表明芍药PsASMT基因影响了花色苷合成。
烟草植株花瓣中花色苷合成相关基因的表达水平检测：分别以野生型烟草和转PsASMT基因烟草的花瓣为材料，采用RNAiso Plus(Total RNA提取)(TaKaRa)试剂盒来提取总RNA，并采用PrimerScriptTM RT reagent Kit with gDNAEraser(TaKaRa)试剂盒将总RNA反转录成cDNA，反应体系为：1.0μLRNA、2.0μL 5×gDNAEraser Buffer、1.0μL gDNA Eraser和6.0μL RNase Free dH2O；反应条件为：42℃反应2min。反应完成后，再在第一步的反应液中依次加入4.0μL 5×Buffer 2(for Real Time)、1.0μLRTPrimer Mix、4.0μLRNase Free dH2O和1.0μL/>RT Enzyme Mix I；反应条件为：37℃反应15min，85℃反应5s。将反转录所获得的cDNA采用/>Tip Green qPCR SuperMix试剂盒(Trans)进行qRT-PCR检测，以烟草Actin(AB158612)为内参基因(Actin-F：5'-TCCTCATGCAATTCTTCG-3'(SEQ ID NO.7)；Actin-R：5'-ACCTGCCCATCTGGTAAC-3'(SEQ IDNO.8))，检测花色苷合成相关基因CHS1(NM_001325705.1)、CHS2(XM_016634418.1)、CHI(AB213651.1)、DFR1(EF421429.1)、DFR2(EF421430.1)、ANS1(JQ866630.1)和ANS2(JQ866631.1)的表达水平；上述7种基因的反应体系均为：2.0μL cDNA(1000ng/μL)、12.5μL2×/>Tip Green qPCR、上游引物与下游引物各1.0μL(10mM)、8.5μL ddH2O；反应程序均为：94℃预变性30s，然后94℃变性5s，52℃退火30s，72℃延伸30s，反应45个循环，溶解曲线65℃-95℃，每5s升温0.5℃；它们的特异性引物分别为：CHS1-F：5'-CAAACTCTTCTCCCCGAT-3'(SEQ ID NO.9)、CHS1-R：5'-CCACAAGGCTTTTCTCAAT-3'(SEQ IDNO.10)；CHS2-F：5'-CCAAGATTACCCATTTAGTC-3'(SEQ ID NO.11)、CHS2-R：5'-CTTAGCCAATCGGAGAAC-3'(SEQ ID NO.12)；CHI-F：5'-GTGCCTCCATTCTTTTTAC-3'(SEQ IDNO.13)、CHI-R：5'-GCGATACTACACTTTGCTG-3'(SEQ ID NO.14)；DFR1-F：5'-GATACTGGCAGAGAAGGC-3'(SEQ ID NO.15)、DFR1-R：5'-AGTGAAAGGGCAGTGATT-3'(SEQ IDNO.16)；DFR2-F：5'-GTTTTCACTTCATCGGCT-3'(SEQ ID NO.17)、DFR2-R：5'-CCATCCTGTCATCTTCTTAG-3'(SEQ ID NO.18)；ANS1-F：5'-GCAAATAGTGCTTGTGGT-3'(SEQ IDNO.19)、ANS1-R：5'-TGCTGGAATGTAGTCTGTAG-3'(SEQ ID NO.20)；ANS2-F：5'-TCCAGGCTATCCCTAAAG-3'(SEQ ID NO.21)、ANS2-R：5'-CATAACACCCCACTCCAT-3'(SEQ IDNO.22)。采用公式2-△△CT法计算基因的相对表达量。从图8可以看出，与野生型烟草相比，CHS1、CHS2、CHI、DFR1、DFR2、ANS1、ANS2在转PlASMT基因烟草花瓣中的表达水平较低。
综上所述，本发明提供了一种芍药PsASMT基因在改变植物花色中的应用，通过将构建的PsASMT基因过量表达载体转化到烟草中进行表达，降低了烟草花瓣中花色苷含量，抑制了花色苷合成相关基因表达，创制了淡粉色烟草新种质。
Claims (10)
1.牡丹PsASMT基因或其载体在改变植物花色中的应用。
2.根据权利要求1所述的应用，其特征在于，所述应用包括改变植物花瓣色泽参数a*值。
3.根据权利要求1所述的应用，其特征在于，所述应用包括影响植物花色苷合成。
4.根据权利要求1所述的应用，其特征在于，所述应用包括改变植物花色苷合成相关基因的表达情况，所述相关基因包括CHS1、CHS2、CHI、DFR1、DFR2、ANS1或ANS2中的一种或几种。
5.根据权利要求1～4任一项所述的应用，其特征在于，所述植物包括烟草。
6.一种利用牡丹PsASMT基因改变烟草花色的方法，其特征在于，包括以下步骤：构建牡丹PsASMT基因过量表达载体，转染至烟草中，即得改变花色后的烟草。
7.根据权利要求6所述的方法，其特征在于，所述构建牡丹PsASMT基因过量表达载体包括以下步骤：
(1)以牡丹叶片DNA为模板进行PCR扩增，将扩增产物进行凝胶电泳分析，回收含酶切位点的PsASMT大片段；
(2)分别用BamHI和Kpn I对pCAMBIA1301质粒进行双酶切，进行凝胶电泳分析，回收纯化质粒pCAMBIA1301大片段；
(3)用连接酶连接步骤(1)中的PsASMT大片段和步骤(2)中的pCAMBIA1301大片段，得到连接产物；
(4)将步骤(3)中的连接产物转化至感受态细胞，培养，挑取阳性单克隆扩大培养，获得牡丹PsASMT基因过量表达载体pCAMBIA1301-PsASMT。
8.根据权利要求7所述的方法，其特征在于，步骤(1)中所述PCR扩增的引物对序列如SEQ ID NO.5和SEQ ID NO.6所示。
9.根据权利要求7所述的方法，其特征在于，步骤(1)中所述扩增产物的序列如SEQ IDNO.23所示。
10.根据权利要求7所述的方法，其特征在于，步骤(2)中所述双酶切的体系为：CutSmart Buffer、pCAMBIA1301质粒、BamHI、Kpn I和ddH2O。
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