CN114774427B - 一种提高金银花中木犀草素含量的重组基因及其应用 - Google Patents
一种提高金银花中木犀草素含量的重组基因及其应用 Download PDFInfo
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Abstract
本发明提供了一种提高金银花中木犀草素含量的重组基因及其应用，涉及基因工程领域，本发明提供一种提高金银花中木犀草素含量的重组基因及其应用，所述重组基因的核苷酸序列如SEQ ID No 2所示。本发明还提供一种提高金银花中木犀草素含量的方法，所述方法包括：构建重组质粒，所述重组质粒包含核苷酸序列如SEQ ID No 2所示的重组基因；采用农杆菌介导法将重组质粒转入到金银花叶片中，进行培养，获得目的植株。将含有目的基因的重组质粒在金银花中表达，能够明显提高金银花叶片中木犀草素含量，现对于未处理的金银花，使得金银花叶片中木犀草素含量提高了109.7％。
Description
技术领域
本发明涉及基因工程领域，具体而言，涉及一种提高金银花中木犀草素含量的重组基因及其应用。
背景技术
金银花是常用名贵中药，为忍冬科植物忍冬(Lonicera japonica Thunb)的干燥花蕾及初开的花，其性甘寒、味甘气芳香，自古以来就被誉为清热解毒、疏风散热的良药；现代药理学研究表明其提取物具有抗菌、抗炎、抗病毒、抗氧化、保肝、抗肿瘤等多种药理活性。金银花含有的绿原酸和木犀草苷是衡量金银花品质的指标性成分，木犀草素是木犀草苷的前体物质。
近年来，调控植物积累特异黄酮醇的关键酶及转录因子的研究在拟南芥、矮牵牛、苹果、柑橘等重要模式植物或园艺植物中取得很大进展。但由于金银花物种的基因组信息少，关于金银花活性成分生物合成途径的报道尚不多见。金银花以花蕾和初开的花入药，研究发现金银花中黄酮类化合物在花生育期含量差异变化很大，给金银花药材高品质采收带来困难。
基于次，通过转基因金银花的表基因表达提高金银花中黄酮类化合物的含量是亟需发展的技术。
发明内容
本发明的目的在于提供一种提高金银花中木犀草素含量的重组基因及其应用，解决背景所提出的至少一个技术问题。
本发明提供一种提高金银花中木犀草素含量的重组基因，所述重组基因的核苷酸序列如SEQ ID No.2所示。
本发明还提供一种上述的重组基因编码的蛋白质。
本发明还提供一种上述的重组基因的植物表达载体。
本发明还提供一种上述的重组基因在提高金银花中木犀草素含量方面的应用。
本发明还提供一种重组基因的植物表达载体在提高金银花中木犀草素含量方面的应用。
本发明还提供一种提高金银花中木犀草素含量的方法，所述方法包括：
构建重组质粒，所述重组质粒包含核苷酸序列如SEQ ID No.2所示的重组基因；
采用农杆菌介导法将重组质粒转入到金银花叶片中，进行培养，获得目的植株。
优选的，构建重组质粒的步骤为：
提取金银花RNA，反转录获得cDNA，以cDNA为模板，设计引物，在MYB12基因的上下游均引入KpnI酶切位点，利用高保真酶从cDNA模板中扩增MYB12基因序列，回收扩增出来的含有MYB12基因的PCR产物；
使用KpnI内切酶将PCambia2301-JC酶切成线性化载体，用同源重组法将MYB12基因与线性化PCambia2301-JC载体连接，热激法将重组载体导入大肠杆菌DH5ɑ感受态，菌液PCR检测阳性菌落，提取阳性质粒，测序后获得含有目的基因的重组质粒。
优选的，将构建成功的重组质粒导入农杆菌中，进行培养；
将培养好的农杆菌在室温下离心，弃掉废液，加入渗透培养基重悬菌体，室温下搅拌；
渗透培养基重悬后的农杆菌，注射到金银花叶片中。
技术效果：
将含有目的基因的重组质粒在金银花中表达，能够明显提高金银花叶片中木犀草素含量，现对于未处理的金银花，使得金银花叶片中木犀草素含量提高了109.7％。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为本发明实施例pCambia1301-JC载体质粒图谱；
图2为本发明实施例检测目的基因的表达量的结果图；
图3为本发明实施例检测木犀草素含量的变化图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，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。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
本发明实施例，结合多项研究表明黄酮类化合物生物合成主要受到MYB家族转录因子的调控作用。拟南芥中调控花青苷合成的MYBs主要为MYBA、MYB1、MYB10、MYB110、MYB75和MYB90等转录因子成员。除了作为激活子，MYB也可能作为抑制子参与黄酮生物合成的调控。在烟草中过量表达草莓FaMYB1，会抑制黄酮醇积累，同时也抑制花青苷积累。在拟南芥中过量表达大豆GmMYB100，其转基因植株黄酮含量显著降低。除了MYB外，其他转录因子参与黄酮生物合成的调控也陆续见报道。玉米R2R3型MYB转录因子C1和bHLH转录因子LC需要通过形成MYB-bHLH转录复合体来调控黄酮合成，在番茄中同时过量表达C1和LC能提高果实山柰酚含量，而C1或LC单个转录因子的转基因植株却不能显著提高黄酮含量。拟南芥中属于bHLH转录因子家族的TCP3能与AtMYB12和AtMYB111进行蛋白互作，转基因mTCP3植株黄酮含量改变显著。
本发明实施例，通过以下实施例将含有目的基因的重组质粒在金银花中表达，用于提高金银花叶片中木犀草素含量：
载体构建和表达的过程
使用RNA提取试剂盒(AG公司)提取金银花RNA，使用反转录试剂盒(AG公司)获得cDNA，以cDNA为模板，设计引物，在MYB12基因的上下游均引入KpnI酶切位点，利用高保真酶从cDNA模板中扩增MYB12基因序列，回收扩增出来的含有MYB12基因的PCR产物。使用KpnI内切酶将PCambia2301-JC酶切成线性化载体，用同源重组法将MYB12基因与线性化PCambia2301-JC载体连接，热激法将重组载体导入大肠杆菌DH5ɑ感受态，菌液PCR检测阳性菌落，提取阳性质粒，测序后获得含有目的基因的重组质粒。
具体的，使用RNA提取试剂盒(AG公司)提取金银花RNA，反转录获得cDNA，利用高保真酶从cDNA模板中扩增MYB12基因序列，其核苷酸序列如SEQ ID No.1所示。
正向引物：5’-ATGGGTAGAGCACCTTGTT-3’
反向引物：5’-TTATCCTTGAGACAAATTTCCTA-3’
PCR扩增体系为：
反应程序：95℃变性3min；95℃15S，52℃30S，72℃1min，35个循环；72℃延伸5min，4℃保存。PCR产物经电泳、回收、测序后-20℃保存备用。
利用SteadyPure DNA凝胶回收试剂盒(AG公司)对已连接同源臂的MYB12基因进行胶回收。利用内切酶BamHI对PCR纯化产物和pCambia1301-JC质粒进行酶切，酶切体系为：
pCambia1301-JC载体质粒图谱如图1所示。
使用同源重组法将目的基因与载体pCambia1301-JC连接，反应体系为：
上述反应液使用移液器轻轻吸打混匀，短暂离心将反应液收集至管底。放置于37℃反应30min，随后立即置于冰上冷却。
重组质粒基因序列，如SEQ ID No.2所示。
重组质粒热激法导入大肠杆菌中：
转化步骤：
(1)在100μl大肠杆菌感受态细胞中加入10μL连接产物；
(2)冰浴30min；
(3)42℃热激60-90s；
(4)冰浴2min；
(5)加入800μl LB液体培养基；
(6)37℃摇床培养30min；
(7)6000rpm离心3min，弃上清，涂布Kana(50mg/L)抗性培养基平板；
(8)37℃倒置培养12-16h后，挑取抗性菌落；
(9)在96孔板中，每孔加入100μL LB(含Kana)液体培养基；
(10)各平板取4-8个菌落，37℃，2h，180rpm扩大培养；
(11)取1μL菌液进行PCR阳性检测。
挑取PCR阳性的转化子摇菌培养提取质粒，同时扩增产物送测序。扩增和测序引物为插入目的基因两侧的载体序列，分别为：
35S-F：5’-GACGCACAATCCCACTATCC-3’
2301-F：5’-GCTTCCGGCTCGTATGTTG-3’。
经菌液PCR扩增、电泳、测序后获得含有目的基因的重组质粒。
将构建成功的重组载体导入农杆菌中，农杆菌选择为农杆菌GV3101：
1、在冰上解冻感受态大肠杆菌后加入10μl重组质粒，轻弹混匀；
2、冰上静置5min，液氮中静置5min；
3、37℃水浴5min，冰浴5min；
4、加1ml LB培养基至离心管中，37℃，200rpm培养1.5h；
5、8000r/min离心1min，去除900μl上清液，重悬菌体；
6、涂布至含有Kana+Rif+Gen的LB平板中；
7、28℃倒置培养2-3d后，挑取抗性菌落；
8、28℃，1d，200rpm扩大培养；
9、取1μL菌液进行PCR阳性检测。
农杆菌侵染金银花叶片
1、取300μl菌液至30ml LB液体培养基中，28℃，200rpm培养至OD600＝0.6。
2、将培养好的农杆菌倒入50ml离心管中，室温8000rpm离心10min，弃掉废液，加入渗透培养基重悬菌体至OD600＝0.6，室温下搅拌2-3h；
3、渗透培养基重悬后的农杆菌，并注射到金银花叶片中，暗培养12-24小时后转正常培养，2天后取样，液氮冻存样本后，-80℃保存；
渗透培养基：MS、10mM MES(2-(N-吗啡啉)乙磺酸)、10mM MgCl2和100μM AC(乙酰丁香酮)
样本检测
使用RNA提取试剂盒(AG公司)提取金银花RNA，反转录获得cDNA，使用荧光定量检测试剂盒(AG公司)检测目的基因的表达量，结果如图2所示，目的基因表达量上升
木犀草素提取
1、105℃，15min杀青样本，50℃烘至恒重
2、使用研钵和研棒将样本研磨成粉末
3、称取0.1g粉末，加入20ml甲醇
4、超声提取30min
旋蒸后使用1ml甲醇复溶，所得溶液进行HPLC检测，如图3所示，木犀草素含量增加：
其中，对照为常规栽培的金银花；本发明上述是合理将目的基因注射到金银花叶片后目的基因表达量增加了3154.8％，木犀草素含量增加了109.7％。
以上描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等同物界定。
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序列表
<110> 浙江理工大学
<120> 一种提高金银花中木犀草素含量的重组基因及其应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 987
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgggtagag caccttgttg cgataaagtg gggctgaaga aaggaagatg gacagctgaa 60
gaagatgaga ctttgactaa atacattcaa gcccatggtg aaggctcttg gaggtcttta 120
cccaagaatg caggtttgtt aaggtgcgga aagagttgca gactcagatg gataaactat 180
ttgagagccg acttgaagag agggaacatc actgctgaag aagaagatct tattgtgcaa 240
ttgcatagat ccatgggaaa taggtggtct ttgatagctg gccatttacc cgggcgaaca 300
gataatgaga taaagaatta ctggaactct tatttaagca gaaaagttag cagcatcttt 360
acgccgacca atccatcact gccaacaagc gccatcagcc ccgtcagtcc ttcacacaag 420
cgtaaagttg gccgagtgag ccgttcagta gctagaaagt acaacaacaa aagctcccac 480
cgactctttg gtgaaatccc aatgagaaac tcaaatgatg ctgatcccca aaactgggcg 540
ccggtgcagc ccgtggccac agtaaatgaa aacccaccaa tgggttcagt gcctgtggag 600
cagagtgagg agaaagacaa gggtgcagtt atgttgggac catttgaaga gatgaatgat 660
caggtgatgg gactcaacta tttgctggag ggtgggtctg gggaatcaag tggggttaag 720
taccttaatg aggagaaaga aaatgcacta atgggtgtgg acagtagtac agtgataagt 780
aatgatgaga gagaaagtgg tggttggggg tcaagttgtg ggattggggt gtcatttggg 840
tgttcttctt ctcccatgaa tgttgcatgc tttggttttg agtgggatga tcaagatctt 900
gcattttgtg atgaagggga atacgcgttg ctttggccat gggaggagag tgacactttg 960
gaggtaggaa atttgtctca aggataa 987
<210> 2
<211> 12836
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gatctgaggg taaatttcta gtttttctcc ttcattttct tggttaggac ccttttctct 60
ttttattttt ttgagctttg atctttcttt aaactgatct attttttaat tgattggtta 120
tggtgtaaat attacatagc tttaactgat aatctgatta ctttatttcg tgtgtctatg 180
atgatgatga tagttacaga accgacgact cgtccgtcct gtagaaaccc caacccgtga 240
aatcaaaaaa ctcgacggcc tgtgggcatt cagtctggat cgcgaaaact gtggaattga 300
tcagcgttgg tgggaaagcg cgttacaaga aagccgggca attgctgtgc caggcagttt 360
taacgatcag ttcgccgatg cagatattcg taattatgcg ggcaacgtct ggtatcagcg 420
cgaagtcttt ataccgaaag gttgggcagg ccagcgtatc gtgctgcgtt tcgatgcggt 480
cactcattac ggcaaagtgt gggtcaataa tcaggaagtg atggagcatc agggcggcta 540
tacgccattt gaagccgatg tcacgccgta tgttattgcc gggaaaagtg tacgtatcac 600
cgtttgtgtg aacaacgaac tgaactggca gactatcccg ccgggaatgg tgattaccga 660
cgaaaacggc aagaaaaagc agtcttactt ccatgatttc tttaactatg ccggaatcca 720
tcgcagcgta atgctctaca ccacgccgaa cacctgggtg gacgatatca ccgtggtgac 780
gcatgtcgcg caagactgta accacgcgtc tgttgactgg caggtggtgg ccaatggtga 840
tgtcagcgtt gaactgcgtg atgcggatca acaggtggtt gcaactggac aaggcactag 900
cgggactttg caagtggtga atccgcacct ctggcaaccg ggtgaaggtt atctctatga 960
actcgaagtc acagccaaaa gccagacaga gtctgatatc tacccgcttc gcgtcggcat 1020
ccggtcagtg gcagtgaagg gccaacagtt cctgattaac cacaaaccgt tctactttac 1080
tggctttggt cgtcatgaag atgcggactt acgtggcaaa ggattcgata acgtgctgat 1140
ggtgcacgac cacgcattaa tggactggat tggggccaac tcctaccgta cctcgcatta 1200
cccttacgct gaagagatgc tcgactgggc agatgaacat ggcatcgtgg tgattgatga 1260
aactgctgct gtcggctttc agctgtcttt aggcattggt ttcgaagcgg gcaacaagcc 1320
gaaagaactg tacagcgaag aggcagtcaa cggggaaact cagcaagcgc acttacaggc 1380
gattaaagag ctgatagcgc gtgacaaaaa ccacccaagc gtggtgatgt ggagtattgc 1440
caacgaaccg gatacccgtc cgcaaggtgc acgggaatat ttcgcgccac tggcggaagc 1500
aacgcgtaaa ctcgacccga cgcgtccgat cacctgcgtc aatgtaatgt tctgcgacgc 1560
tcacaccgat accatcagcg atctctttga tgtgctgtgc ctgaaccgtt attacggatg 1620
gtatgtccaa agcggcgatt tggaaacggc agagaaggta ctggaaaaag aacttctggc 1680
ctggcaggag aaactgcatc agccgattat catcaccgaa tacggcgtgg atacgttagc 1740
cgggctgcac tcaatgtaca ccgacatgtg gagtgaagag tatcagtgtg catggctgga 1800
tatgtatcac cgcgtctttg atcgcgtcag cgccgtcgtc ggtgaacagg tatggaattt 1860
cgccgatttt gcgacctcgc aaggcatatt gcgcgttggc ggtaacaaga aagggatctt 1920
cactcgcgac cgcaaaccga agtcggcggc ttttctgctg caaaaacgct ggactggcat 1980
gaacttcggt gaaaaaccgc agcagggagg caaacaagct agccaccacc accaccacca 2040
cgtgtgaatt acaggtgacc agctcgaatt tccccgatcg ttcaaacatt tggcaataaa 2100
gtttcttaag attgaatcct gttgccggtc ttgcgatgat tatcatataa tttctgttga 2160
attacgttaa gcatgtaata attaacatgt aatgcatgac gttatttatg agatgggttt 2220
ttatgattag agtcccgcaa ttatacattt aatacgcgat agaaaacaaa atatagcgcg 2280
caaactagga taaattatcg cgcgcggtgt catctatgtt actagatcgg gaattaaact 2340
atcagtgttt gacaggatat attggcgggt aaacctaaga gaaaagagcg tttattagaa 2400
taacggatat ttaaaagggc gtgaaaaggt ttatccgttc gtccatttgt atgtgcatgc 2460
caaccacagg gttcccctcg ggatcaaagt actttgatcc aacccctccg ctgctatagt 2520
gcagtcggct tctgacgttc agtgcagccg tcttctgaaa acgacatgtc gcacaagtcc 2580
taagttacgc gacaggctgc cgccctgccc ttttcctggc gttttcttgt cgcgtgtttt 2640
agtcgcataa agtagaatac ttgcgactag aaccggagac attacgccat gaacaagagc 2700
gccgccgctg gcctgctggg ctatgcccgc gtcagcaccg acgaccagga cttgaccaac 2760
caacgggccg aactgcacgc ggccggctgc accaagctgt tttccgagaa gatcaccggc 2820
accaggcgcg accgcccgga gctggccagg atgcttgacc acctacgccc tggcgacgtt 2880
gtgacagtga ccaggctaga ccgcctggcc cgcagcaccc gcgacctact ggacattgcc 2940
gagcgcatcc aggaggccgg cgcgggcctg cgtagcctgg cagagccgtg ggccgacacc 3000
accacgccgg ccggccgcat ggtgttgacc gtgttcgccg gcattgccga gttcgagcgt 3060
tccctaatca tcgaccgcac ccggagcggg cgcgaggccg ccaaggcccg aggcgtgaag 3120
tttggccccc gccctaccct caccccggca cagatcgcgc acgcccgcga gctgatcgac 3180
caggaaggcc gcaccgtgaa agaggcggct gcactgcttg gcgtgcatcg ctcgaccctg 3240
taccgcgcac ttgagcgcag cgaggaagtg acgcccaccg aggccaggcg gcgcggtgcc 3300
ttccgtgagg acgcattgac cgaggccgac gccctggcgg ccgccgagaa tgaacgccaa 3360
gaggaacaag catgaaaccg caccaggacg gccaggacga accgtttttc attaccgaag 3420
agatcgaggc ggagatgatc gcggccgggt acgtgttcga gccgcccgcg cacgtctcaa 3480
ccgtgcggct gcatgaaatc ctggccggtt tgtctgatgc caagctggcg gcctggccgg 3540
ccagcttggc cgctgaagaa accgagcgcc gccgtctaaa aaggtgatgt gtatttgagt 3600
aaaacagctt gcgtcatgcg gtcgctgcgt atatgatgcg atgagtaaat aaacaaatac 3660
gcaaggggaa cgcatgaagg ttatcgctgt acttaaccag aaaggcgggt caggcaagac 3720
gaccatcgca acccatctag cccgcgccct gcaactcgcc ggggccgatg ttctgttagt 3780
cgattccgat ccccagggca gtgcccgcga ttgggcggcc gtgcgggaag atcaaccgct 3840
aaccgttgtc ggcatcgacc gcccgacgat tgaccgcgac gtgaaggcca tcggccggcg 3900
cgacttcgta gtgatcgacg gagcgcccca ggcggcggac ttggctgtgt ccgcgatcaa 3960
ggcagccgac ttcgtgctga ttccggtgca gccaagccct tacgacatat gggccaccgc 4020
cgacctggtg gagctggtta agcagcgcat tgaggtcacg gatggaaggc tacaagcggc 4080
ctttgtcgtg tcgcgggcga tcaaaggcac gcgcatcggc ggtgaggttg ccgaggcgct 4140
ggccgggtac gagctgccca ttcttgagtc ccgtatcacg cagcgcgtga gctacccagg 4200
cactgccgcc gccggcacaa ccgttcttga atcagaaccc gagggcgacg ctgcccgcga 4260
ggtccaggcg ctggccgctg aaattaaatc aaaactcatt tgagttaatg aggtaaagag 4320
aaaatgagca aaagcacaaa cacgctaagt gccggccgtc cgagcgcacg cagcagcaag 4380
gctgcaacgt tggccagcct ggcagacacg ccagccatga agcgggtcaa ctttcagttg 4440
ccggcggagg atcacaccaa gctgaagatg tacgcggtac gccaaggcaa gaccattacc 4500
gagctgctat ctgaatacat cgcgcagcta ccagagtaaa tgagcaaatg aataaatgag 4560
tagatgaatt ttagcggcta aaggaggcgg catggaaaat caagaacaac caggcaccga 4620
cgccgtggaa tgccccatgt gtggaggaac gggcggttgg ccaggcgtaa gcggctgggt 4680
tgtctgccgg ccctgcaatg gcactggaac ccccaagccc gaggaatcgg cgtgacggtc 4740
gcaaaccatc cggcccggta caaatcggcg cggcgctggg tgatgacctg gtggagaagt 4800
tgaaggccgc gcaggccgcc cagcggcaac gcatcgaggc agaagcacgc cccggtgaat 4860
cgtggcaagc ggccgctgat cgaatccgca aagaatcccg gcaaccgccg gcagccggtg 4920
cgccgtcgat taggaagccg cccaagggcg acgagcaacc agattttttc gttccgatgc 4980
tctatgacgt gggcacccgc gatagtcgca gcatcatgga cgtggccgtt ttccgtctgt 5040
cgaagcgtga ccgacgagct ggcgaggtga tccgctacga gcttccagac gggcacgtag 5100
aggtttccgc agggccggcc ggcatggcca gtgtgtggga ttacgacctg gtactgatgg 5160
cggtttccca tctaaccgaa tccatgaacc gataccggga agggaaggga gacaagcccg 5220
gccgcgtgtt ccgtccacac gttgcggacg tactcaagtt ctgccggcga gccgatggcg 5280
gaaagcagaa agacgacctg gtagaaacct gcattcggtt aaacaccacg cacgttgcca 5340
tgcagcgtac gaagaaggcc aagaacggcc gcctggtgac ggtatccgag ggtgaagcct 5400
tgattagccg ctacaagatc gtaaagagcg aaaccgggcg gccggagtac atcgagatcg 5460
agctagctga ttggatgtac cgcgagatca cagaaggcaa gaacccggac gtgctgacgg 5520
ttcaccccga ttactttttg atcgatcccg gcatcggccg ttttctctac cgcctggcac 5580
gccgcgccgc aggcaaggca gaagccagat ggttgttcaa gacgatctac gaacgcagtg 5640
gcagcgccgg agagttcaag aagttctgtt tcaccgtgcg caagctgatc gggtcaaatg 5700
acctgccgga gtacgatttg aaggaggagg cggggcaggc tggcccgatc ctagtcatgc 5760
gctaccgcaa cctgatcgag ggcgaagcat ccgccggttc ctaatgtacg gagcagatgc 5820
tagggcaaat tgccctagca ggggaaaaag gtcgaaaagg tctctttcct gtggatagca 5880
cgtacattgg gaacccaaag ccgtacattg ggaaccggaa cccgtacatt gggaacccaa 5940
agccgtacat tgggaaccgg tcacacatgt aagtgactga tataaaagag aaaaaaggcg 6000
atttttccgc ctaaaactct ttaaaactta ttaaaactct taaaacccgc ctggcctgtg 6060
cataactgtc tggccagcgc acagccgaag agctgcaaaa agcgcctacc cttcggtcgc 6120
tgcgctccct acgccccgcc gcttcgcgtc ggcctatcgc ggccgctggc cgctcaaaaa 6180
tggctggcct acggccaggc aatctaccag ggcgcggaca agccgcgccg tcgccactcg 6240
accgccggcg cccacatcaa ggcaccctgc ctcgcgcgtt tcggtgatga cggtgaaaac 6300
ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc 6360
agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc 6420
cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg 6480
tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc 6540
gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 6600
ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 6660
acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 6720
cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 6780
caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 6840
gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 6900
tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 6960
aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 7020
ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 7080
cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 7140
tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc 7200
tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 7260
ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 7320
aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 7380
aagggatttt ggtcatgcat tctaggtact aaaacaattc atccagtaaa atataatatt 7440
ttattttctc ccaatcaggc ttgatcccca gtaagtcaaa aaatagctcg acatactgtt 7500
cttccccgat atcctccctg atcgaccgga cgcagaaggc aatgtcatac cacttgtccg 7560
ccctgccgct tctcccaaga tcaataaagc cacttacttt gccatctttc acaaagatgt 7620
tgctgtctcc caggtcgccg tgggaaaaga caagttcctc ttcgggcttt tccgtcttta 7680
aaaaatcata cagctcgcgc ggatctttaa atggagtgtc ttcttcccag ttttcgcaat 7740
ccacatcggc cagatcgtta ttcagtaagt aatccaattc ggctaagcgg ctgtctaagc 7800
tattcgtata gggacaatcc gatatgtcga tggagtgaaa gagcctgatg cactccgcat 7860
acagctcgat aatcttttca gggctttgtt catcttcata ctcttccgag caaaggacgc 7920
catcggcctc actcatgagc agattgctcc agccatcatg ccgttcaaag tgcaggacct 7980
ttggaacagg cagctttcct tccagccata gcatcatgtc cttttcccgt tccacatcat 8040
aggtggtccc tttataccgg ctgtccgtca tttttaaata taggttttca ttttctccca 8100
ccagcttata taccttagca ggagacattc cttccgtatc ttttacgcag cggtattttt 8160
cgatcagttt tttcaattcc ggtgatattc tcattttagc catttattat ttccttcctc 8220
ttttctacag tatttaaaga taccccaaga agctaattat aacaagacga actccaattc 8280
actgttcctt gcattctaaa accttaaata ccagaaaaca gctttttcaa agttgttttc 8340
aaagttggcg tataacatag tatcgacgga gccgattttg aaaccgcggt gatcacaggc 8400
agcaacgctc tgtcatcgtt acaatcaaca tgctaccctc cgcgagatca tccgtgtttc 8460
aaacccggca gcttagttgc cgttcttccg aatagcatcg gtaacatgag caaagtctgc 8520
cgccttacaa cggctctccc gctgacgccg tcccggactg atgggctgcc tgtatcgagt 8580
ggtgattttg tgccgagctg ccggtcgggg agctgttggc tggctggtgg caggatatat 8640
tgtggtgtaa acaaattgac gcttagacaa cttaataaca cattgcggac gtttttaatg 8700
tactgaatta acgccgaatt aattcggggg atctggattt tagtactgga ttttggtttt 8760
aggaattaga aattttattg atagaagtat tttacaaata caaatacata ctaagggttt 8820
cttatatgct caacacatga gcgaaaccct ataggaaccc taattccctt atctgggaac 8880
tactcacaca ttattatgga gaaactcgag cttgtcgatc gacagatccg gtcggcatct 8940
actctatttc tttgccctcg gacgagtgct ggggcgtcgg tttccactat cggcgagtac 9000
ttctacacag ccatcggtcc agacggccgc gcttctgcgg gcgatttgtg tacgcccgac 9060
agtcccggct ccggatcgga cgattgcgtc gcatcgaccc tgcgcccaag ctgcatcatc 9120
gaaattgccg tcaaccaagc tctgatagag ttggtcaaga ccaatgcgga gcatatacgc 9180
ccggagtcgt ggcgatcctg caagctccgg atgcctccgc tcgaagtagc gcgtctgctg 9240
ctccatacaa gccaaccacg gcctccagaa gaagatgttg gcgacctcgt attgggaatc 9300
cccgaacatc gcctcgctcc agtcaatgac cgctgttatg cggccattgt ccgtcaggac 9360
attgttggag ccgaaatccg cgtgcacgag gtgccggact tcggggcagt cctcggccca 9420
aagcatcagc tcatcgagag cctgcgcgac ggacgcactg acggtgtcgt ccatcacagt 9480
ttgccagtga tacacatggg gatcagcaat cgcgcatatg aaatcacgcc atgtagtgta 9540
ttgaccgatt ccttgcggtc cgaatgggcc gaacccgctc gtctggctaa gatcggccgc 9600
agcgatcgca tccatagcct ccgcgaccgg ttgtagaaca gcgggcagtt cggtttcagg 9660
caggtcttgc aacgtgacac cctgtgcacg gcgggagatg caataggtca ggctctcgct 9720
aaactcccca atgtcaagca cttccggaat cgggagcgcg gccgatgcaa agtgccgata 9780
aacataacga tctttgtaga aaccatcggc gcagctattt acccgcagga catatccacg 9840
ccctcctaca tcgaagctga aagcacgaga ttcttcgccc tccgagagct gcatcaggtc 9900
ggagacgctg tcgaactttt cgatcagaaa cttctcgaca gacgtcgcgg tgagttcagg 9960
ctttttcata tctcattgcc ccccgggatc tgcgaaagct cgagagagat agatttgtag 10020
agagagactg gtgatttcag cgtgtcctct ccaaatgaaa tgaacttcct tatatagagg 10080
aaggtcttgc gaaggatagt gggattgtgc gtcatccctt acgtcagtgg agatatcaca 10140
tcaatccact tgctttgaag acgtggttgg aacgtcttct ttttccacga tgctcctcgt 10200
gggtgggggt ccatctttgg gaccactgtc ggcagaggca tcttgaacga tagcctttcc 10260
tttatcgcaa tgatggcatt tgtaggtgcc accttccttt tctactgtcc ttttgatgaa 10320
gtgacagata gctgggcaat ggaatccgag gaggtttccc gatattaccc tttgttgaaa 10380
agtctcaata gccctttggt cttctgagac tgtatctttg atattcttgg agtagacgag 10440
agtgtcgtgc tccaccatgt tatcacatca atccacttgc tttgaagacg tggttggaac 10500
gtcttctttt tccacgatgc tcctcgtggg tgggggtcca tctttgggac cactgtcggc 10560
agaggcatct tgaacgatag cctttccttt atcgcaatga tggcatttgt aggtgccacc 10620
ttccttttct actgtccttt tgatgaagtg acagatagct gggcaatgga atccgaggag 10680
gtttcccgat attacccttt gttgaaaagt ctcaatagcc ctttggtctt ctgagactgt 10740
atctttgata ttcttggagt agacgagagt gtcgtgctcc accatgttgg caagctgctc 10800
tagccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac 10860
gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc 10920
actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt 10980
gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacga attcgagctc 11040
ggtacccggg gatccatggg tagagcacct tgttgcgata aagtggggct gaagaaagga 11100
agatggacag ctgaagaaga tgagactttg actaaataca ttcaagccca tggtgaaggc 11160
tcttggaggt ctttacccaa gaatgcaggt ttgttaaggt gcggaaagag ttgcagactc 11220
agatggataa actatttgag agccgacttg aagagaggga acatcactgc tgaagaagaa 11280
gatcttattg tgcaattgca tagatccatg ggaaataggt ggtctttgat agctggccat 11340
ttacccgggc gaacagataa tgagataaag aattactgga actcttattt aagcagaaaa 11400
gttagcagca tctttacgcc gaccaatcca tcactgccaa caagcgccat cagccccgtc 11460
agtccttcac acaagcgtaa agttggccga gtgagccgtt cagtagctag aaagtacaac 11520
aacaaaagct cccaccgact ctttggtgaa atcccaatga gaaactcaaa tgatgctgat 11580
ccccaaaact gggcgccggt gcagcccgtg gccacagtaa atgaaaaccc accaatgggt 11640
tcagtgcctg tggagcagag tgaggagaaa gacaagggtg cagttatgtt gggaccattt 11700
gaagagatga atgatcaggt gatgggactc aactatttgc tggagggtgg gtctggggaa 11760
tcaagtgggg ttaagtacct taatgaggag aaagaaaatg cactaatggg tgtggacagt 11820
agtacagtga taagtaatga tgagagagaa agtggtggtt gggggtcaag ttgtgggatt 11880
ggggtgtcat ttgggtgttc ttcttctccc atgaatgttg catgctttgg ttttgagtgg 11940
gatgatcaag atcttgcatt ttgtgatgaa ggggaatacg cgttgctttg gccatgggag 12000
gagagtgaca ctttggaggt aggaaatttg tctcaaggat aatctagagt cgacctgcag 12060
gcatgcaagc ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt 12120
tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga 12180
ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gctagagcag 12240
cttgagcttg gatcagattg tcgtttcccg ccttcagttt agcttcatgg agtcaaagat 12300
tcaaatagag gacctaacag aactcgccgt aaagactggc gaacagttca tacagagtct 12360
cttacgactc aatgacaaga agaaaatctt cgtcaacatg gtggagcacg acacacttgt 12420
ctactccaaa aatatcaaag atacagtctc agaagaccaa agggcaattg agacttttca 12480
acaaagggta atatccggaa acctcctcgg attccattgc ccagctatct gtcactttat 12540
tgtgaagata gtggaaaagg aaggtggctc ctacaaatgc catcattgcg ataaaggaaa 12600
ggccatcgtt gaagatgcct ctgccgacag tggtcccaaa gatggacccc cacccacgag 12660
gagcatcgtg gaaaaagaag acgttccaac cacgtcttca aagcaagtgg attgatgtga 12720
tatctccact gacgtaaggg atgacgcaca atcccactat ccttcgcaag acccttcctc 12780
tatataagga agttcatttc atttggagag aacacggggg actcttgacc atggta 12836
Claims (5)
1.一种提高金银花中木犀草素含量的重组基因在提高金银花中木犀草素含量方面的应用，所述重组基因的核苷酸序列如SEQ ID No.2所示。
2.一种提高金银花中木犀草素含量的重组基因的植物表达载体在提高金银花中木犀草素含量方面的应用，所述重组基因的核苷酸序列如SEQ ID No.2所示。
3.一种提高金银花中木犀草素含量的方法，其特征在于，所述方法包括：
构建重组质粒，所述重组质粒包含核苷酸序列如SEQ ID No.2所示的重组基因；
采用农杆菌介导法将重组质粒转入到金银花叶片中，进行培养，获得目的植株。
4.根据权利要求3所述的提高金银花中木犀草素含量的方法，其特征在于，构建重组质粒的步骤为：
提取金银花RNA，反转录获得cDNA，以cDNA为模板，设计引物，在MYB12基因的上下游均引入KpnI酶切位点，利用高保真酶从cDNA模板中扩增MYB12基因序列，回收扩增出来的含有MYB12基因的PCR产物；
使用KpnI内切酶将PCambia2301-JC酶切成线性化载体，用同源重组法将MYB12基因与线性化PCambia2301-JC载体连接，热激法将重组载体导入大肠杆菌DH5ɑ感受态，菌液PCR检测阳性菌落，提取阳性质粒，测序后获得含有目的基因的重组质粒。
5.根据权利要求4所述的提高金银花中木犀草素含量的方法，其特征在于,将构建成功的重组质粒导入农杆菌中，进行培养；
将培养好的农杆菌在室温下离心，弃掉废液，加入渗透培养基重悬菌体，室温下搅拌；
渗透培养基重悬后的农杆菌，注射到金银花叶片中。
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