CN109321594B - 一种以黄花蒿悬浮细胞系为受体的转iaaM基因提高黄花蒿中青蒿素含量的方法 - Google Patents
一种以黄花蒿悬浮细胞系为受体的转iaaM基因提高黄花蒿中青蒿素含量的方法 Download PDFInfo
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Abstract
本发明公开了一种以黄花蒿悬浮细胞系为受体的转iaaM基因提高黄花蒿中青蒿素含量的方法，构建iaaM基因在黄花蒿腺毛细胞特异表达的载体，利用根癌农杆菌介导法转化黄花蒿悬浮细胞系，获得转基因黄花蒿植株。对转基因黄花蒿植株进行PCR基因扩增检测，并对转基因黄花蒿腺毛细胞发育情况及青蒿素含量进行观察与分析，筛选青蒿素含量显著提高的转基因黄花蒿植株，通过组织培养繁殖建立转基因高青蒿素含量的黄花蒿品系。本发明获得的转基因黄花蒿中青蒿素含量显著提高，转iaaM基因的黄花蒿中青蒿素的含量最高可达21.6mg/g DW，是非转化黄花蒿含量的1.8倍。本发明对于为青蒿素的规模化生产提供高产、稳定新药源具有重要意义。
Description
技术领域
本发明属于植物基因工程技术领域，具体涉及一种以黄花蒿悬浮细胞系为受体的转iaaM基因提高黄花蒿中青蒿素含量的方法。
背景技术
青蒿素是从植物黄花蒿中提取的含过氧基团的倍半萜内酯药物，是当今疟疾治疗的最理想药物，能有效治疗对传统药物氯奎产生抗药性的疟原虫感染。青蒿素主要从黄花蒿(Artemisia annua L.)中直接提取，或提取黄花蒿中含量较高的青蒿酸，然后半合成而成。目前除黄花蒿外，尚未发现含有青蒿素的其它天然植物资源。因此黄花蒿仍是青蒿素药物生产的惟一药源。
虽然黄花蒿系世界广泛分布品种，但青蒿素含量随产地不同差异极大。研究证明黄花蒿中合成青蒿素的主要部位是其叶片表面的腺毛细胞。由于其合成部位的特异性，黄花蒿中青蒿素的含量仅占叶片干重的0.8％—1.4％，因而使其分离成本高、产量低、废弃物多。
野生黄花蒿中青蒿素含量很低，主要集中在0.1％—0.60％之间，高者也仅为0.8％左右。通过杂交和定向育种开展了大量提高黄花蒿中青蒿素含量的尝试，但目前报道的最高青蒿素含量也未超过1.4％，说明了常规选育方法的局限性。
青蒿素主要在黄花蒿腺毛中合成、分泌及储存，通过增大黄花蒿腺毛的密度及大小，能够有效地增加黄花蒿中青蒿素的含量。因此，将生长素合成酶关键基因iaaM转入能够促进黄花蒿腺毛细胞的生长发育，增大其密度或大小；提高腺毛细胞代谢水平从而增加黄花蒿中青蒿素的合成，进而提高黄花蒿中青蒿素的含量。
发明内容
本发明旨在克服现有技术的不足，提供一种以黄花蒿悬浮细胞系为受体的转iaaM基因提高黄花蒿中青蒿素含量的方法。
为了达到上述目的，本发明提供的技术方案为：
所述以黄花蒿悬浮细胞系为受体的转iaaM基因提高黄花蒿中青蒿素含量的方法包括如下步骤：
(1)黄花蒿腺毛细胞特异表达基因的载体重组构建：
将黄花蒿腺毛特异启动子AaGTpro和iaaM基因构建到Ti质粒的T-DNA区，以HPT基因为选择标记基因，以pWM101作为出发载体进行改造和载体构建；得到重组Ti质粒pWM101-AaGTpro::iaaM，所述黄花蒿腺毛特异启动子AaGTpro的序列如SEQ ID NO.1所示；
(2)工程化根癌农杆菌的制备：
构建的重组Ti质粒通过电激转化法转化根癌农杆菌LBA4404(市售)，经过抗生素筛选和分子检测验证工程农杆菌，培养工程农杆菌至对数生长后期；
(3)黄花蒿悬浮细胞系的建立：
将深绿色或淡黄色生长快、结构疏松颗粒状的黄花蒿愈伤组织置于100mLMS液体培养基中，将愈伤轻轻夹碎，摇床150r/min，光照周期为16h/8h光暗交替，温度为25±2℃振荡培养；培养15—20d左右为悬浮细胞的快速增长且细胞活性较高期，选取该时期期的直径在5mm以内的小细胞团及含有单细胞的黄花蒿悬浮细胞系悬浮液，备用；
(4)黄花蒿悬浮细胞的共培浸染转化：
将步骤(3)所得的黄花蒿悬浮细胞系悬浮液与步骤(2)中生长至对数生长后期的工程农杆菌共培2.5—3.5d，优选为3d，然后转入筛选培养基上培养；
(5)转化细胞的筛选和获得抗性愈伤组织：
经步骤(4)共培2.5—3.5d，优选为3d后的黄花蒿悬浮细胞经MS液体培养基冲洗(洗3遍)后，置于含200mg/L羧苄青霉素和30mg/LHPT抗性的诱导愈伤及丛生芽分化的培养基上，培养至愈伤上有丛生芽的分化(6周左右)，获得抗性丛生芽并继代增殖；
(6)诱导根分化和获得转基因苗：
当抗性丛生芽生长至3—5cm大小时，将抗性丛生芽转移到生根培养基诱导根分化，分化后幼苗经过炼苗后移栽至营养基质中，获得转基因植株；
(7)转基因植株的分子检测和腺毛细胞发育观察：
提取转基因植株叶片的DNA，用PCR法进行目的基因的分子检测验证，通过体视荧光显微镜观察转基因植株叶片腺毛细胞的密度、大小及荧光强度，通过与对照的比较判断腺毛的发育情况，对腺毛发育具有显著促进的植株进行青蒿素含量测定；
(8)建立转基因黄花蒿品系：
筛选青蒿素含量显著提高的转基因黄花蒿植株，通过组织培养繁殖建立转基因高青蒿素含量的黄花蒿品系。
其中，步骤(1)是用HindIII和BamH1双酶切pWM101载体，将AaGTpro替换pWM101中的35Spro，完成PWM101-AaGTpro重组载体构建，再用BamH1和PSt1酶切重组载体PWM101-AaGTpro，将iaaM基因插入，构建得到腺毛特异表达的重组Ti质粒pWM101-AaGTpro::iaaM。步骤(1)是从GenBank中的Nucleotide数据库中获得黄花蒿腺毛特异启动子AaDB2序列信息，设计克隆和同源重组引物，以黄花蒿基因组DNA为模板，通过PCR扩增技术，获得黄花蒿腺毛特异启动子AaGTpro序列。
下面对本发明作进一步说明：
本发明通过转基因技术，将生长素合成酶关键基因iaaM转入黄花蒿，利用黄花蒿腺毛特异性启动子AaGTpro驱动iaaM在黄花蒿腺毛细胞中特异表达，促进黄花蒿腺毛细胞发育和代谢，增加腺毛的密度和长度，从而提高其青蒿素含量。
具体而言，本发明采用黄花蒿叶片诱导愈伤，对黄花蒿愈伤进行振荡培养，获得大量的黄花蒿单细胞及小细胞团(悬浮细胞系)，通过农杆菌介导，将生长素合成酶关键基因iaaM导入黄花蒿单细胞及小细胞团中，经过共培转化后，诱导黄花蒿抗性愈伤组织、分化丛生芽和诱导生根的过程，从而获得转基因黄花蒿植株。
利用黄花蒿单细胞及小细胞团为转化受体，转化生长素合成酶关键基因iaaM(iaaM基因是植物通过色氨酸途径合成生长素(IAA)的关键酶)，使用黄花蒿腺毛细胞特异性表达的基因启动子AaGTpro，能在黄花蒿腺毛细胞中有效作用，保证其功能基因在腺毛细胞中表达，从而不影响植株的整体发育。
本发明独特之处在于利用黄花蒿单细胞及小细胞团(悬浮细胞系)进行生长素合成相关基因的遗传转化。
总之，本发明采用基因工程的植物遗传转化技术开展黄花蒿的转基因，涉及到生长素合成酶关键基因在其腺毛细胞中特异表达，是一种运用现代生物技术开展的黄花蒿育种新方法。
具体实施方式
1.腺毛细胞特异表达载体的构建
从GenBank中的Nucleotide数据库中获得黄花蒿腺毛特异启动子AaDB2序列信息，设计克隆和同源重组引物，以黄花蒿基因组DNA为模板，通过PCR扩增技术，获得黄花蒿腺毛特异启动子AaGTpro序列，构建腺毛特异表达的重组Ti质粒pWM101-AaGTpro::iaaM。
(1)黄花蒿腺毛特异启动子AaDB2序列的获得
从GenBank中的Nucleotide数据库中获得黄花蒿腺毛特异启动子AaDB2序列(登录号为：KC347592.1)，通过PCR技术扩增获得黄花蒿腺毛特异启动子AaDB2序列，并与pMD18-T连接，转化至E.coli DH5α中保存，经测序确定所克隆基因的正确性，构建pWM101-AaGTpro::iaaM Ti质粒表达载体。其中所克隆的AaDB2启动子序列与GenBank报道的同源性为95％(与所用黄花蒿的基因型不同)，因此自命名为AaGTpro。
具体操作步骤为：
设计AaGTpro特异性引物。
上游引物AaGTpro UP1 CCCAAGCTTTGAAGGATGACCAAAAGCATAA(SEQ ID NO.2)
下游引物AaGTpro DN1 AGTCATAGGAGCTAACACCACCCT(SEQ ID NO.3)
以黄花蒿DNA为模版，利用上述AaGTpro特异性引物，PCR扩增得到AaGTpro基因片段，PCR反应体系别为：
黄花蒿DNA(约100-200ng)1μL，10×Buffer 2.5μL，dNTPs(10mmol/L)2μL，AaGTproUP1(10μmol/L)1μL，AaGTpro DN1(10μmol/L)1μL，PfuTaqDNA聚合酶(1U/μL)0.5μL，补ddH2O至25μL。
AaGTpro PCR反应程序为：95℃预变性5min，94℃变性1min、56℃退火2min、72℃延伸2min，共30个循环，终72℃延伸10min。
PCR产物经1％琼脂糖凝胶电泳检测、切胶回收后，与pMD18-T连接，连接体系为：pMD18-T Vector 1μL，ddH2O补至10μL。16℃连接过夜，转化大肠杆菌E.coli DH5α。
(2)构建pWM101-AaGTpro::iaaM Ti质粒表达载体
以pWM101为出发载体，利用载体上的HindIII和BamHⅠ酶切位点，设计AaGTpro扩增引物。
上游引物AaGTpro UP2：(SEQ ID NO.4)
下游引物AaGTpro DN2：(SEQ ID NO.5)
iaaM基因扩增引物(注：iaaM由深圳华大基因公司合成pMD18-iaaM)
iaaM UP(SEQ ID NO.6)上游引入BamHⅠ酶切位点
iaaM DN(SEQ ID NO.7)下游引入PstⅠ酶切位点
以pMD18-AaGTpro质粒为模板，利用上述AaGTproUP2和DN2基因特异性引物，PCR技术扩增AaGTpro。PCR反应体系为pMD18-AaGTpro(约100ng)1μL，10×Buffer 2.5μL，dNTPs(10mmol/L)2μL，UP2(10μmol/L)1μL，DN2(10μmol/L)1μL，TaqDNA聚合酶(1U/μL)1μL，补ddH2O至25μL。反应程序为：95℃预变性4min，94℃变性1min、56℃退火1min、72℃延伸2min、30个循环，最后72℃延伸7min。
PCR产物经1％琼脂糖凝胶电泳检测并回收AaGTpro片段，将AaGTpro回收片段与双酶切后的pWM101载体连接，用AaGTpro替换pWM101中的35Spro，完成PWM101-AaGTpro重组载体构建，再用BamH1和PSt1酶切重组载体PWM101-AaGTpro，将iaaM基因插入，完成PWM101-AaGTpro::iaaM黄花蒿腺毛特异性表达载体构建。
本实施例获得的AaGTpro基因，构建了黄花蒿腺毛特异性启动子与生长素合成关键酶基因iaaM的重组特异表达载体PWM101-AaGTpro::iaaM。通过转化上述基因，促进黄花蒿腺毛细胞的发育和代谢，从而提高其青蒿素含量。
2.质粒的检测和根癌农杆菌转化
构建的重组Ti质粒经过酶切检测，验证其构建的正确性。然后用电激转化法转化到根癌农杆菌LBA4404。具体方式是：制备的质粒DNA 0.1μg与200μL感受态农杆菌混合后置于电激杯，Bio Rad电激转化仪以细菌转化模式进行电激转化。转化后的根癌农杆菌铺于含卡那霉素50mg/L、利福平50mg/L和链霉素50mg/L的YEB培养基上筛选，将筛选获得的阳性菌落，经菌落PCR验证表明，所构建的在黄花蒿腺毛细胞特异表达的载体AaGTpro::iaaM已成功转化到根癌农杆菌菌株中，可用于后续遗传转化。
3.工程农杆菌共培养转化
将上述筛选鉴定的工程农杆菌接种至YEB液体培养基，28℃摇瓶培养过夜至对数生长后期，按1:50接种到扩大的培养体系继续摇瓶至OD600nm＝1.0。1/2MS液体培养基将菌体重悬，将菌液加入黄花蒿悬浮细胞液中进行转化：按每平皿8mL的细胞悬浮液与200μL的工程农杆菌菌悬液避光共培养3d。
4.农杆菌的抑制、愈伤诱导和丛生芽分化
将共培后的黄花蒿悬浮细胞系以MS液体培养基冲洗3遍，然后转接到含2.0mg/L 6-BA+1.0mg/L NAA+30g/L蔗糖+7g/L琼脂的MS固体培养基上。其中加入200mg/L羧苄青霉素和30mg/LHPT(潮霉素)，分别抑制农杆菌和诱导并筛选抗性愈伤组织及丛生芽。25±2℃，16h/8h进行光暗交替培养。约6周后可以诱导出抗性的丛生芽。
5.诱导根分化与炼苗
待抗性丛生芽生长至3-5cm后，将其从愈伤组织基部切下，移入1/2MS+0.1mg/L NAA/0.1mg/L NAA+30g/L蔗糖+7g/L琼脂的生根培养基中诱导其生根，此时对每一株阳性植株进行编号，25±2℃左右，16h/8h进行光暗交替培养。诱导根分化2周后，室温下炼苗1周。然后移栽至营养土中生长。
6.PCR检测转基因黄花蒿植株、黄花蒿腺毛发育观察及青蒿素含量分析
提取转基因黄花蒿植株叶片DNA，利用转入的目的基因引物对其进行PCR分子检测验证；并对转基因黄花蒿植株叶片腺毛发育情况进行观察与分析，具体操作为：取转基因黄花蒿植株叶片，置于体视荧光显微镜下进行荧光显微观察，统计单位叶面积的腺毛细胞数，与对照非转基因黄花蒿比较，可以估测青蒿素含量提高情况，采用高效液相色谱法及蒸发光散射检测器测定转基因黄花蒿中青蒿素的含量。
7.建立转基因黄花蒿品系
测定转基因黄花蒿中的青蒿素含量，筛选青蒿素显著提高的转基因黄花蒿植株，通过组织培养或有性繁殖，筛选稳定遗传的转基因高青蒿素含量的黄花蒿品系。
以上实施例利用带有特异性表达载体的根癌农杆菌菌株转化黄花蒿悬浮细胞系，获得经PCR检测的转基因黄花蒿植株，成功建立了黄花蒿悬浮培养细胞遗传转化体系。通过荧光显微观察黄花蒿腺毛细胞的发育情况，结果表明黄花蒿叶片上的腺毛密度和腺毛长度明显增加。利用黄花蒿悬浮细胞系与根癌农杆菌共培转化，经筛选诱导分化可实现同期获得大量均一的转化植株，提高转基因植株的效率。本发明获得的转基因黄花蒿中青蒿素含量显著提高，转iaaM基因的黄花蒿中青蒿素的含量最高可达21.6mg/g DW，是非转化黄花蒿含量的1.8倍。
SEQ ID NO.1：
SEQ ID NO.8：
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序列表
<110> 湖南农业大学
<120> 一种以黄花蒿悬浮细胞系为受体的转iaaM基因提高黄花蒿中青蒿素含量的方法
<141> 2022-09-14
<160> 8
<170> SIPOSequenceListing 1.0
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ttcactagct ttcttgaaga gtcccgtgcg tcgtttggag aaggtgactt gacgactgct 180
tgtctcttct atcttcttga tagggatctt ctttcgccct ttgtttggct gcttcatcat 240
attgccttcc gaaaagttca ttaatgtaat ggtttaggta ataaaatggg ctcgtatata 300
tatatagttt caagaagata agaatatagg gtaaaagatt tcatatttaa tgccttgtag 360
gttaaatatg gtaccttgct acatagttgt agggatttcg tagttattta cgtactagct 420
tagcatttcc aacgatatgg aatttcattt ggattaaaga aatttagcca aaatgaatcg 480
tggtccaaaa tatttacgtg ggccataagc ccgcaaataa tagttgctca agaacaacgt 540
ggcttaaatt tatttccaac aacttttctt ccaccggcca aagaaagcaa atttctataa 600
tctatagttc tatactacat tctaaatgtg tcatcttcgt aatgttcact tgttaaaagc 660
tactcaaata tacataataa aacaaaacta caagtataat gtccaaacaa tcgaccaagc 720
gaaagctatt gacgtagacg gaatttgata agtttaattg aagggccaaa ttgcaatgta 780
ctacgtattt gttttatagg gtcaatttac attttcattt tacattccta ctaataatgt 840
ctacttatac aaagtgtaag ggttaaaaat gtacctatga ctaagaattt tgaaagaatc 900
aggacacatc tcggtccaaa ataagtaccg tctgtaacta ttgacacaag aatcgtaaaa 960
atgaacacca aacaacaacc cacaaccatc gcttcgtagc catcttgacc catcaagtta 1020
tcatggtgtc actgatacat caacaaagag tttataaaaa tatatctaca taataatttg 1080
tcaattaaat gtgaaagata aagaaaagaa ataaaagatt atgatacaat atgtttagac 1140
cattatactt gacaaatcaa tgtatgaata taattttcta aattttttgt atatgtagca 1200
tcattcgatg atctttatca aagccaaaca tgataggtga taaggggtga gcataacgga 1260
tcgtggaccg gaccaaaccg agaaaccgaa ctggaccgac cggaccggat cgaacctatt 1320
tcggtccggt cctcggtcca cactttactt tatgttcggt cctcggtccg gttcggtctg 1380
gtccagtctt acgttcaacc gaccatttta tcttctgaga ccggaccgga ctggaccgaa 1440
cacattcggt ccggttcctc ggttcatgat tttgggctaa atcgatcttg gttcagtccg 1500
ggtttttagg tccgggccgc gtttaacccg atgctcattc atttatctac cttgttatta 1560
atgaaaatta ctatgacata taaacccaaa aaagtaaaac aagtagtaca cgtttatttt 1620
atgttcgaga cacggtgtct aagactaaaa tggtgatagt ttgactctgc acccaaaaaa 1680
aaaatgttga tagtttgact ttgaaacaca agtcaaattc cacgttttca tactcatata 1740
ctactattta aatattcaac attggatcac tctttgtagt gctttttaat attctgtatt 1800
caacacatca cactttacta actccatcac tgatctctga tcctgatcaa catcaaactc 1860
aata 1864
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gggggatctg gattttagta ctggattttg gttttaggaa ttagaaattt tattgataga 60
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accctatagg aaccctaatt cccttatctg ggaactactc acacattatt atggagaaac 180
tcgagcttgc atgcctgcag acttaatttc tattgcggta gttatatctc ttccaagggt 240
gtttaagagg attgtccttc gctaaaatgc ctccacaatt gtggataatt gcgcagacgg 300
cgttacacgc ggtctgaata gcgccctcca cccatccacc ggtgaaggaa caactgcaac 360
ccgccaagta aactccagta tcattagtcg tgtccagcgc ttgaaagaaa agttcttcag 420
aataaaaatc ttcgccgcgt cggttaagtt tgaaacgtcc gccggcattc tcgtctgtaa 480
gccaatcatg ttgaacaaca ttttggtcgt aatcagcgca ggcgggaact agatgctggg 540
caaacaccgg gaaagatttt gaaattgcgt cacgcagcag acataatctc tcttttttgt 600
cggggaccgc caatagcttg tgggagtcgt cctcccatgt ataactgatg agcaccagac 660
ctttaccgtt cggatcctgc ggctcatagt ccaggcaata gactgctttt gcgaacccgt 720
ccatgaggac acaggacggg agcatatggt caaaccaaaa ttttcgttca gtcagcagga 780
agagttttga agagcctgtc atgtggctgt tatcaaccgc ttggttcact ggtgcacgaa 840
aaatggtggt atcgcatgtc agacaatgcc tgagttggat atttgcgagt ccagatgtaa 900
ccaccacttt atcataaagt tctgatatcc cgctcttaag ccttatcttt atttttgtct 960
tttccttctc gatcgccctg acttgaacat ggcgtatacg ctggcttaca gacacaccgt 1020
taaccacttg agaggctatt cgacgtggaa gttctgagat tccttcagag cacatccgct 1080
gattttcttc atatccgttt atgaccaagc gaaggatctc agtaaacccg ctttcaaaaa 1140
ctggaccaaa cccgccagat cctattccca ttagcttgaa taggtcccaa tcatgaggga 1200
aactccatgt ttctccacca ggaggatgcg tgcccagaaa gatcctctct atcgctgagg 1260
agaacgactc cctcccgaaa cggttcagcc aaatttgcca ggagtcatga gcccgcctaa 1320
tgtcccctga tttcaaggcc tcagtgatag catcaggcga cgccaacaca atatctccct 1380
caaggaaacc gtccttcaag aacgcatgcc acccgctgta aacgcgatgg aacaacttcg 1440
gtggctgctg cccggctttc cacatgtatc ggcaaccctc gtagaccaag tcagtgtcga 1500
ctgtgccggg atttgggaac ggcctcatcg aagacagacc gtatcgctcg aggaagaaaa 1560
acaagcacga tgcagcagga ggaaatcgca tcgcccccat ttcggccacg acgcccggag 1620
cgtccttgaa ggcatgtgac caaagcttac ctccaacccg atcacctgct tcatatattg 1680
taacatcgtc tacgccagca tgaagcagtt cgcttgccac cacgagcccg gaaatgccag 1740
ccccaatgac cgctactttt ggcctgggaa catcttccgg aaagaagccg atccgcccac 1800
tatcggaaca tttgtcgaga aacaatctgt agtcatagag caagtcgatc gttggaaagg 1860
accctgccga ggctttcagc atcaggtttc gaccataagc taccatgtca aatggtactg 1920
cctcttcaat gattacggta aacccggtaa cgcttttcaa cgagccttct tcgtacggcg 1980
ccatttgcag ggttcgggag cgggcagtct cagagattga aatggccacg aaatgactaa 2040
ggtcgctggg tgcacatctt ttaccactca gcaaattcat ggtttggaga agttgagctc 2100
tggaaatatc catgcaagat ggtgcaaggt cgatggcaac gagaccactc actcttgttc 2160
gcaccgaagc ttctggttcg agtatccacc tcagaatttc ctcgccaaca tagatgtaag 2220
cggagaggac tgcgaccctc tgaccggctg atatctccgg aaagcgaccg tcggccagcc 2280
ttttgcaagc taacccagcg ttgcactcgc cggaaatttg actaattctt ctttccctcg 2340
aagcttctcg ttctgagaag gctgcggcaa cgcggcggtc caactcatca gccttgtcga 2400
ccattatcag atccacaatt ttggtagaga aatgatcgca ttgtttatca agaagagatg 2460
aggctgacat ggatccttat tgagtttgat gttgatcagg atcagagatc agtgatggag 2520
ttagtaaagt gtgatgtgtt gaatacagaa tattaaaaag cactacaaag agtgatccaa 2580
tgttgaatat ttaaatagta gtatatgagt atgaaaacgt ggaatttgac ttgtgtttca 2640
aagtcaaact atcaacattt tttttttggg tgcagagtca aactatcacc attttagtct 2700
tagacaccgt gtctcgaaca taaaataaac gtgtactact tgttttactt ttttgggttt 2760
atatgtcata gtaattttca ttaataacaa ggtagataaa tgaatgagca tcgggttaaa 2820
cgcggcccgg acctaaaaac ccggactgaa ccaagatcga tttagcccaa aatcatgaac 2880
cgaggaaccg gaccgaatgt gttcggtcca gtccggtccg gtctcagaag ataaaatggt 2940
cggttgaacg taagactgga ccagaccgaa ccggaccgag gaccgaacat aaagtaaagt 3000
gtggaccgag gaccggaccg aaataggttc gatccggtcc ggtcggtcca gttcggtttc 3060
tcggtttggt ccggtccacg atccgttatg ctcacccctt atcacctatc atgtttggct 3120
ttgataaaga tcatcgaatg atgctacata tacaaaaaat ttagaaaatt atattcatac 3180
attgatttgt caagtataat ggtctaaaca tattgtatca taatctttta tttcttttct 3240
ttatctttca catttaattg acaaattatt atgtagatat atttttataa actctttgtt 3300
gatgtatcag tgacaccatg ataacttgat gggtcaagat ggctacgaag cgatggttgt 3360
gggttgttgt ttggtgttca tttttacgat tcttgtgtca atagttacag acggtactta 3420
ttttggaccg agatgtgtcc tgattctttc aaaattctta gtcataggta catttttaac 3480
ccttacactt tgtataagta gacattatta gtaggaatgt aaaatgaaaa tgtaaattga 3540
ccctataaaa caaatacgta gtacattgca atttggccct tcaattaaac ttatcaaatt 3600
ccgtctacgt caatagcttt cgcttggtcg attgtttgga cattatactt gtagttttgt 3660
tttattatgt atatttgagt agcttttaac aagtgaacat tacgaagatg acacatttag 3720
aatgtagtat agaactatag attatagaaa tttgctttct ttggccggtg gaagaaaagt 3780
tgttggaaat aaatttaagc cacgttgttc ttgagcaact attatttgcg ggcttatggc 3840
ccacgtaaat attttggacc acgattcatt ttggctaaat ttctttaatc caaatgaaat 3900
tccatatcgt tggaaatgct aagctagtac gtaaataact acgaaatccc tacaactatg 3960
tagcaaggta ccatatttaa cctacaaggc attaaatatg aaatctttta ccctatattc 4020
ttatcttctt gaaactatat atatatacga gcccatttta ttacctaaac cattacatta 4080
atgaactttt cggaaggcaa tatgatgaag cagccaaaca aagggcgaaa gaagatccct 4140
atcaagaaga tagaagagac aagcagtcgt caagtcacct tctccaaacg acgcacggga 4200
ctcttcaaga aagctagtga actttgtgtc ttgacggggg ctgaaatggc aatccttgtt 4260
caatcacccg gtggccattg ttatgctttt ggtcatcctt caanaagctt aagcttagct 4320
cgaatttccc cgatcgttca aacatttggc aataaagttt cttaagattg aatcctgttg 4380
ccggtcttgc gatgattatc atataatttc tgttgaatta cgttaagcat gtaataatta 4440
acatgtaatg catgacgtta tttatgagat gggtttttat gattagagtc ccgcgcgcgc 4500
ggtgtcatct atgttactag atcgggaatt aaactatcag tgtttgacag gatatattgg 4560
cgggtaaacc taagagaaaa gagcgtttat tagaataacg gatatttaaa agggcgtgaa 4620
aaggtttatc cgttcgtcca tttgtatgtg catgccaacc acagggttcc cctcgggatc 4680
aaagtacttt gatccaaccc ctccgctgct atagtgcagt cggcttctga cgttcagtgc 4740
agccgtcttc tgaaaacgac atgtcgcaca agtcctaagt tacgcgacag gctgccgccc 4800
tgcccttttc ctggcgtttt cttgtcgcgt gttttagtcg cataaagtag aatacttgcg 4860
actagaaccg gagacattac gccatgaaca agagcgccgc cgctggcctg ctgggctatg 4920
cccgcgtcag caccgacgac caggacttga ccaaccaacg ggccgaactg cacgcggccg 4980
gctgcaccaa gctgttttcc gagaagatca ccggcaccag gcgcgaccgc ccggagctgg 5040
ccaggatgct tgaccaccta cgccctggcg acgttgtgac agtgaccagg ctagaccgcc 5100
tggcccgcag cacccgcgac ctactggaca ttgccgagcg catccaggag gccggcgcgg 5160
gcctgcgtag cctggcagag ccgtgggccg acaccaccac gccggccggc cgcatggtgt 5220
tgaccgtgtt cgccggcatt gccgagttcg agcgttccct aatcatcgac cgcacccgga 5280
gcgggcgcga ggccgccaag gcccgaggcg tgaagtttgg cccccgccct accctcaccc 5340
cggcacagat cgcgcacgcc cgcgagctga tcgaccagga aggccgcacc gtgaaagagg 5400
cggctgcact gcttggcgtg catcgctcga ccctgtaccg cgcacttgag cgcagcgagg 5460
aagtgacgcc caccgaggcc aggcggcgcg gtgccttccg tgaggacgca ttgaccgagg 5520
ccgacgccct ggcggccgcc gagaatgaac gccaagagga acaagcatga aaccgcacca 5580
ggacggccag gacgaaccgt ttttcattac cgaagagatc gaggcggaga tgatcgcggc 5640
cgggtacgtg ttcgagccgc ccgcgcacgt ctcaaccgtg cggctgcatg aaatcctggc 5700
cggtttgtct gatgccaagc tggcggcctg gccggccagc ttggccgctg aagaaaccga 5760
gcgccgccgt ctaaaaaggt gatgtgtatt tgagtaaaac agcttgcgtc atgcggtcgc 5820
tgcgtatatg atgcgatgag taaataaaca aatacgcaag gggaacgcat gaaggttatc 5880
gctgtactta accagaaagg cgggtcaggc aagacgacca tcgcaaccca tctagcccgc 5940
gccctgcaac tcgccggggc cgatgttctg ttagtcgatt ccgatcccca gggcagtgcc 6000
cgcgattggg cggccgtgcg ggaagatcaa ccgctaaccg ttgtcggcat cgaccgcccg 6060
acgattgacc gcgacgtgaa ggccatcggc cggcgcgact tcgtagtgat cgacggagcg 6120
ccccaggcgg cggacttggc tgtgtccgcg atcaaggcag ccgacttcgt gctgattccg 6180
gtgcagccaa gcccttacga catatgggcc accgccgacc tggtggagct ggttaagcag 6240
cgcattgagg tcacggatgg aaggctacaa gcggcctttg tcgtgtcgcg ggcgatcaaa 6300
ggcacgcgca tcggcggtga ggttgccgag gcgctggccg ggtacgagct gcccattctt 6360
gagtcccgta tcacgcagcg cgtgagctac ccaggcactg ccgccgccgg cacaaccgtt 6420
cttgaatcag aacccgaggg cgacgctgcc cgcgaggtcc aggcgctggc cgctgaaatt 6480
aaatcaaaac tcatttgagt taatgaggta aagagaaaat gagcaaaagc acaaacacgc 6540
taagtgccgg ccgtccgagc gcacgcagca gcaaggctgc aacgttggcc agcctggcag 6600
acacgccagc catgaagcgg gtcaactttc agttgccggc ggaggatcac accaagctga 6660
agatgtacgc ggtacgccaa ggcaagacca ttaccgagct gctatctgaa tacatcgcgc 6720
agctaccaga gtaaatgagc aaatgaataa atgagtagat gaattttagc ggctaaagga 6780
ggcggcatgg aaaatcaaga acaaccaggc accgacgccg tggaatgccc catgtgtgga 6840
ggaacgggcg gttggccagg cgtaagcggc tgggttgtct gccggccctg caatggcact 6900
ggaaccccca agcccgagga atcggcgtga cggtcgcaaa ccatccggcc cggtacaaat 6960
cggcgcggcg ctgggtgatg acctggtgga gaagttgaag gccgcgcagg ccgcccagcg 7020
gcaacgcatc gaggcagaag cacgccccgg tgaatcgtgg caagcggccg ctgatcgaat 7080
ccgcaaagaa tcccggcaac cgccggcagc cggtgcgccg tcgattagga agccgcccaa 7140
gggcgacgag caaccagatt ttttcgttcc gatgctctat gacgtgggca cccgcgatag 7200
tcgcagcatc atggacgtgg ccgttttccg tctgtcgaag cgtgaccgac gagctggcga 7260
ggtgatccgc tacgagcttc cagacgggca cgtagaggtt tccgcagggc cggccggcat 7320
ggccagtgtg tgggattacg acctggtact gatggcggtt tcccatctaa ccgaatccat 7380
gaaccgatac cgggaaggga agggagacaa gcccggccgc gtgttccgtc cacacgttgc 7440
ggacgtactc aagttctgcc ggcgagccga tggcggaaag cagaaagacg acctggtaga 7500
aacctgcatt cggttaaaca ccacgcacgt tgccatgcag cgtacgaaga aggccaagaa 7560
cggccgcctg gtgacggtat ccgagggtga agccttgatt agccgctaca agatcgtaaa 7620
gagcgaaacc gggcggccgg agtacatcga gatcgagcta gctgattgga tgtaccgcga 7680
gatcacagaa ggcaagaacc cggacgtgct gacggttcac cccgattact ttttgatcga 7740
tcccggcatc ggccgttttc tctaccgcct ggcacgccgc gccgcaggca aggcagaagc 7800
cagatggttg ttcaagacga tctacgaacg cagtggcagc gccggagagt tcaagaagtt 7860
ctgtttcacc gtgcgcaagc tgatcgggtc aaatgacctg ccggagtacg atttgaagga 7920
ggaggcgggg caggctggcc cgatcctagt catgcgctac cgcaacctga tcgagggcga 7980
agcatccgcc ggttcctaat gtacggagca gatgctaggg caaattgccc tagcagggga 8040
aaaaggtcga aaaggtctct ttcctgtgga tagcacgtac attgggaacc caaagccgta 8100
cattgggaac cggaacccgt acattgggaa cccaaagccg tacattggga accggtcaca 8160
catgtaagtg actgatataa aagagaaaaa aggcgatttt tccgcctaaa actctttaaa 8220
acttattaaa actcttaaaa cccgcctggc ctgtgcataa ctgtctggcc agcgcacagc 8280
cgaagagctg caaaaagcgc ctacccttcg gtcgctgcgc tccctacgcc ccgccgcttc 8340
gcgtcggcct atcgcggccg ctggccgctc aaaaatggct ggcctacggc caggcaatct 8400
accagggcgc ggacaagccg cgccgtcgcc actcgaccgc cggcgcccac atcaaggcac 8460
cctgcctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga 8520
cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag 8580
cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt 8640
atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg 8700
tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc 8760
gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 8820
ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 8880
aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 8940
ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 9000
aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 9060
gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc 9120
tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg 9180
tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga 9240
gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag 9300
cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta 9360
cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag 9420
agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg 9480
caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac 9540
ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgcattctag 9600
gtactaaaac aattcatcca gtaaaatata atattttatt ttctcccaat caggcttgat 9660
ccccagtaag tcaaaaaata gctcgacata ctgttcttcc ccgatatcct ccctgatcga 9720
ccggacgcag aaggcaatgt cataccactt gtccgccctg ccgcttctcc caagatcaat 9780
aaagccactt actttgccat ctttcacaaa gatgttgctg tctcccaggt cgccgtggga 9840
aaagacaagt tcctcttcgg gcttttccgt ctttaaaaaa tcatacagct cgcgcggatc 9900
tttaaatgga gtgtcttctt cccagttttc gcaatccaca tcggccagat cgttattcag 9960
taagtaatcc aattcggcta agcggctgtc taagctattc gtatagggac aatccgatat 10020
gtcgatggag tgaaagagcc tgatgcactc cgcatacagc tcgataatct tttcagggct 10080
ttgttcatct tcatactctt ccgagcaaag gacgccatcg gcctcactca tgagcagatt 10140
gctccagcca tcatgccgtt caaagtgcag gacctttgga acaggcagct ttccttccag 10200
ccatagcatc atgtcctttt cccgttccac atcataggtg gtccctttat accggctgtc 10260
cgtcattttt aaatataggt tttcattttc tcccaccagc ttatatacct tagcaggaga 10320
cattccttcc gtatctttta cgcagcggta tttttcgatc agttttttca attccggtga 10380
tattctcatt ttagccattt attatttcct tcctcttttc tacagtattt aaagataccc 10440
caagaagcta attataacaa gacgaactcc aattcactgt tccttgcatt ctaaaacctt 10500
aaataccaga aaacagcttt ttcaaagttg ttttcaaagt tggcgtataa catagtatcg 10560
acggagccga ttttgaaacc gcggtgatca caggcagcaa cgctctgtca tcgttacaat 10620
caacatgcta ccctccgcga gatcatccgt gtttcaaacc cggcagctta gttgccgttc 10680
ttccgaatag catcggtaac atgagcaaag tctgccgcct tacaacggct ctcccgctga 10740
cgccgtcccg gactgatggg ctgcctgtat cgagtggtga ttttgtgccg agctgccggt 10800
cggggagctg ttggctggct ggtggcagga tatattgtgg tgtaaacaaa ttgacgctta 10860
gacaacttaa taacacattg cggacgtttt taatgtactg aattaacgcc gaattaattc 10920
gggggatctg gattttagta ctggattttg gttttaggaa ttagaaattt tattgataga 10980
agtattttac aaatacaaat acatactaag ggtttcttat atgctcaaca catgagcgaa 11040
accctatagg aaccctaatt cccttatctg ggaactactc acacattatt atggagaaac 11100
tcgagcttgt cgatcgacag atccggtcgg catctactct atttctttgc cctcggacga 11160
gtgctggggc gtcggtttcc actatcggcg agtacttcta cacagccatc ggtccagacg 11220
gccgcgcttc tgcgggcgat ttgtgtacgc ccgacagtcc cggctccgga tcggacgatt 11280
gcgtcgcatc gaccctgcgc ccaagctgca tcatcgaaat tgccgtcaac caagctctga 11340
tagagttggt caagaccaat gcggagcata tacgcccgga gtcgtggcga tcctgcaagc 11400
tccggatgcc tccgctcgaa gtagcgcgtc tgctgctcca tacaagccaa ccacggcctc 11460
cagaagaaga tgttggcgac ctcgtattgg gaatccccga acatcgcctc gctccagtca 11520
atgaccgctg ttatgcggcc attgtccgtc aggacattgt tggagccgaa atccgcgtgc 11580
acgaggtgcc ggacttcggg gcagtcctcg gcccaaagca tcagctcatc gagagcctgc 11640
gcgacggacg cactgacggt gtcgtccatc acagtttgcc agtgatacac atggggatca 11700
gcaatcgcgc atatgaaatc acgccatgta gtgtattgac cgattccttg cggtccgaat 11760
gggccgaacc cgctcgtctg gctaagatcg gccgcagcga tcgcatccat agcctccgcg 11820
accggttgta gaacagcggg cagttcggtt tcaggcaggt cttgcaacgt gacaccctgt 11880
gcacggcggg agatgcaata ggtcaggctc tcgctaaact ccccaatgtc aagcacttcc 11940
ggaatcggga gcgcggccga tgcaaagtgc cgataaacat aacgatcttt gtagaaacca 12000
tcggcgcagc tatttacccg caggacatat ccacgccctc ctacatcgaa gctgaaagca 12060
cgagattctt cgccctccga gagctgcatc aggtcggaga cgctgtcgaa cttttcgatc 12120
agaaacttct cgacagacgt cgcggtgagt tcaggctttt tcatatctca ttgccccccg 12180
ggatctgcga aagctcgaga gagatagatt tgtagagaga gactggtgat ttcagcgtgt 12240
cctctccaaa tgaaatgaac ttccttatat agaggaaggt cttgcgaagg atagtgggat 12300
tgtgcgtcat cccttacgtc agtggagata tcacatcaat ccacttgctt tgaagacgtg 12360
gttggaacgt cttctttttc cacgatgctc ctcgtgggtg ggggtccatc tttgggacca 12420
ctgtcggcag aggcatcttg aacgatagcc tttcctttat cgcaatgatg gcatttgtag 12480
gtgccacctt ccttttctac tgtccttttg atgaagtgac agatagctgg gcaatggaat 12540
ccgaggaggt ttcccgatat taccctttgt tgaaaagtct caatagccct ttggtcttct 12600
gagactgtat ctttgatatt cttggagtag acgagagtgt cgtgctccac catgttatca 12660
catcaatcca cttgctttga agacgtggtt ggaacgtctt ctttttccac gatgctcctc 12720
gtgggtgggg gtccatcttt gggaccactg tcggcagagg catcttgaac gatagccttt 12780
cctttatcgc aatgatggca tttgtaggtg ccaccttcct tttctactgt ccttttgatg 12840
aagtgacaga tagctgggca atggaatccg aggaggtttc ccgatattac cctttgttga 12900
aaagtctcaa tagccctttg gtcttctgag actgtatctt tgatattctt ggagtagacg 12960
agagtgtcgt gctccaccat gttggcaagc tgctctagcc aatacgcaaa ccgcctctcc 13020
ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg 13080
gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac 13140
actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag 13200
gaaacagcta tgaccatgat tacgaattc 13229
Claims (4)
1.一种以黄花蒿悬浮细胞系为受体的转iaaM基因提高黄花蒿中青蒿素含量的方法，其特征在于，所述方法包括如下步骤：
(1)黄花蒿腺毛细胞特异表达基因的载体重组构建：
将黄花蒿腺毛特异启动子AaGTpro和iaaM基因构建到Ti质粒的T-DNA区，以HPT基因为选择标记基因，以pWM101作为出发载体进行改造和载体构建；得到重组Ti质粒pWM101-AaGTpro::iaaM，所述黄花蒿腺毛特异启动子AaGTpro的序列如SEQ ID NO.1所示，所述重组Ti质粒pWM101-AaGTpro::iaaM的序列如SEQ ID NO.8所示；
(2)工程化根癌农杆菌的制备：
构建的重组Ti质粒通过电激转化法转化根癌农杆菌LBA4404，经过抗生素筛选和分子检测验证工程农杆菌，培养工程农杆菌至对数生长后期；
(3)黄花蒿悬浮细胞系的建立：
将深绿色或淡黄色生长快、结构疏松颗粒状的黄花蒿愈伤组织置于MS液体培养基中，将愈伤轻轻夹碎，摇床150r/min，光照周期为16h/8h光暗交替，温度为25±2℃振荡培养；培养15—20d为悬浮细胞的快速增长且细胞活性较高期，选取该时期的直径在5mm以内的小细胞团及含有单细胞的黄花蒿悬浮细胞系悬浮液，备用；
(4)黄花蒿悬浮细胞的共培浸染转化：
将步骤(3)所得的黄花蒿悬浮细胞系悬浮液与步骤(2)中生长至对数生长后期的工程农杆菌共培2.5—3.5d，然后转入筛选培养基上培养；
(5)转化细胞的筛选和获得抗性愈伤组织：
经步骤(4)共培2.5—3.5d后的黄花蒿悬浮细胞经MS液体培养基冲洗后，置于含200mg/L羧苄青霉素和30mg/LHPT抗性的诱导愈伤及丛生芽分化的培养基上，培养至愈伤上有丛生芽的分化，获得抗性丛生芽并继代增殖；
(6)诱导根分化和获得转基因植株：
当抗性丛生芽生长至3—5cm大小时，将抗性丛生芽转移到生根培养基诱导根分化，分化后幼苗经过炼苗后移栽至营养基质中，获得转基因植株；
(7)转基因植株的分子检测和腺毛细胞发育观察：
提取转基因植株叶片的DNA，用PCR法进行目的基因的分子检测验证，通过体视荧光显微镜观察转基因植株叶片腺毛细胞的密度、大小及荧光强度，通过与对照比较，判断腺毛的发育情况，对腺毛发育具有显著促进的植株进行青蒿素含量测定；
(8)建立转基因黄花蒿品系：
筛选青蒿素含量显著提高的转基因黄花蒿植株，通过组织培养繁殖建立转基因高青蒿素含量的黄花蒿品系。
2.如权利要求1所述的方法，其特征在于，步骤(1)是用HindIII和BamH1双酶切pWM101载体，将AaGTpro替换pWM101中的35Spro，完成PWM101-AaGTpro重组载体构建，再用BamH1和PSt1酶切重组载体PWM101-AaGTpro，将iaaM基因插入，构建得到腺毛特异表达的重组Ti质粒pWM101-AaGTpro::iaaM。
3.如权利要求1所述的方法，其特征在于，步骤(1)是从GenBank中的Nucleotide数据库中获得黄花蒿腺毛特异启动子AaDB2序列信息，设计克隆和同源重组引物，以黄花蒿基因组DNA为模板，通过PCR扩增技术，获得黄花蒿腺毛特异启动子AaGTpro序列。
4.如权利要求2所述的方法，其特征在于，构建腺毛特异表达的重组Ti质粒pWM101-AaGTpro::iaaM所用引物如下：
上游引物AaGTpro UP1：CCCAAGCTTTGAAGGATGACCAAAAGCATAA；
下游引物AaGTpro DN1：AGTCATAGGAGCTAACACCACCCT；
上游引物AaGTpro UP2：CCCAAGCTTTGAAGGATGACCAAAAGCATAA；
下游引物AaGTpro DN2：CGGGATCCTATTGAATTTGATGTTGATCAGG；
iaaM UP：GCGGATCCATGTCAGCCTCATCTCTTCT；
iaaM DN：AACTGCAGTTAATTTCTATTGCGGTAGTTATATC。
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