CN115386589A - 一种玉米自交系双载体遗传转化系统、其构建方法及其应用 - Google Patents
一种玉米自交系双载体遗传转化系统、其构建方法及其应用 Download PDFInfo
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Abstract
本发明涉及一种玉米自交系双载体遗传转化系统、其构建方法及其应用,能优化玉米自交系遗传转化效率的双载体遗传转化体系的建立,该体系使难以转化且重要的玉米品系能进行高效转化。该体系首先构建pHB‑ZmBBM‑ZmWUS诱导载体和pCAMBIA3301‑GFP报告载体,并利用基因枪轰击结合农杆菌介导法转化玉米自交系,使玉米自交系郑58、PH4CV和Mo17实现转化并获得转基因植株。其中,转基因郑58转化率最高,且其整个转化周期仅52天,比传统转化周期缩短30天。双载体遗传转化体系的建立大大缩短了转化周期,从而有助于玉米优质性状的遗传改良。
Description
技术领域
本发明涉及一种优化玉米自交系遗传转化效率的双载体转化体系及其应用,该体系使难以转化且重要的玉米品系能进行高效转化。
技术背景
玉米(Zea mays L.)作为全世界广泛种植的禾本科作物之一,主要用于粮食、动物饲料及各种行业原料。随着世界人口的增长和食品消费的增加,人们对玉米产量及质量的需求日夜增大。玉米分子育种的快速发展,使得这种需求成为现实,尤其遗传转化技术的应用,从基因层面为改良玉米品种、保留优势性状及育种提供新策略。
遗传转化技术中,组织培养是玉米遗传转化技术的关键环节。不同玉米品种的遗传背景复杂且不同,玉米基因型也差异很大,致使不同基因型的再生能力也千差万别。传统遗传转化技术已改善许多难以转化的玉米自交系,但更有价值的自交系的遗传转化问题仍是巨大的挑战。借用特异的启动子及新型启动子诱导基因组成型表达,建立高效的遗传转化体系,以期为改良玉米新品种发挥重要的作用。
植物胚胎细胞从分化出各种组织,再发育产生根茎叶等各器官;或由植物体分生组织直接分化器官再形成植株的过程。整个植物形态发生的过程受到内在基因协同控制和外界环境因素的影响。近些年,研究者发现形态发生的相关基因过度表达可刺激转基因植物的生长,进而改善植物遗传转化。
BABYBOOM(BBM)是一种形态发生基因,通过其在发育中的种子中的表达模式和过度表达表型的结合,在胚胎发生过程中促进细胞增殖和形态发生。WUSCHEL(WUS)是调控茎和花分生组织形态发生的因子,在维持结构和功能完整性方面起着重要作用。BBM/WUS的共表达可成功诱导高粱未成熟胚、甘蔗愈伤组织和籼稻愈伤组织获得转基因植株。然而,许多形态发生基因的强烈表达往往导致产生多效性和有害表型,导致转基因TO植株及其后代的低生育率。目前,有效缓解这一局面具有挑战性。
发明内容
本发明的目的之一在于提供一种玉米自交系双载体遗传转化系统的设计和构建方法。
本发明的目的之二在于提供一种双载体遗传转化体系构建的培养流程和应用。本发明还提供双载体体系转基因玉米后代基因型鉴定与分离分析方法。
为达到上述目的,本发明采用如下技术方案:
一种玉米自交系双载体遗传转化系统,该双载体遗传转化系统包含pHB-ZmBBM-ZmWUS诱导载体和pCAMBIA3301-GFP报告载体,其中,在愈伤中胚胎发育调控因子(BBM)强表达,分生组织形态发生因子(WUS)正常表达。
一种本发明所述玉米自交系双载体遗传转化系统的构建方法,采用如下玉米自交系双载体遗传转化体系的具体培养流程:
利用基因枪轰击,结合农杆菌介导法转化玉米自交系,将双载体导入玉米未成熟胚中,通过共培养、恢复培养、无筛选培养、预再生培养、暗再生培养、光再生培养等过程,获得转基因植株。
一种本发明所述玉米自交系双载体遗传转化系统的应用,使玉米自交系郑58、PH4CV、Mo17、昌7-2和B73成功转化,并获得转基因植株。
一种本发明所述的双载体转基因玉米的分离分析,设计特异性引物对所获取的转基因玉米进行鉴定及其后代分离分析。
优选地,本发明双载体遗传转化体系的构建,该载体包含pHB-ZmBBM-ZmWUS共表达的诱导载体和pCAMBIA3301-GFP报告载体,在愈伤中BBM保持强表达,WUS正常表达。其中玉米愈伤组织特异性磷脂转移酶蛋白基因(ZmPLTP)、玉米几丁质酶A1基因(ZmCTA1)在愈伤组织中的表达量最高,故采用ZmCTA1pro和ZmPLTPpro驱动ZmBBM(ZmCTA1pro::BBM andZmPLTPpro::BBM);玉米生长素诱导启动子(Axig1pro)与Nopaline合成酶启动子(NOSpro)在不产生任何激素的情况下正常表达,因此采用NOSpro和ZmAxig1pro驱动的ZmWUS(NOSpro::WUS and ZmAxig1pro::WUS)。报告pCAMBIA3301载体(pCA-GFP),是花椰菜花叶病毒35S启动子(35Spro)驱动绿色荧光蛋白GFP(35Spro::GFP)。
基因序列(NOSpro-WUS-RbsT)如下:
AAGCTTGATCATGAGCGGAGAATTAAGGGAGTCACGTTATGACCCCCGCCGATGACGCGGGACAAGCCGTTTTACGTTTGGAACTGACAGAACCGCAACGTTGAAGGAGCCACTCAGCCGCGGGTTTCTGGAGTTTAATGAGCTAAGCACATACGTCAGAAACCATTATTGCGCGTTCAAAAGTCGCCTAAGGTCACTATCAGCTAGCAAATATTTCTTGTCAAAAATGCTCCACTGACGTTCCATAAATTCCCCTCGGTATCCAATTAGAGTCTCATATTCACTCTCAATCCAAATAATCTGCACCGGATCTGTCGACATGGCGGCCAATGCGGGCGGCGGTGGAGCGGGAGGAGGCAGCGGCAGCGGCAGCGTGGCTGCGCCGGCGGTGTGCCGCCCCAGCGGCTCGCGGTGGACGCCGACGCCGGAGCAGATCAGGATGCTGAAGGAGCTCTACTACGGCTGCGGCATCCGGTCGCCCAGCTCGGAGCAGATCCAGCGCATCACCGCCATGCTGCGGCAGCACGGCAAGATCGAGGGCAAGAACGTCTTCTACTGGTTCCAGAACCACAAGGCCCGCGAGCGCCAGAAGCGCCGCCTCACCAGCCTCGACGTCAACGTGCCCGCCGCCGGCGCGGCCGACGCCACCACCAGCCAACTCGGCGTCCTCTCGCTGTCGTCGCCGCCGCCTTCAGGCGCGGCGCCTCCCTCGCCCACCCTCGGCTTCTACGCCGCCGGCAATGGCGGCGGATCGGCTGTGCTGCTGGACACGAGTTCCGACTGGGGCAGCAGCGGCGCTGCCATGGCCACCGAGACATGCTTCCTGCAGGACTACATGGGCGTGACGGACACGGGCAGCTCGTCGCAGTGGCCACGCTTCTCGTCGTCGGACACGATAATGGCGGCGGCCGCGGCGCGGGCGGCGACGACGCGGGCGCCCGAGACGCTCCCTCTCTTCCCGACCTGCGGCGACGACGGCGGCAGCGGTAGCAGCAGCTACTTGCCGTTCTGGGGTGCCGCGTCCACAACTGCCGGCGCCACTTCTTCCGTTGCGATCCAGCAGCAACACCAGCTGCAGGAGCAGTACAGCTTTTACAGCAACAGCAACAGCACCCAGCTGGCCGGCACCGGCAACCAAGACGTATCGGCAACAGCAGCAGCAGCCGCCGCCCTGGAGCTGAGCCTCAGCTCATGGTGCTCCCCTTACCCTGCTGCAGGGAGTATGTGACCCGGGCTTTCGTTCGTATCATCGGTTTCGACAACGTTCGTCAAGTTCAATGCATCAGTTTCATTGCGCACACACCAGAATCCTACTGAGTTTGAGTATTATGGCATTGGGAAAACTGTTTTTCTTGTACCATTTGTTGTGCTTGTAATTTACTGTGTTTTTTATTCGGTTTTCGCTATCGAACTGTGAAATGGAAATGGATGGAGAAGAGTTAATGAATGATATGGTCCTTTTGTTCATTCTCAAATTAATATTATTTGTTTTTTCTCTTATTTGTTGTGTGTTGAATTTGAAATTATAAGAGATATGCAAACATTTTGTTTTGAGTAAAAATGTGTCAAATCGTGGCCTCTAATGACCGAAGTTAATATGAGGAGTAAAACACTTGTAGTTGTACCATTATGCTTATTCACTAGGCAACAAATATATTTTCAGACCTAGAAAAGCTGCAAATGTTACTGAATACAAGTATGTCCTCTTGTGTTTTAGACATTTATGAACTTTCCTTTATGTAATTTTCCAGAATCCTTGTCAGATTCTAATCATTGCTTTATAATTATAGTTATACTCATGGATTTGTAGTTGAGTATGAAAATATTTTTTAATGCATTTTATGACTTGCCAATTGATTGACAACATGGAATTC
1-6bp:HindⅢ
7-313bp:NOS启动子
314-319bp:SalⅠ
320-1228bp:WUS
1229-1234bp:SmaⅠ
1235-1868:RbsT终止子
1869-1874bp:EcoRⅠ。
基因序列(Axig1pro-Wus-RbsT)如下:
AAGCTTCCCATCGCTGCTTTGTCTACATCATGTTCTTCATCATCCTCCCCAGGCGACGCGTGCTGCTGTTCTTATTCAGACTACCGTTCGAGTGACTGCATGGCGTACATCTTTCTGCATCGACTTTGTACGGCTACATCGAACATATACACGAGATGTCTCGTGTGAATAGAGTCACTAAGCCTTAAGCATCGGTTACTCCGTAGGGTACATTCTGTTTCTTCTTATTTGTGCATATTTTTATTGTTATTTACTGATTATACGAGTAGTTATACATACATGCACATACATATCATCACATATATCACAATATTTTTCTAAATTAAATTAAAACTAAAAATGACTAAATTTCTAACACCAACGACATTGTAATGTTTTCTCCAACAACTTTACCTATTCTACATTGTTCTATTTCGAATTTCACTCTATAAACAACATAGTCTACAATGGAAAACAGTGCTTTGTACGACTATATACGCGATGTGTGGCTACAACATAAGACAATATAGTCGTTTGAAGATTGAACCTATATATCGGTACGGTTAATCCGTCTATGTACGTGGGCATGACGAACACCCGTGATAACGAAGGATTAACGTGCACAATCATAAATCCAAAGTAGGAGCGGTGCATGATGAGAATCGCTCTCAGTACTCGACATAATGAACCTTACGAGGTACAACAGGCAGGCAGGCAGGGACCAGGGGCCGCCTTTATTTCAGGCTCGCTGGCCCCACGGGCGTGCTGCGTGCACGAAGGGCACTACCCCAACCTCTCACCGAAAAACCGCGCTGGATCGGCAAATCAAACGAGGTGGTGCCCCGTGCCCACTCTCCACGTCCACGGCACCATCCCTCTGCAGCCGCTCACCAGCCATGCCGTGTCGCGGAACGGCACAACCACCCCCAACCCACTCACGAAACCCCGTCCCGGCCGTGCCCGTGTCGGTCCGCGCTCGGCAACGAGGCGGCCCGCGCTGCTGAGTCCCCTGGACACCCGACACCCTGTCGGCCCTTTGTTTATTCATCCCGAAATCTCATCTGCCCCCACGGCCGACTGCGCTGCGCTGCGCTGCGCCGCCCGGATATATATACCCATCGTTATCGATCGATCGATCGCGTCACTCACGGGTAGCTCATGGTCGAGCGTAGCATGCAGGAACTTATTTGCCGTGCGCTCCCAGGTCTCCGCTCGCGTGCCTTCCAGTCTGTCTCACACTAGCTGCTGTGGGACGATCGAAGTGGGTGTGTCAGCTAGCTAGCTGCGCCGTGACCACGCACATGACCGCAGTGCGCGCGGGGCTGATCAAGGGAAAGTGATCCCGTCGACATGGCGGCCAATGCGGGCGGCGGTGGAGCGGGAGGAGGCAGCGGCAGCGGCAGCGTGGCTGCGCCGGCGGTGTGCCGCCCCAGCGGCTCGCGGTGGACGCCGACGCCGGAGCAGATCAGGATGCTGAAGGAGCTCTACTACGGCTGCGGCATCCGGTCGCCCAGCTCGGAGCAGATCCAGCGCATCACCGCCATGCTGCGGCAGCACGGCAAGATCGAGGGCAAGAACGTCTTCTACTGGTTCCAGAACCACAAGGCCCGCGAGCGCCAGAAGCGCCGCCTCACCAGCCTCGACGTCAACGTGCCCGCCGCCGGCGCGGCCGACGCCACCACCAGCCAACTCGGCGTCCTCTCGCTGTCGTCGCCGCCGCCTTCAGGCGCGGCGCCTCCCTCGCCCACCCTCGGCTTCTACGCCGCCGGCAATGGCGGCGGATCGGCTGTGCTGCTGGACACGAGTTCCGACTGGGGCAGCAGCGGCGCTGCCATGGCCACCGAGACATGCTTCCTGCAGGACTACATGGGCGTGACGGACACGGGCAGCTCGTCGCAGTGGCCACGCTTCTCGTCGTCGGACACGATAATGGCGGCGGCCGCGGCGCGGGCGGCGACGACGCGGGCGCCCGAGACGCTCCCTCTCTTCCCGACCTGCGGCGACGACGGCGGCAGCGGTAGCAGCAGCTACTTGCCGTTCTGGGGTGCCGCGTCCACAACTGCCGGCGCCACTTCTTCCGTTGCGATCCAGCAGCAACACCAGCTGCAGGAGCAGTACAGCTTTTACAGCAACAGCAACAGCACCCAGCTGGCCGGCACCGGCAACCAAGACGTATCGGCAACAGCAGCAGCAGCCGCCGCCCTGGAGCTGAGCCTCAGCTCATGGTGCTCCCCTTACCCTGCTGCAGGGAGTATGTGACCCGGGCTTTCGTTCGTATCATCGGTTTCGACAACGTTCGTCAAGTTCAATGCATCAGTTTCATTGCGCACACACCAGAATCCTACTGAGTTTGAGTATTATGGCATTGGGAAAACTGTTTTTCTTGTACCATTTGTTGTGCTTGTAATTTACTGTGTTTTTTATTCGGTTTTCGCTATCGAACTGTGAAATGGAAATGGATGGAGAAGAGTTAATGAATGATATGGTCCTTTTGTTCATTCTCAAATTAATATTATTTGTTTTTTCTCTTATTTGTTGTGTGTTGAATTTGAAATTATAAGAGATATGCAAACATTTTGTTTTGAGTAAAAATGTGTCAAATCGTGGCCTCTAATGACCGAAGTTAATATGAGGAGTAAAACACTTGTAGTTGTACCATTATGCTTATTCACTAGGCAACAAATATATTTTCAGACCTAGAAAAGCTGCAAATGTTACTGAATACAAGTATGTCCTCTTGTGTTTTAGACATTTATGAACTTTCCTTTATGTAATTTTCCAGAATCCTTGTCAGATTCTAATCATTGCTTTATAATTATAGTTATACTCATGGATTTGTAGTTGAGTATGAAAATATTTTTTAATGCATTTTATGACTTGCCAATTGATTGACAACATGGAATTC
1-6bp:HindⅢ
7-1323bp:Axig1启动子
1324-1329bp:SalⅠ
1330-2238bp:WUS
2239-2244bp:SmaⅠ
2245-2878:RbsT终止子
2879-2884bp:EcoRⅠ。
基因序列(CTA1pro-BBM-RbsT)如下:
AAGCTTTGCGTACGATTACATCGAACATGCACACGAGATATCTCGTGTGAATGAAGCCACTTTTGCCTTGAGCATCGGAGCATCCGTAGGGCACACTTTGTTCTAAGTATTTGTGCATATTTTACTGTTGTTTACTGTTTACGTGAGTAGTAATACACATACATATACATGTTGTCACATATATCACTGTGATTTTCTAGATTAAATTAAAACTGAAAATATCTATTTCTTGAACAATATAAGTGCTAAAACAACTAATATTTTGGTCTGGAGGGAGTTTTACATAACAAGGGGGGAGTATGTCGTTGTATAGACGAGGCAATTTATTGTTTTAGCTCAAGCCCCACGAAACTAACTCCGTCTCAAGAAAATCTTCTAAATTCCTCGCGAGAGAACACAATACTCTATTATACTCCCTTTTTATTTTCTTTACTTGACGTTTTGTAGTTCATTTTTGTACTATTTAGCGTCAGATATAAAAAGATGAATATACTCTAAGAGTAGCGAAACCATGAATAGTGAATAAGGAAGAAAAACTCTAAAGAGCTTTCTAGAGAAATACTAAAAATATATAAAAAGGGAAAATCCTAGTCTTCGTTTGATCGTGCATGCATATGTGTGGCCCAACCGCCCAATCCATCACCGGTACGTGCTCCAGGCTGTAGCTGCTTGGAAGTCTTCCGTTCTTGCTTGACCGATTCCTGCTAGCCTTTGGAACACGGAGACCGAGGCCACCAATAAATCCTCACATGCGTGGTACTGGTACGTGGAATTGAAGCAAACAAAATACGGCTGCCCGCGGCGGCCGGATAATATATAAGATGATGGCGGCAAATTGTCTTCGCAGACGAGAAAAATCGCAGGAAAGCGGTGACTTCTAGCAGGTAGCATGTGCGGATTTTCCACCGCGATGGGCGGCGGCGGCGGCAATCCAAATCTGATGATATCTCAACCTAGGCTAGAGTGCTTGTGTCTCGATACATGGTGCGTTGCATGCATTGCTGACCCATGCGTCAAAAGTTCATCATAAAACGTGGGACAGCTGTACGTTCCCGCGTGCAATGGTGGACATCACACCTAGCCACTCCACGTCGCATGCCATGCATCGACAGGATGCACGTTCCGATCCCTATATAAAGGGGCACCCTTGTGACACCTCAAATCAGTCGACATGGCCACTGTGAACAACTGGCTCGCTTTCTCCCTCTCCCCGCAGGAGCTGCCGCCCTCCCAGACGACGGACTCCACGCTCATCTCGGCCGCCACCGCCGACCATGTCTCCGGCGATGTCTGCTTCAACATCCCCCAAGATTGGAGCATGAGGGGATCAGAGCTTTCGGCGCTCGTCGCGGAGCCGAAGCTGGAGGACTTCCTCGGCGGCATCTCCTTCTCCGAGCAGCATCACAAGTCCAACTGCAACTTGATACCCAGCACTAGCAGCACAGTTTGCTACGCGAGCTCAGCTGCTAGCACCGGCTACCATCACCAGCTGTACCAGCCCACCAGCTCCGCGCTCCACTTCGCGGACTCCGTCATGGTGGCCTCCTCGGCCGGTGTCCACGACGGCGGTGCCATGCTCAGCGCGGCCGCCGCTAACGGTGTCGCTGGCGCTGCCAGTGCCAACGGCGGCGGCATCGGGCTGTCCATGATCAAGAACTGGCTGCGGAGCCAACCGGCGCCCATGCAGCCGAGGGCGGCGGCGGCTGAGGGCGCGCAGGGGCTCTCTTTGTCCATGAACATGGCGGGGACGACCCAAGGCGCTGCTGGCATGCCACTTCTCGCTGGAGAGCGCGCACGGGCGCCCGAGAGTGTATCGACGTCAGCACAGGGTGGTGCCGTCGTCGTCACGGCGCCGAAGGAGGATAGCGGTGGCAGCGGTGTTGCCGGTGCTCTAGTAGCCGTGAGCACGGACACGGGTGGCAGCGGCGGCGCGTCGGCTGACAACACGGCAAGGAAGACGGTGGACACGTTCGGGCAGCGCACGTCGATTTACCGTGGCGTGACAAGGCATAGATGGACTGGGAGATATGAGGCACATCTTTGGGATAACAGTTGCAGAAGGGAAGGACAAACTCGTAAGGGTCGTCAAGTCTATTTAGGTGGCTATGATAAAGAGGAGAAAGCTGCTAGGGCTTATGATCTTGCTGCTCTGAAGTACTGGGGTCCCACAACAACAACAAATTTCCCAGTGAGTAACTACGAAAAGGAGCTCGAGGACATGAAGCACATGACAAGGCAGGAGTTTGTAGCGTCTCTGAGAAGGAAGAGCAGTGGTTTCTCCAGAGGTGCATCCATTTACAGGGGAGTGACTAGGCATCACCAACATGGAAGATGGCAAGCACGGATTGGACGAGTTGCAGGGAACAAGGATCTTTACTTGGGCACCTTCAGCACCCAGGAGGAGGCAGCGGAGGCGTACGACATCGCGGCGATCAAGTTCCGCGGCCTAAACGCCGTCACCAACTTCGACATGAGCCGCTACGACGTGAAGAGCATCCTGGACAGCAGCGCCCTCCCCATCGGCAGCGCCGCCAAGCGCCTCAAGGAGGCCGAGGCCGCAGCGTCCGCGCAGCACCACCACGCCGGCGTGGTGAGTTACGACGTCGGCCGCATCGCCTCGCAGCTCGGCGACGGCGGAGCCCTGGCGGCGGCGTACGGCGCGCACTACCACGGCGCCGCCTGGCCGACCATCGCGTTCCAGCCGGGCGCCGCCACCACAGGCCTGTACCACCCGTACGCGCAGCAGCCAATGCGCGGCGGCGGGTGGTGCAAGCAGGAGCAGGACCACGCGGTGATCGCGGCCGCGCACAGCCTGCAGGACCTCCACCACCTGAACCTGGGCGCGGCCGGCGCGCACGACTTTTTCTCGGCAGGGCAGCAGGCCGCCGCCGCTGCGATGCACGGCCTGGGTAGCATCGACAGTGCGTCGCTCGAGCACAGCACCGGCTCCAACTCCGTCGTCTACAACGGCGGGGTCGGCGACAGCAACGGCGCCAGCGCCGTCGGCGGCAGTGGCGGTGGCTACATGATGCCGATGAGCGCTGCCGGAGCAACCACTACATCGGCAATGGTGAGCCACGAGCAGGTGCATGCACGGGCCTACGACGAAGCCAAGCAGGCTGCTCAGATGGGGTACGAGAGCTACCTGGTGAACGCGGAGAACAATGGTGGCGGAAGGATGTCTGCATGGGGGACTGTCGTGTCTGCAGCCGCGGCGGCAGCAGCAAGCAGCAACGACAACATGGCCGCCGACGTCGGCCATGGCGGCGCGCAGCTCTTCAGTGTCTGGAACGACACTTAAGTCGACCTTTCGTTCGTATCATCGGTTTCGACAACGTTCGTCAAGTTCAATGCATCAGTTTCATTGCGCACACACCAGAATCCTACTGAGTTTGAGTATTATGGCATTGGGAAAACTGTTTTTCTTGTACCATTTGTTGTGCTTGTAATTTACTGTGTTTTTTATTCGGTTTTCGCTATCGAACTGTGAAATGGAAATGGATGGAGAAGAGTTAATGAATGATATGGTCCTTTTGTTCATTCTCAAATTAATATTATTTGTTTTTTCTCTTATTTGTTGTGTGTTGAATTTGAAATTATAAGAGATATGCAAACATTTTGTTTTGAGTAAAAATGTGTCAAATCGTGGCCTCTAATGACCGAAGTTAATATGAGGAGTAAAACACTTGTAGTTGTACCATTATGCTTATTCACTAGGCAACAAATATATTTTCAGACCTAGAAAAGCTGCAAATGTTACTGAATACAAGTATGTCCTCTTGTGTTTTAGACATTTATGAACTTTCCTTTATGTAATTTTCCAGAATCCTTGTCAGATTCTAATCATTGCTTTATAATTATAGTTATACTCATGGATTTGTAGTTGAGTATGAAAATATTTTTTAATGCATTTTATGACTTGCCAATTGATTGACAACATGGGATCC
1-6bp:HindⅢ
7-1165bp:CTA1启动子
1166-1171bp:SalⅠ
1172-3301bp:BBM
3302-3307bp:SalⅠ
3308-3941bp:Rbst终止子
3942-3947bp:BamHⅠ。
基因序列(PLTPpro-BBM-RbsT)如下:
AAGCTTATCTATCCCCCACAACCACGCTCTAGCTTTTCAGAGAACATGTTGTTATATATAACCCCGAATTGTTCAACTATATATTGCAGCTAATGACACTTTAACTTTGTTAAGAATAGCTATTTGGTTTACTTGTGACTTTTTTTAATGCCACTAGTACTGACTTATTAGACAAATGTTCATTCTTTAATCTAGCTCTAATTCTAATATCTACCAGACTATATATCTTGTGTTGCTTACTTAATAAATGCTTTTATAAAAAAACATTTTAAAATGTTGATCTGTTTATTACTGAACATTGATTTGATTATTAGTGTTAACTATAGAGTAGACGAAAGTATGTTTCTTTGTAATTTAAAGGTTTTTTTACCACACGTGTAGTTCATATTTTTAGATCAAAAAAGTGAACAGCCTAGTGGCTACGGCCAAAGAAGCTCCCAACTCAAAAGTGTTAGGCATTGCTTCGTAATTTAAAGCATGAATTTCTTCGTGTGTCATCTCTTCACGCGGTCGCTCCCGGTCCGCACGCACGCCCCCACACAGGAAAAAAATAGTCCGCGCCCTCGCACCAGAAAATAAAAATAGAGGCACTAGGGTTCGAATCCAACCCTAGTTACTAGGGAAGCGATTGCTCGGCTGCTACCGCAGGTTCGATTATATTGCACAAGGATTGATATTGTAAATATAAATATATAGTAATAATTTAAAAATTAAAAATATAATAATACAAACCTAGCAATGTATGGGAACTAACTAGTATCCCCTCAGTCCTAAAATATAATTCTTTCTAGCCTATTTTTTTGTCCACATTCGTTCAAATGATAATGAATATAGATATATATGGAAACTATATTTATATGTTACTCAATGAATATGTAATTAATCTAAAACGAATTATATTTTAGGATAGAGGGAAGTATAAATAAAAGAAAGGAAATTCAAGAGACAAGTACGAGGAGTCGGGGACTTGGGGGAGGCAATTCCCTCAAACGACTAGCTGGCCTCCTCATTCGTTACGCAGGCCAGGCCGCACTGCGCTCACGGCTTTTAGGTTGCACCAGTCCGGTAGCTTGGTCCACGAACGGCCTAGCGGATCCGTTACCGGCAATGCCGACTTGGGCCACGTTCACAGCAGCCCAGCAATGACGGCTCGTCATGTTAAAAATCCGCTACAACTTTGTAAATTCCATACCTCTAGTATTCATGTGCCATGAATCATGAACCTATTGTTTGTCTTGGGAGTTTTCTGGATTCAAACTTACTAATTTATTTTCTGTCTATAAATACCCTGTAAAACTACTGGTAAATAAAAAGGAGAAACCAATCATTTTGTTCCATTTGTCTCGTTGCTTCCTTGTTTTAGAGAAAAATGTATCAGCAATTCTTAAACTTGACACACCGTCATTTGGACCCTAAACTTAGAAAATCAGGAAAACGAGTTTGGAGCTCTTGGACTCTGATGCAAATAAAGCATGAGATTTGCACGGGATAGAGGAAATTAATATATCACAGGTCCCGTTTAGTGTCCGTTCGGTTACCTAGAATTCGACCCCGTTCTACCTCATTAAGAGTATACGTATATACTCTCTTTATTTGTCGCGTTTTAGTTTAAAAATGAACTAGCAAGCCATAAGTATTGGCCCTAGTAGTAATAAACCATTTCAGCCAGGAATCATTCCAGTATTCAATCCGAAGGAACCGGAGAGGGTACATCAGCCCCTCGCCCAGCTTTGTTTTATAGTTTTTGTTTTGTATTTTCTGAATTATTAAGACAAATTTTGTAGTATCCGAGTAGCACATGCAGTGCGTGTCGTCGTACGTGTCATGAAAAAAAGGCGTGTGGACATATGGAACACACGGTCACATATGCGTCGACATGGCCACTGTGAACAACTGGCTCGCTTTCTCCCTCTCCCCGCAGGAGCTGCCGCCCTCCCAGACGACGGACTCCACGCTCATCTCGGCCGCCACCGCCGACCATGTCTCCGGCGATGTCTGCTTCAACATCCCCCAAGATTGGAGCATGAGGGGATCAGAGCTTTCGGCGCTCGTCGCGGAGCCGAAGCTGGAGGACTTCCTCGGCGGCATCTCCTTCTCCGAGCAGCATCACAAGTCCAACTGCAACTTGATACCCAGCACTAGCAGCACAGTTTGCTACGCGAGCTCAGCTGCTAGCACCGGCTACCATCACCAGCTGTACCAGCCCACCAGCTCCGCGCTCCACTTCGCGGACTCCGTCATGGTGGCCTCCTCGGCCGGTGTCCACGACGGCGGTGCCATGCTCAGCGCGGCCGCCGCTAACGGTGTCGCTGGCGCTGCCAGTGCCAACGGCGGCGGCATCGGGCTGTCCATGATCAAGAACTGGCTGCGGAGCCAACCGGCGCCCATGCAGCCGAGGGCGGCGGCGGCTGAGGGCGCGCAGGGGCTCTCTTTGTCCATGAACATGGCGGGGACGACCCAAGGCGCTGCTGGCATGCCACTTCTCGCTGGAGAGCGCGCACGGGCGCCCGAGAGTGTATCGACGTCAGCACAGGGTGGTGCCGTCGTCGTCACGGCGCCGAAGGAGGATAGCGGTGGCAGCGGTGTTGCCGGTGCTCTAGTAGCCGTGAGCACGGACACGGGTGGCAGCGGCGGCGCGTCGGCTGACAACACGGCAAGGAAGACGGTGGACACGTTCGGGCAGCGCACGTCGATTTACCGTGGCGTGACAAGGCATAGATGGACTGGGAGATATGAGGCACATCTTTGGGATAACAGTTGCAGAAGGGAAGGACAAACTCGTAAGGGTCGTCAAGTCTATTTAGGTGGCTATGATAAAGAGGAGAAAGCTGCTAGGGCTTATGATCTTGCTGCTCTGAAGTACTGGGGTCCCACAACAACAACAAATTTCCCAGTGAGTAACTACGAAAAGGAGCTCGAGGACATGAAGCACATGACAAGGCAGGAGTTTGTAGCGTCTCTGAGAAGGAAGAGCAGTGGTTTCTCCAGAGGTGCATCCATTTACAGGGGAGTGACTAGGCATCACCAACATGGAAGATGGCAAGCACGGATTGGACGAGTTGCAGGGAACAAGGATCTTTACTTGGGCACCTTCAGCACCCAGGAGGAGGCAGCGGAGGCGTACGACATCGCGGCGATCAAGTTCCGCGGCCTAAACGCCGTCACCAACTTCGACATGAGCCGCTACGACGTGAAGAGCATCCTGGACAGCAGCGCCCTCCCCATCGGCAGCGCCGCCAAGCGCCTCAAGGAGGCCGAGGCCGCAGCGTCCGCGCAGCACCACCACGCCGGCGTGGTGAGTTACGACGTCGGCCGCATCGCCTCGCAGCTCGGCGACGGCGGAGCCCTGGCGGCGGCGTACGGCGCGCACTACCACGGCGCCGCCTGGCCGACCATCGCGTTCCAGCCGGGCGCCGCCACCACAGGCCTGTACCACCCGTACGCGCAGCAGCCAATGCGCGGCGGCGGGTGGTGCAAGCAGGAGCAGGACCACGCGGTGATCGCGGCCGCGCACAGCCTGCAGGACCTCCACCACCTGAACCTGGGCGCGGCCGGCGCGCACGACTTTTTCTCGGCAGGGCAGCAGGCCGCCGCCGCTGCGATGCACGGCCTGGGTAGCATCGACAGTGCGTCGCTCGAGCACAGCACCGGCTCCAACTCCGTCGTCTACAACGGCGGGGTCGGCGACAGCAACGGCGCCAGCGCCGTCGGCGGCAGTGGCGGTGGCTACATGATGCCGATGAGCGCTGCCGGAGCAACCACTACATCGGCAATGGTGAGCCACGAGCAGGTGCATGCACGGGCCTACGACGAAGCCAAGCAGGCTGCTCAGATGGGGTACGAGAGCTACCTGGTGAACGCGGAGAACAATGGTGGCGGAAGGATGTCTGCATGGGGGACTGTCGTGTCTGCAGCCGCGGCGGCAGCAGCAAGCAGCAACGACAACATGGCCGCCGACGTCGGCCATGGCGGCGCGCAGCTCTTCAGTGTCTGGAACGACACTTAAGTCGACCTTTCGTTCGTATCATCGGTTTCGACAACGTTCGTCAAGTTCAATGCATCAGTTTCATTGCGCACACACCAGAATCCTACTGAGTTTGAGTATTATGGCATTGGGAAAACTGTTTTTCTTGTACCATTTGTTGTGCTTGTAATTTACTGTGTTTTTTATTCGGTTTTCGCTATCGAACTGTGAAATGGAAATGGATGGAGAAGAGTTAATGAATGATATGGTCCTTTTGTTCATTCTCAAATTAATATTATTTGTTTTTTCTCTTATTTGTTGTGTGTTGAATTTGAAATTATAAGAGATATGCAAACATTTTGTTTTGAGTAAAAATGTGTCAAATCGTGGCCTCTAATGACCGAAGTTAATATGAGGAGTAAAACACTTGTAGTTGTACCATTATGCTTATTCACTAGGCAACAAATATATTTTCAGACCTAGAAAAGCTGCAAATGTTACTGAATACAAGTATGTCCTCTTGTGTTTTAGACATTTATGAACTTTCCTTTATGTAATTTTCCAGAATCCTTGTCAGATTCTAATCATTGCTTTATAATTATAGTTATACTCATGGATTTGTAGTTGAGTATGAAAATATTTTTTAATGCATTTTATGACTTGCCAATTGATTGACAACATGTCTAGA
1-6bp:HindⅢ
7-1870bp:PLTP启动子
1871-1876bp:SalⅠ
1877-4006bp:BBM
3302-3307bp:SalⅠ
3308-4646bp:RbsT终止子
4647-4652bp:XbalⅠ。
优选地,本发明用于检测双载体体系后代分离的方法,设计特异性引物进行PCR反应鉴定,计算玉米自交系郑58、PH4CV、PH6WV、B73、Mo17和昌7-2的转化率,并对转基因后代进行基因型鉴定与分离分析。
优选地,本发明利用双载体体系转化玉米自交系郑58、PH4CV、PH6WC、Mo17、B73和昌72,其中玉米自交系郑58、PH4CV和Mo1实现转化并获得转基因植株。利用pHB-CB-NW载体和pCA-GFP载体,使郑58和PH4CV获得转基因植株,转化率分别为2.94%和2.64%。利用pHB-CB-AW载体和pCA-GFP载体,使郑58和Mo17获得转基因植株,转化率为0.14%和0.66%。其中,运用双载体遗传转化体系,转基因郑58(含ZmCTAlpro::BBM-NOSpro::WUS基因和35Spro::GFP基因)的转化率最高,其整个转化周期仅52天,比传统转化周期缩短30天。此外,在转基因郑58的后代中,GFP可稳定遗传并获得诱导载体分离现象,有效消除诱导载体中形态发生基因(ZmBBM-ZmWUS)的干扰。
本发明与现有技术相比较,具有如下显而易见的突出实质性特点和显著优点:
1.本发明建立优化的高效的双载体遗传转化体系,可帮助难以转化的玉米自交系获得再生植株,为探究具有抗病、抗虫等高质量的转基因新品系奠定基础;以难以转化的玉米自交系郑58、PH4CV、PH6WC、Mo17、昌7-2和B73的未成熟胚为受体材料,采用玉米愈伤组织特异性磷脂转移酶蛋白基因启动子ZmCTAlpro、磷脂转移酶蛋白启动子ZmPLTPpro驱动胚胎发育调控因子ZmBBM,Nopaline合成酶启动子及玉米生长素诱导启动子ZmAxiglpr驱动分生组织形态发生因子ZmWUS,并结合报告基因GFP,在基因枪轰击和农杆菌介导下转化未成熟胚,将基因转移、整合到玉米基因组中,并表达发挥作用,收获转基因阳性植株;
2.本发明运用高效的遗传转化体系可缩短转化周期间,帮助更多的玉米自交系获得转基因植株,改良并保留优势性状自交系;同时,通过后代基因分离,有效消除形态发生基因,避免其有害影响;
3.本发明为玉米遗传转化提供新思路,改良优质自交系,为研究抗性转基因新品系提供技术支持,加快开发更多未知玉米品系农艺和优质性状;
4.本发明是对现有玉米遗传转化技术的优化,为玉米转化提供技术支持,可用于玉米优质性状的遗传改良及育种的研究;本发明双载体遗传转化体系的建立可帮助许多难以转化的玉米品系实现稳定遗传转化获得转基因植株,推动开发高品质和具有抵抗各种胁迫能力的玉米新品系,提高玉米产量与质量,发掘更多玉米品系在农业和商业上的潜力。
附图说明
图1是双载体遗传转化体系载体构建示意图。
图2是双载体遗传转化流程。
图3是T0代转基因植株的转化率。
图4是转基因郑58的Southern blot拷贝数鉴定分析。
图5是T1代转基因郑58的GFP表达水平图
图6是转基因郑58的后代分离率。
具体实施方式
下面结合具体实施事例,进一步阐述本发明。应理解,这些实例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体实验条件的实验方法,通常按照常规条件,如分子克隆(Molecular Cloning:A Laboratory Manual,3rd ed.)或植物分子生物学-实验手册(Plant Molecular Biology-A Laboratory Manual,Melody S.Clark编,Springer-verlag Berlin Heidelberg,1997)中所述条件,或按照制造厂商所建议的条件。
实施例一
在本实施例中,双载体遗传转化体系的载体构建。
本发明采用强表达ZmBBM和正常表达ZmWUS的策略,构建诱导载体pHB-ZmBBM-ZmWUS。根据载体构建策略,分别从玉米基因组中克隆到ZmBBM、ZmWUS、ZmCTALpro、ZmPLTPpro、NOSpro、Axiglpro、RbsT终止子,从tair质粒中克隆到NOSpro。如在pHB-CB-NW载体中,ZmCTA1pro驱动ZmBBM,NOSpro驱动ZmWUS(ZmCTA1pro::BBM-NOSpro::WUS),二者均以RbsT终止,通过重组及酶切连接分别构建在pMD18-T中,再通过酶切连接依次转移到pHB载体中。在pCA-GFP中,花椰菜花叶病毒35S启动子(35Spro)驱动GFP(35Spro::GFP),RbsT进行终止,通过与pCAMBIA3301载体连接形成报告载体。
利用相同的原理,分别构建pHB-CB-AW和pHB-PB-AW双元表达载体。在pHB-CB-AW载体中,ZmCTA1pro驱动ZmBBM,ZmAxig1pro驱动ZmWUS(ZmCTA1pro::BBM-Axig1pro::WUS)。在pHB-PB-AW载体中,ZmPLTPpro驱动ZmBBM,ZmAxig1pro驱动ZmWUS(ZmPLTPpro::BBM-Axig1pro::WUS),如图1所示。
实施例二
在本实施例中,遗传转化流程。
实验选择基因枪法结合农杆菌介导的转化技术,选取六种玉米自交系(郑58、PH4CV、PH6WC、Mo17、B73和昌72),姊妹交授粉14-16天的幼胚(大小为1.5mm)作为受体材料,进行幼胚转化,如图2所示,具体流程如下:
1.未成熟胚在高渗培养基培养3小时、25℃、黑暗;
2.基因枪轰击未成熟胚,转入pHB-CB-NW或pHB-CB-AW或pHB-PB-AW质粒;
3.高渗培养基培养3小时、25℃、黑暗;
4.pCA-GFP载体进行农杆菌侵染;
5.转基因胚胎共培养3天、20℃、黑暗;
6.恢复培养7天、28℃、黑暗;
7.无筛选培养7天、28℃、黑暗;本步骤非必要步骤,本步骤可以不进行;
8.预再生培养14天、28℃、黑暗;
9.暗再生培养14天、25℃、黑暗;
10.光再生培养14-16天、25℃、光强80-100mE\m2\s、光周期16:8;
11.待发根、长出3-5cm的幼苗后,开盖、加ddH2O于光照培养箱培养2-3天;控制光周期16:8,温度day26℃\night22℃,光强350Em-2s-1;
12.小苗转到小盆培养10-14天;
13.鉴定为阳性的小苗移入大盆中培养,授粉并获得后代。
本实施例双载体遗传转化体系用基因枪法结合农杆菌介导的转化技术,使郑58、PH4CV和Mo17成功获得转基因植株。利用pHB-CB-NW载体和pCA-GFP载体,使郑58和PH4CV获得转基因植株,转化率分别为2.94%和2.64%。利用pHB-CB-AW载体和pCA-GFP载体,使郑58和Mo17获得转基因植株,转化率为0.14%和0.66%。对比发现,难以转化的自交系郑58用双载体遗传转化体系,整个培养转化周期为52-64天,最短可52天获得转基因植株,比传统转化所需时间短30天。
实施例三
在本实施例中,转基因阳性事件的叶片基因组大量抽提。
1.首先向50mL离心管中加入15mL基因组大量抽提液,并在离心管盖和离心管壁上做相应的标记;
2.取6叶期的双载体转基因玉米嫩叶,放入液氮中进行速冻,研磨成泛白的粉末;
3.将粉末放进相应编号的50mL离心管中(含有DNA大抽提取液),并加入20uL的10mg/mL的RNAase,上下颠倒,混合均匀,室温静置10min;
4.每管加入15mL的酚:氯仿:异戊醇(25:24:1),剧烈震荡,充分混匀,4℃,12000rpm离心10min;
5.取上清于新的50mL离心管中,加入0.7倍体积的异丙醇,上下轻轻颠倒,放置于-20℃,30min,使基因组DNA充分沉淀;
6.将絮状沉淀挑出,移到2mL EP管中,用70%乙醇洗3次;
7.吸尽乙醇,将沉淀放在65℃条件下烘干,加入1mL 1×TE溶液和20uL的10mg/mLRNAase,于37℃,1h,消化RNA;
8.电泳检测:取1uL的样品加3uL的染色液进行琼脂糖凝胶电泳,有明显亮带且无拖带即为RNA被完全消化;
9.确定样品中RNA降解完全后,向其中加入与溶液等体积的酚:氯仿:异戊醇(25:24:1),充分混匀,4℃,12000rpm,离心10min;
10.将上清转移到另一个新的2mL EP管中(注意不要吸到下层杂质),加入0.7体积的异丙醇和0.1体积的醋酸钠(PH=5.2),轻柔混匀,待出现絮状沉淀;
11.取沉淀于新的2mL EP管中,用70%乙醇洗3遍,除尽乙醇;
12.将沉淀在37℃条件下烘干,加入100μL的1×TE溶解沉淀,用于做Southernblot实验。
实施例四
在本实施例中,转基因阳性事件的Southern blot拷贝数分析
为了研究转基因玉米中转化拷贝数的情况,对转基因阳性植株用EcoRⅠ和HindⅢ两种酶进行酶切,并进行Southern blot分析。选取了转基因郑的1、2、3号事件进行了Southern blot实验分析拷贝数,如图4所示。具体步骤如下:
1.酶切体系50μL:2μL的EcoRⅠ或HindⅢ、5μL 10×Buffer、10μg基因组DNA,加ddH2O定容至50μL;37℃水浴,酶切10h;
2.配制0.8%的大孔琼脂糖凝胶(无EB),向酶切完全的体系中加入8μL染色液,在恒定4℃条件下,36V,电泳14h;
3.准备转膜装置,在装置方槽中加入400mL的ddH2O和20μL的EB染料,混合均匀后将电泳好的凝胶浸泡其中,10min后拍照显影,用ddH2O冲洗2遍;
5.在装置的另一个方槽中加入500mL的0.2M盐酸,将凝胶轻轻放入其中,浸泡10min;
6.倒掉盐酸溶液,再加入500mL的变性液,浸泡15min,重复一次;变性期间剪取一张尼龙膜(11×12)和滤纸(12×12),并将尼龙膜放在碱转液中浸泡5-10min;
7.组装转膜装置:先在转膜台上倒少许的碱转液,铺1层滤纸,再在滤纸中间铺上尼龙膜,然后用黑皮垫子压住尼龙膜,最后在中间放置凝胶;每铺一层都要用玻璃棒赶气泡,确保形成转膜的封闭环境;
8.启动真空泵进行真空抽提,加入少量碱转液,100mbar,预抽5min,然后向转膜装置中加入适量的碱转液,注意不要漫过凝胶,75min;
9.转膜结束后,关闭电源;用镊子将尼龙膜取出,放在适量的中和液中浸泡10min,然后将尼龙膜放置在滤纸上约25-35min左右,晾干,紫外交联固定;
10.将尼龙膜放到杂交管中(与凝胶接触的那一面朝里),加入10mL的预杂液,42℃的杂交炉,杂交2h;
11.取约250ng制备好的探针,加1×TE溶液补足至40μL,封口膜封口,沸水浴,10min,反应结束后立刻冰浴5min,瞬离,待用;
12.将变性的探针加到预热的10mL杂交液中,轻轻吹打混匀;将混匀的杂交液用玻璃棒导流入杂交管中,50℃杂交炉,低转速孵育16-20小时,18-19小时最佳;
13.杂交后加入10mL的洗膜液I(25℃度预热),25℃杂交炉,15min,重复一遍;
14.加入10mL洗膜液II(65℃预热),65℃杂交炉,30min,重复一遍;
15.配制洗膜液20mL,25℃,每次10mL,5-10min,共洗两次;
16.加10mL封闭液(用马来酸将试剂盒中10×Blocking Solution稀释至1×),25℃,30-40min;
17.加10mL抗体溶液,避光,25℃,30-60min;
18.加20mL洗膜液,每次10mL,25℃,10min;
19.加10mL检测液,25℃,5-10min;
20.将保鲜膜平铺在水平实验台上,最好不要有褶皱,把CSPD显色液均匀滴在保鲜膜上,将尼龙膜的内侧一面与其接触,显影保存。
结果显示杂交背景低,样本的拷贝数清晰,如图4所示。通过统计发现,转基因郑58的1、2、3号事件拷贝数分别有3、3、1,而且两种酶切结果一致。
实施例五
在本实施例中,T1代转基因郑58植株的GFP表达水平分析
转基因植株进行阳性鉴定后,用RT-qPCR分析GFP表达量。抽提转基因郑58植株叶片的RNA,反转为cDNA,运用GFP的特定引物,通过RT-qPCR实验进行检测。
结果表明GFP基因在1#、2#、3#和4#转基因郑58中均表达,如图5所示。
实施例六
在本实施例中,转基因郑58的后代分离分析
选转基因郑58的T2代的4个事件的籽粒(T2-1#、T2-2#、T2-3#、T2-4#),鉴定基因型,并分析后代的分离情况。本发明用GFP的特异性引物检测报告载体;用诱导载体筛选标记基因HPT的特异性引物检测诱导载体。仅存在报告载体时后代成功分离,而诱导载体也存在时没有成功获得分离后代,还需要进一步杂交分离。
检测报告载体的特异性引物及碱基序列为(5'-3'):
GFP-F:GGGCACAAGCTGGAGTACAA
GFP-R:GAACGTTGTCGAAACCGATGA
检测诱导载体的特异性引物及碱基序列为(5'-3'):
HPT-F:CAAGACCTGCCTGAAACCGA
HPT-R:CAAGACCTGCCTGAAACCGA
结果表明,在部分T2代种子中仅检测到报告载体而未检测到诱导载体,在T2-1#、T2-2#、T2-3#、T2-4#中,获得分离的籽粒数分别为4、3、3和3个,如图6所示。统计分析,转基因郑58的后代分离率在2.03-6.78%之间。
上述实施例能优化玉米自交系遗传转化效率的双载体遗传转化体系的建立,该体系使难以转化且重要的玉米品系能进行高效转化。该体系首先构建pHB-ZmBBM-ZmWUS诱导载体和pCAMBIA3301-GFP报告载体,并利用基因枪轰击结合农杆菌介导法转化玉米自交系,使玉米自交系郑58、PH4CV和Mo17实现转化并获得转基因植株。其中,转基因郑58转化率最高,且其整个转化周期仅52天,比传统转化周期缩短30天。双载体遗传转化体系的建立大大缩短了转化周期,从而有助于玉米优质性状的遗传改良。
上面对本发明实施例结合附图进行了说明,但本发明不限于上述实施例,还可以根据本发明的发明创造的目的做出多种变化,凡依据本发明技术方案的精神实质和原理下做的改变、修饰、替代、组合或简化,均应为等效的置换方式,只要符合本发明的发明目的,只要不背离本发明的技术原理和发明构思,都属于本发明的保护范围。
序列表
<110> 上海大学
<120> 优化玉米自交系遗传转化效率的双载体转化体系及其应用
<141> 2022-05-09
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<213> 基因序列(Axig1pro-Wus-RbsT)
<220>
<223> AAGCTTCCCATCGCTGCTTTGTCTACATCATGTTCTTCATCATCCTCCCCAGGCGACGCGTGCTGCTGTTCTTATTCAGACTACCGTTCGAGTGACTGCATGGCGTACATCTTTCTGCATCGACTTTGTACGGCTACATCGAACATATACACGAGATGTCTCGTGTGAATAGAGTCACTAAGCCTTAAGCATCGGTTACTCCGTAGGGTACATTCTGTTTCTTCTTATTTGTGCATATTTTTATTGTTATTTACTGATTATACGAGTAGTTATACATACATGCACATACATATCATCACATATATCACAATATTTTTCTAAATTAAATTAAAACTAAAAATGACTAAATTTCTAACACCAACGACATTGTAATGTTTTCTCCAACAACTTTACCTATTCTACATTGTTCTATTTCGAATTTCACTCTATAAACAACATAGTCTACAATGGAAAACAGTGCTTTGTACGACTATATACGCGATGTGTGGCTACAACATAAGACAATATAGTCGTTTGAAGATTGAACCTATATATCGGTACGGTTAATCCGTCTATGTACGTGGGCATGACGAACACCCGTGATAACGAAGGATTAACGTGCACAATCATAAATCCAAAGTAGGAGCGGTGCATGATGAGAATCGCTCTCAGTACTCGACATAATGAACCTTACGAGGTACAACAGGCAGGCAGGCAGGGACCAGGGGCCGCCTTTATTTCAGGCTCGCTGGCCCCACGGGCGTGCTGCGTGCACGAAGGGCACTACCCCAACCTCTCACCGAAAAACCGCGCTGGATCGGCAAATCAAACGAGGTGGTGCCCCGTGCCCACTCTCCACGTCCACGGCACCATCCCTCTGCAGCCGCTCACCAGCCATGCCGTGTCGCGGAACGGCACAACCACCCCCAACCCACTCACGAAACCCCGTCCCGGCCGTGCCCGTGTCGGTCCGCGCTCGGCAACGAGGCGGCCCGCGCTGCTGAGTCCCCTGGACACCCGACACCCTGTCGGCCCTTTGTTTATTCATCCCGAAATCTCATCTGCCCCCACGGCCGACTGCGCTGCGCTGCGCTGCGCCGCCCGGATATATATACCCATCGTTATCGATCGATCGATCGCGTCACTCACGGGTAGCTCATGGTCGAGCGTAGCATGCAGGAACTTATTTGCCGTGCGCTCCCAGGTCTCCGCTCGCGTGCCTTCCAGTCTGTCTCACACTAGCTGCTGTGGGACGATCGAAGTGGGTGTGTCAGCTAGCTAGCTGCGCCGTGACCACGCACATGACCGCAGTGCGCGCGGGGCTGATCAAGGGAAAGTGATCCCGTCGACATGGCGGCCAATGCGGGCGGCGGTGGAGCGGGAGGAGGCAGCGGCAGCGGCAGCGTGGCTGCGCCGGCGGTGTGCCGCCCCAGCGGCTCGCGGTGGACGCCGACGCCGGAGCAGATCAGGATGCTGAAGGAGCTCTACTACGGCTGCGGCATCCGGTCGCCCAGCTCGGAGCAGATCCAGCGCATCACCGCCATGCTGCGGCAGCACGGCAAGATCGAGGGCAAGAACGTCTTCTACTGGTTCCAGAACCACAAGGCCCGCGAGCGCCAGAAGCGCCGCCTCACCAGCCTCGACGTCAACGTGCCCGCCGCCGGCGCGGCCGACGCCACCACCAGCCAACTCGGCGTCCTCTCGCTGTCGTCGCCGCCGCCTTCAGGCGCGGCGCCTCCCTCGCCCACCCTCGGCTTCTACGCCGCCGGCAATGGCGGCGGATCGGCTGTGCTGCTGGACACGAGTTCCGACTGGGGCAGCAGCGGCGCTGCCATGGCCACCGAGACATGCTTCCTGCAGGACTACATGGGCGTGACGGACACGGGCAGCTCGTCGCAGTGGCCACGCTTCTCGTCGTCGGACACGATAATGGCGGCGGCCGCGGCGCGGGCGGCGACGACGCGGGCGCCCGAGACGCTCCCTCTCTTCCCGACCTGCGGCGACGACGGCGGCAGCGGTAGCAGCAGCTACTTGCCGTTCTGGGGTGCCGCGTCCACAACTGCCGGCGCCACTTCTTCCGTTGCGATCCAGCAGCAACACCAGCTGCAGGAGCAGTACAGCTTTTACAGCAACAGCAACAGCACCCAGCTGGCCGGCACCGGCAACCAAGACGTATCGGCAACAGCAGCAGCAGCCGCCGCCCTGGAGCTGAGCCTCAGCTCATGGTGCTCCCCTTACCCTGCTGCAGGGAGTATGTGACCCGGGCTTTCGTTCGTATCATCGGTTTCGACAACGTTCGTCAAGTTCAATGCATCAGTTTCATTGCGCACACACCAGAATCCTACTGAGTTTGAGTATTATGGCATTGGGAAAACTGTTTTTCTTGTACCATTTGTTGTGCTTGTAATTTACTGTGTTTTTTATTCGGTTTTCGCTATCGAACTGTGAAATGGAAATGGATGGAGAAGAGTTAATGAATGATATGGTCCTTTTGTTCATTCTCAAATTAATATTATTTGTTTTTTCTCTTATTTGTTGTGTGTTGAATTTGAAATTATAAGAGATATGCAAACATTTTGTTTTGAGTAAAAATGTGTCAAATCGTGGCCTCTAATGACCGAAGTTAATATGAGGAGTAAAACACTTGTAGTTGTACCATTATGCTTATTCACTAGGCAACAAATATATTTTCAGACCTAGAAAAGCTGCAAATGTTACTGAATACAAGTATGTCCTCTTGTGTTTTAGACATTTATGAACTTTCCTTTATGTAATTTTCCAGAATCCTTGTCAGATTCTAATCATTGCTTTATAATTATAGTTATACTCATGGATTTGTAGTTGAGTATGAAAATATTTTTTAATGCATTTTATGACTTGCCAATTGATTGACAACATGGAATTC
<400> 2
000
000
000
000
000
000
000
000
<210> 3
<211> 23
<212> DNA
<213> 基因序列(CTA1pro-BBM-RbsT)
<220>
<223> AAGCTTTGCGTACGATTACATCGAACATGCACACGAGATATCTCGTGTGAATGAAGCCACTTTTGCCTTGAGCATCGGAGCATCCGTAGGGCACACTTTGTTCTAAGTATTTGTGCATATTTTACTGTTGTTTACTGTTTACGTGAGTAGTAATACACATACATATACATGTTGTCACATATATCACTGTGATTTTCTAGATTAAATTAAAACTGAAAATATCTATTTCTTGAACAATATAAGTGCTAAAACAACTAATATTTTGGTCTGGAGGGAGTTTTACATAACAAGGGGGGAGTATGTCGTTGTATAGACGAGGCAATTTATTGTTTTAGCTCAAGCCCCACGAAACTAACTCCGTCTCAAGAAAATCTTCTAAATTCCTCGCGAGAGAACACAATACTCTATTATACTCCCTTTTTATTTTCTTTACTTGACGTTTTGTAGTTCATTTTTGTACTATTTAGCGTCAGATATAAAAAGATGAATATACTCTAAGAGTAGCGAAACCATGAATAGTGAATAAGGAAGAAAAACTCTAAAGAGCTTTCTAGAGAAATACTAAAAATATATAAAAAGGGAAAATCCTAGTCTTCGTTTGATCGTGCATGCATATGTGTGGCCCAACCGCCCAATCCATCACCGGTACGTGCTCCAGGCTGTAGCTGCTTGGAAGTCTTCCGTTCTTGCTTGACCGATTCCTGCTAGCCTTTGGAACACGGAGACCGAGGCCACCAATAAATCCTCACATGCGTGGTACTGGTACGTGGAATTGAAGCAAACAAAATACGGCTGCCCGCGGCGGCCGGATAATATATAAGATGATGGCGGCAAATTGTCTTCGCAGACGAGAAAAATCGCAGGAAAGCGGTGACTTCTAGCAGGTAGCATGTGCGGATTTTCCACCGCGATGGGCGGCGGCGGCGGCAATCCAAATCTGATGATATCTCAACCTAGGCTAGAGTGCTTGTGTCTCGATACATGGTGCGTTGCATGCATTGCTGACCCATGCGTCAAAAGTTCATCATAAAACGTGGGACAGCTGTACGTTCCCGCGTGCAATGGTGGACATCACACCTAGCCACTCCACGTCGCATGCCATGCATCGACAGGATGCACGTTCCGATCCCTATATAAAGGGGCACCCTTGTGACACCTCAAATCAGTCGACATGGCCACTGTGAACAACTGGCTCGCTTTCTCCCTCTCCCCGCAGGAGCTGCCGCCCTCCCAGACGACGGACTCCACGCTCATCTCGGCCGCCACCGCCGACCATGTCTCCGGCGATGTCTGCTTCAACATCCCCCAAGATTGGAGCATGAGGGGATCAGAGCTTTCGGCGCTCGTCGCGGAGCCGAAGCTGGAGGACTTCCTCGGCGGCATCTCCTTCTCCGAGCAGCATCACAAGTCCAACTGCAACTTGATACCCAGCACTAGCAGCACAGTTTGCTACGCGAGCTCAGCTGCTAGCACCGGCTACCATCACCAGCTGTACCAGCCCACCAGCTCCGCGCTCCACTTCGCGGACTCCGTCATGGTGGCCTCCTCGGCCGGTGTCCACGACGGCGGTGCCATGCTCAGCGCGGCCGCCGCTAACGGTGTCGCTGGCGCTGCCAGTGCCAACGGCGGCGGCATCGGGCTGTCCATGATCAAGAACTGGCTGCGGAGCCAACCGGCGCCCATGCAGCCGAGGGCGGCGGCGGCTGAGGGCGCGCAGGGGCTCTCTTTGTCCATGAACATGGCGGGGACGACCCAAGGCGCTGCTGGCATGCCACTTCTCGCTGGAGAGCGCGCACGGGCGCCCGAGAGTGTATCGACGTCAGCACAGGGTGGTGCCGTCGTCGTCACGGCGCCGAAGGAGGATAGCGGTGGCAGCGGTGTTGCCGGTGCTCTAGTAGCCGTGAGCACGGACACGGGTGGCAGCGGCGGCGCGTCGGCTGACAACACGGCAAGGAAGACGGTGGACACGTTCGGGCAGCGCACGTCGATTTACCGTGGCGTGACAAGGCATAGATGGACTGGGAGATATGAGGCACATCTTTGGGATAACAGTTGCAGAAGGGAAGGACAAACTCGTAAGGGTCGTCAAGTCTATTTAGGTGGCTATGATAAAGAGGAGAAAGCTGCTAGGGCTTATGATCTTGCTGCTCTGAAGTACTGGGGTCCCACAACAACAACAAATTTCCCAGTGAGTAACTACGAAAAGGAGCTCGAGGACATGAAGCACATGACAAGGCAGGAGTTTGTAGCGTCTCTGAGAAGGAAGAGCAGTGGTTTCTCCAGAGGTGCATCCATTTACAGGGGAGTGACTAGGCATCACCAACATGGAAGATGGCAAGCACGGATTGGACGAGTTGCAGGGAACAAGGATCTTTACTTGGGCACCTTCAGCACCCAGGAGGAGGCAGCGGAGGCGTACGACATCGCGGCGATCAAGTTCCGCGGCCTAAACGCCGTCACCAACTTCGACATGAGCCGCTACGACGTGAAGAGCATCCTGGACAGCAGCGCCCTCCCCATCGGCAGCGCCGCCAAGCGCCTCAAGGAGGCCGAGGCCGCAGCGTCCGCGCAGCACCACCACGCCGGCGTGGTGAGTTACGACGTCGGCCGCATCGCCTCGCAGCTCGGCGACGGCGGAGCCCTGGCGGCGGCGTACGGCGCGCACTACCACGGCGCCGCCTGGCCGACCATCGCGTTCCAGCCGGGCGCCGCCACCACAGGCCTGTACCACCCGTACGCGCAGCAGCCAATGCGCGGCGGCGGGTGGTGCAAGCAGGAGCAGGACCACGCGGTGATCGCGGCCGCGCACAGCCTGCAGGACCTCCACCACCTGAACCTGGGCGCGGCCGGCGCGCACGACTTTTTCTCGGCAGGGCAGCAGGCCGCCGCCGCTGCGATGCACGGCCTGGGTAGCATCGACAGTGCGTCGCTCGAGCACAGCACCGGCTCCAACTCCGTCGTCTACAACGGCGGGGTCGGCGACAGCAACGGCGCCAGCGCCGTCGGCGGCAGTGGCGGTGGCTACATGATGCCGATGAGCGCTGCCGGAGCAACCACTACATCGGCAATGGTGAGCCACGAGCAGGTGCATGCACGGGCCTACGACGAAGCCAAGCAGGCTGCTCAGATGGGGTACGAGAGCTACCTGGTGAACGCGGAGAACAATGGTGGCGGAAGGATGTCTGCATGGGGGACTGTCGTGTCTGCAGCCGCGGCGGCAGCAGCAAGCAGCAACGACAACATGGCCGCCGACGTCGGCCATGGCGGCGCGCAGCTCTTCAGTGTCTGGAACGACACTTAAGTCGACCTTTCGTTCGTATCATCGGTTTCGACAACGTTCGTCAAGTTCAATGCATCAGTTTCATTGCGCACACACCAGAATCCTACTGAGTTTGAGTATTATGGCATTGGGAAAACTGTTTTTCTTGTACCATTTGTTGTGCTTGTAATTTACTGTGTTTTTTATTCGGTTTTCGCTATCGAACTGTGAAATGGAAATGGATGGAGAAGAGTTAATGAATGATATGGTCCTTTTGTTCATTCTCAAATTAATATTATTTGTTTTTTCTCTTATTTGTTGTGTGTTGAATTTGAAATTATAAGAGATATGCAAACATTTTGTTTTGAGTAAAAATGTGTCAAATCGTGGCCTCTAATGACCGAAGTTAATATGAGGAGTAAAACACTTGTAGTTGTACCATTATGCTTATTCACTAGGCAACAAATATATTTTCAGACCTAGAAAAGCTGCAAATGTTACTGAATACAAGTATGTCCTCTTGTGTTTTAGACATTTATGAACTTTCCTTTATGTAATTTTCCAGAATCCTTGTCAGATTCTAATCATTGCTTTATAATTATAGTTATACTCATGGATTTGTAGTTGAGTATGAAAATATTTTTTAATGCATTTTATGACTTGCCAATTGATTGACAACATGGGATCC
<400> 3
000
000
000
000
000
000
000
000
<210> 4
<211> 23
<212> DNA
<213> 基因序列(PLTPpro-BBM-RbsT)
<220>
<223> AAGCTTATCTATCCCCCACAACCACGCTCTAGCTTTTCAGAGAACATGTTGTTATATATAACCCCGAATTGTTCAACTATATATTGCAGCTAATGACACTTTAACTTTGTTAAGAATAGCTATTTGGTTTACTTGTGACTTTTTTTAATGCCACTAGTACTGACTTATTAGACAAATGTTCATTCTTTAATCTAGCTCTAATTCTAATATCTACCAGACTATATATCTTGTGTTGCTTACTTAATAAATGCTTTTATAAAAAAACATTTTAAAATGTTGATCTGTTTATTACTGAACATTGATTTGATTATTAGTGTTAACTATAGAGTAGACGAAAGTATGTTTCTTTGTAATTTAAAGGTTTTTTTACCACACGTGTAGTTCATATTTTTAGATCAAAAAAGTGAACAGCCTAGTGGCTACGGCCAAAGAAGCTCCCAACTCAAAAGTGTTAGGCATTGCTTCGTAATTTAAAGCATGAATTTCTTCGTGTGTCATCTCTTCACGCGGTCGCTCCCGGTCCGCACGCACGCCCCCACACAGGAAAAAAATAGTCCGCGCCCTCGCACCAGAAAATAAAAATAGAGGCACTAGGGTTCGAATCCAACCCTAGTTACTAGGGAAGCGATTGCTCGGCTGCTACCGCAGGTTCGATTATATTGCACAAGGATTGATATTGTAAATATAAATATATAGTAATAATTTAAAAATTAAAAATATAATAATACAAACCTAGCAATGTATGGGAACTAACTAGTATCCCCTCAGTCCTAAAATATAATTCTTTCTAGCCTATTTTTTTGTCCACATTCGTTCAAATGATAATGAATATAGATATATATGGAAACTATATTTATATGTTACTCAATGAATATGTAATTAATCTAAAACGAATTATATTTTAGGATAGAGGGAAGTATAAATAAAAGAAAGGAAATTCAAGAGACAAGTACGAGGAGTCGGGGACTTGGGGGAGGCAATTCCCTCAAACGACTAGCTGGCCTCCTCATTCGTTACGCAGGCCAGGCCGCACTGCGCTCACGGCTTTTAGGTTGCACCAGTCCGGTAGCTTGGTCCACGAACGGCCTAGCGGATCCGTTACCGGCAATGCCGACTTGGGCCACGTTCACAGCAGCCCAGCAATGACGGCTCGTCATGTTAAAAATCCGCTACAACTTTGTAAATTCCATACCTCTAGTATTCATGTGCCATGAATCATGAACCTATTGTTTGTCTTGGGAGTTTTCTGGATTCAAACTTACTAATTTATTTTCTGTCTATAAATACCCTGTAAAACTACTGGTAAATAAAAAGGAGAAACCAATCATTTTGTTCCATTTGTCTCGTTGCTTCCTTGTTTTAGAGAAAAATGTATCAGCAATTCTTAAACTTGACACACCGTCATTTGGACCCTAAACTTAGAAAATCAGGAAAACGAGTTTGGAGCTCTTGGACTCTGATGCAAATAAAGCATGAGATTTGCACGGGATAGAGGAAATTAATATATCACAGGTCCCGTTTAGTGTCCGTTCGGTTACCTAGAATTCGACCCCGTTCTACCTCATTAAGAGTATACGTATATACTCTCTTTATTTGTCGCGTTTTAGTTTAAAAATGAACTAGCAAGCCATAAGTATTGGCCCTAGTAGTAATAAACCATTTCAGCCAGGAATCATTCCAGTATTCAATCCGAAGGAACCGGAGAGGGTACATCAGCCCCTCGCCCAGCTTTGTTTTATAGTTTTTGTTTTGTATTTTCTGAATTATTAAGACAAATTTTGTAGTATCCGAGTAGCACATGCAGTGCGTGTCGTCGTACGTGTCATGAAAAAAAGGCGTGTGGACATATGGAACACACGGTCACATATGCGTCGACATGGCCACTGTGAACAACTGGCTCGCTTTCTCCCTCTCCCCGCAGGAGCTGCCGCCCTCCCAGACGACGGACTCCACGCTCATCTCGGCCGCCACCGCCGACCATGTCTCCGGCGATGTCTGCTTCAACATCCCCCAAGATTGGAGCATGAGGGGATCAGAGCTTTCGGCGCTCGTCGCGGAGCCGAAGCTGGAGGACTTCCTCGGCGGCATCTCCTTCTCCGAGCAGCATCACAAGTCCAACTGCAACTTGATACCCAGCACTAGCAGCACAGTTTGCTACGCGAGCTCAGCTGCTAGCACCGGCTACCATCACCAGCTGTACCAGCCCACCAGCTCCGCGCTCCACTTCGCGGACTCCGTCATGGTGGCCTCCTCGGCCGGTGTCCACGACGGCGGTGCCATGCTCAGCGCGGCCGCCGCTAACGGTGTCGCTGGCGCTGCCAGTGCCAACGGCGGCGGCATCGGGCTGTCCATGATCAAGAACTGGCTGCGGAGCCAACCGGCGCCCATGCAGCCGAGGGCGGCGGCGGCTGAGGGCGCGCAGGGGCTCTCTTTGTCCATGAACATGGCGGGGACGACCCAAGGCGCTGCTGGCATGCCACTTCTCGCTGGAGAGCGCGCACGGGCGCCCGAGAGTGTATCGACGTCAGCACAGGGTGGTGCCGTCGTCGTCACGGCGCCGAAGGAGGATAGCGGTGGCAGCGGTGTTGCCGGTGCTCTAGTAGCCGTGAGCACGGACACGGGTGGCAGCGGCGGCGCGTCGGCTGACAACACGGCAAGGAAGACGGTGGACACGTTCGGGCAGCGCACGTCGATTTACCGTGGCGTGACAAGGCATAGATGGACTGGGAGATATGAGGCACATCTTTGGGATAACAGTTGCAGAAGGGAAGGACAAACTCGTAAGGGTCGTCAAGTCTATTTAGGTGGCTATGATAAAGAGGAGAAAGCTGCTAGGGCTTATGATCTTGCTGCTCTGAAGTACTGGGGTCCCACAACAACAACAAATTTCCCAGTGAGTAACTACGAAAAGGAGCTCGAGGACATGAAGCACATGACAAGGCAGGAGTTTGTAGCGTCTCTGAGAAGGAAGAGCAGTGGTTTCTCCAGAGGTGCATCCATTTACAGGGGAGTGACTAGGCATCACCAACATGGAAGATGGCAAGCACGGATTGGACGAGTTGCAGGGAACAAGGATCTTTACTTGGGCACCTTCAGCACCCAGGAGGAGGCAGCGGAGGCGTACGACATCGCGGCGATCAAGTTCCGCGGCCTAAACGCCGTCACCAACTTCGACATGAGCCGCTACGACGTGAAGAGCATCCTGGACAGCAGCGCCCTCCCCATCGGCAGCGCCGCCAAGCGCCTCAAGGAGGCCGAGGCCGCAGCGTCCGCGCAGCACCACCACGCCGGCGTGGTGAGTTACGACGTCGGCCGCATCGCCTCGCAGCTCGGCGACGGCGGAGCCCTGGCGGCGGCGTACGGCGCGCACTACCACGGCGCCGCCTGGCCGACCATCGCGTTCCAGCCGGGCGCCGCCACCACAGGCCTGTACCACCCGTACGCGCAGCAGCCAATGCGCGGCGGCGGGTGGTGCAAGCAGGAGCAGGACCACGCGGTGATCGCGGCCGCGCACAGCCTGCAGGACCTCCACCACCTGAACCTGGGCGCGGCCGGCGCGCACGACTTTTTCTCGGCAGGGCAGCAGGCCGCCGCCGCTGCGATGCACGGCCTGGGTAGCATCGACAGTGCGTCGCTCGAGCACAGCACCGGCTCCAACTCCGTCGTCTACAACGGCGGGGTCGGCGACAGCAACGGCGCCAGCGCCGTCGGCGGCAGTGGCGGTGGCTACATGATGCCGATGAGCGCTGCCGGAGCAACCACTACATCGGCAATGGTGAGCCACGAGCAGGTGCATGCACGGGCCTACGACGAAGCCAAGCAGGCTGCTCAGATGGGGTACGAGAGCTACCTGGTGAACGCGGAGAACAATGGTGGCGGAAGGATGTCTGCATGGGGGACTGTCGTGTCTGCAGCCGCGGCGGCAGCAGCAAGCAGCAACGACAACATGGCCGCCGACGTCGGCCATGGCGGCGCGCAGCTCTTCAGTGTCTGGAACGACACTTAAGTCGACCTTTCGTTCGTATCATCGGTTTCGACAACGTTCGTCAAGTTCAATGCATCAGTTTCATTGCGCACACACCAGAATCCTACTGAGTTTGAGTATTATGGCATTGGGAAAACTGTTTTTCTTGTACCATTTGTTGTGCTTGTAATTTACTGTGTTTTTTATTCGGTTTTCGCTATCGAACTGTGAAATGGAAATGGATGGAGAAGAGTTAATGAATGATATGGTCCTTTTGTTCATTCTCAAATTAATATTATTTGTTTTTTCTCTTATTTGTTGTGTGTTGAATTTGAAATTATAAGAGATATGCAAACATTTTGTTTTGAGTAAAAATGTGTCAAATCGTGGCCTCTAATGACCGAAGTTAATATGAGGAGTAAAACACTTGTAGTTGTACCATTATGCTTATTCACTAGGCAACAAATATATTTTCAGACCTAGAAAAGCTGCAAATGTTACTGAATACAAGTATGTCCTCTTGTGTTTTAGACATTTATGAACTTTCCTTTATGTAATTTTCCAGAATCCTTGTCAGATTCTAATCATTGCTTTATAATTATAGTTATACTCATGGATTTGTAGTTGAGTATGAAAATATTTTTTAATGCATTTTATGACTTGCCAATTGATTGACAACATGTCTAGA
<400> 4
000
000
000
000
000
000
000
<213> 4647-4652bp:XbalⅠ
Claims (4)
1.一种玉米自交系双载体遗传转化系统,其特征在于:该双载体遗传转化系统包含pHB-ZmBBM-ZmWUS诱导载体和pCAMBIA3301-GFP报告载体,其中,在愈伤中胚胎发育调控因子(BBM)强表达,分生组织形态发生因子(WUS)正常表达。
2.一种权利要求1所述玉米自交系双载体遗传转化系统的构建方法,其特征在于:采用如下玉米自交系双载体遗传转化体系的具体培养流程:
利用基因枪轰击,结合农杆菌介导法转化玉米自交系,将双载体导入玉米未成熟胚中,通过共培养、恢复培养、无筛选培养、预再生培养、暗再生培养、光再生培养等过程,获得转基因植株。
3.一种权利要求1和2所述玉米自交系双载体遗传转化系统的应用,其特征在于:使玉米自交系郑58、PH4CV、Mo17、昌7-2和B73实现转化,并获得转基因植株。
4.一种权利要求1和2所述的双载体遗传转化系统的应用,其特征在于:玉米转基因阳性事件后代的鉴定与分离分析。
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CN111465321A (zh) * | 2017-10-13 | 2020-07-28 | 先锋国际良种公司 | 用于植物细胞的细胞重编程的系统和方法 |
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CN111465321A (zh) * | 2017-10-13 | 2020-07-28 | 先锋国际良种公司 | 用于植物细胞的细胞重编程的系统和方法 |
Non-Patent Citations (2)
Title |
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YAJING GU ET AL: "Establishment of a Bivector Genetic Transformation System in Recalcitrant Maize Inbred Lines", 《AGRICULTURE》, pages 1 - 14 * |
黎裕;王天宇;: "玉米转基因技术研发与应用现状及展望", 玉米科学, no. 02, pages 7 - 21 * |
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