铁棍山药微型块茎遗传转化体系的建立
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摘要
怀山药(Dioscorea opposita)遗传转化是对其进行基因功能分析和遗传改良的基础,但目前国内外尚未见相关报道。以怀山药优良品种铁棍山药(D. opposita cv.‘Tiegun’)的微型块茎为受体材料,对影响遗传转化的因素进行优化,建立了由根癌农杆菌介导的山药遗传转化体系。过表达质粒载体p CAMBIA1301-DoSERK2含GUS标记基因和潮霉素(Hyg)抗性筛选基因,沉默质粒载体pART27-DoSERK2含卡那霉素(Kan)抗性筛选基因。根癌农杆菌抑制剂特美汀(Tim)的最佳浓度为500mg·L~(–1);再生芽和生根时, Hyg的最佳浓度分别为15和20 mg·L~(–1), Kan的最佳浓度分别为120和160 mg·L~(–1)。对转化植株进行PCR和GUS组织化学检测,结果显示外源基因已整合到铁棍山药转基因株系的基因组中并在细胞中表达。该研究建立了一套取材便利的铁棍山药遗传转化方法,对其它品种山药的转化也具有参考价值。
Genetic transformation system is the foundation for gene function study and genetic improvement of Dioscorea opposita. In this study, we established an Agrobacterium-mediated genetic transformation system using microtuber in D. opposita cv. ‘Tiegun'. The transformation was performed with the DoSERK2 gene overexpression plasmid pCAMBIA1301-DoSERK2 that harboring the GUS reporter gene and the hygromycin(Hyg) resistence gene as the selectable marker, and the gene-silencing vector pART27-DoSERK2 that containing kanamycin(Kan) resistence marker. The optimal concentration of timentin(Tim) for Agrobacterium counter selection was 500 mg·L~(–1). For shoot regeneration and rooting, the optimal concentrations of Hyg were 15 mg·L~(–1) and 20 mg·L~(–1), respectively, and the optimal concentration of Kan were 120 mg·L~(–1) and 160 mg·L~(–1), respectively. Results of PCR analysis and GUS histochemical staining indicated that the exogenous genes had been integrated into the genome of transgenic plants, and are expressed in the plants. Our study provides a genetic transformation system in D. opposita cv. ‘Tiegun', which has the advantage that explants are easy available. This method has reference value for the genetic transformation of other varieties of D. opposita.
Genetic transformation system is the foundation for gene function study and genetic improvement of Dioscorea opposita. In this study, we established an Agrobacterium-mediated genetic transformation system using microtuber in D. opposita cv. ‘Tiegun'. The transformation was performed with the DoSERK2 gene overexpression plasmid pCAMBIA1301-DoSERK2 that harboring the GUS reporter gene and the hygromycin(Hyg) resistence gene as the selectable marker, and the gene-silencing vector pART27-DoSERK2 that containing kanamycin(Kan) resistence marker. The optimal concentration of timentin(Tim) for Agrobacterium counter selection was 500 mg·L~(–1). For shoot regeneration and rooting, the optimal concentrations of Hyg were 15 mg·L~(–1) and 20 mg·L~(–1), respectively, and the optimal concentration of Kan were 120 mg·L~(–1) and 160 mg·L~(–1), respectively. Results of PCR analysis and GUS histochemical staining indicated that the exogenous genes had been integrated into the genome of transgenic plants, and are expressed in the plants. Our study provides a genetic transformation system in D. opposita cv. ‘Tiegun', which has the advantage that explants are easy available. This method has reference value for the genetic transformation of other varieties of D. opposita.
引文
范俊强(2008).根癌农杆菌介导的盾叶薯蓣转化体系的建立.硕士论文.武汉:华中农业大学.pp.1-5.
付洪冰,崔崇士,赵曦,刘琦(2010).农杆菌介导南瓜遗传转化体系的建立.植物学报45,472-478.
郭利军,曾炳山,刘英(2013).农杆菌介导巨桉Eg5高效遗传转化.植物学报48,87-93.
韩林林,李俊华,赵喜亭,张晓丽,宋志辉,刘世宇,李明军(2016).农杆菌介导的怀山药叶片瞬时表达方法的建立.河南师范大学学报(自然科学版)44,135-139.
李明军(2013).提高山药商品性栽培技术问答.北京:金盾出版社.pp.36-50.
李明军,陈明霞,洪森荣,徐鑫,张晓丽(2004).NAA、IBA和PP333对怀山药试管苗生长发育的影响.广西植物24,376-379.
李明军,刘世宇,刘雯,李俊华,张晓丽,赵喜亭(2017).怀山药微型块茎形成过程中的生理生化变化.植物生理学报53,807-814.
李明军,张峰,陈明霞,于相丽(2003).怀山药病毒病的研究.中草药34,U003-U005.
李瑞雪,李纪强,蒲腾飞,张晓丽,赵喜亭,李俊华,李明军(2018).怀山药类原球茎的诱导形成与植株再生.植物学报53,334-340.
李卫,郭光沁,郑国锠(2000).根癌农杆菌介导遗传转化研究的若干新进展.科学通报45,798-807.
廖华兰,黎秀琼,李可,李伯凌,熊茜,苏童,李春霞,陈银华,罗丽娟(2016).农杆菌介导的木薯遗传转化体系的优化.热带生物学报7,427-434.
王善平,许智宏,卫志明(1990).毛白杨叶外植体的遗传转化.植物学报32,172-177.
王运英(2016).怀山药脱毒微型块茎管内管外萌发条件及生理生化机制研究.硕士论文.新乡:河南师范大学.pp.9-19.
许云(2014).大薯遗传多样性的AFLP分析和类原球茎遗传转化体系的研究.硕士论文.海口:海南大学.pp.61-66.
杨亚萍,李永兰,梁月荣,陆建良,郑新强(2015).发根农杆菌抑菌剂的抑菌效果及对茶组培苗丛生芽的影响.茶叶科学35,437-442.
张宁,司怀军,李学才,王蒂(2004).根癌农杆菌介导的马铃薯高效遗传转化体系的研究.中国马铃薯18,132-135.
赵喜亭,蒋丽薇,王苗,朱玉婷,张文芳,李明军(2016).怀黄菊间接体胚受体再生体系的建立及CmTGA1的遗传转化.植物学报51,525-532.
Edwards K,Johnstone C,Thompson C(1991).A simple and rapid method for the preparation of plant genomic DNA for PCR analysis.Nucleic Acids Res 19,1349.
Li MJ,Li JH,Wang YP,Liu W,Guo XB,Li SJ,Han LL,Song ZH,Zhao XT,Yang QX(2015).A simple method for microtuber production in Dioscorea opposita using single nodal segments.Pakistan J Bot 47,665-668.
Mignouna HD,Abang MM,Asiedu R(2008).Genomics of yams,a common source of food and medicine in the tropics.In:Moore PH,Ming R,eds.Genomics of Tropical Crop Plants.Plant Genetics and Genomics:Crops and Models,Vol.1.Berlin:Springer.pp.549-570.
Nyaboga E,Tripathi JN,Manoharan R,Tripathi L(2014).Agrobacterium-mediated genetic transformation of yam(Dioscorea rotundata):an important tool for functional study of genes and crop improvement.Front Plant Sci 5,463.
T?r M,Ainsworth C,Mantell SH(1993).Stable transformation of the food yam Dioscorea alata L.by particle bombardment.Plant Cell Rep 12,468-473.
T?r M,Twyford CT,Funes I,Boccon-Gibod J,Ainsworth CC,Mantell SH(1998).Isolation and culture of protoplasts from immature leaves and embryogenic cell suspensions of Dioscorea yams:tools for transient gene expression studies.Plant Cell Tissue Organ Cult 53,113-126.
Zhao XT,Zhang XL,Guo XB,Li SJ,Han LL,Song ZH,Wang YY,Li JH,Li MJ(2016).Identification and validation of reference genes for qRT-PCR studies of gene expression in Dioscorea opposita.Biomed Res Int 2016,3089584.
付洪冰,崔崇士,赵曦,刘琦(2010).农杆菌介导南瓜遗传转化体系的建立.植物学报45,472-478.
郭利军,曾炳山,刘英(2013).农杆菌介导巨桉Eg5高效遗传转化.植物学报48,87-93.
韩林林,李俊华,赵喜亭,张晓丽,宋志辉,刘世宇,李明军(2016).农杆菌介导的怀山药叶片瞬时表达方法的建立.河南师范大学学报(自然科学版)44,135-139.
李明军(2013).提高山药商品性栽培技术问答.北京:金盾出版社.pp.36-50.
李明军,陈明霞,洪森荣,徐鑫,张晓丽(2004).NAA、IBA和PP333对怀山药试管苗生长发育的影响.广西植物24,376-379.
李明军,刘世宇,刘雯,李俊华,张晓丽,赵喜亭(2017).怀山药微型块茎形成过程中的生理生化变化.植物生理学报53,807-814.
李明军,张峰,陈明霞,于相丽(2003).怀山药病毒病的研究.中草药34,U003-U005.
李瑞雪,李纪强,蒲腾飞,张晓丽,赵喜亭,李俊华,李明军(2018).怀山药类原球茎的诱导形成与植株再生.植物学报53,334-340.
李卫,郭光沁,郑国锠(2000).根癌农杆菌介导遗传转化研究的若干新进展.科学通报45,798-807.
廖华兰,黎秀琼,李可,李伯凌,熊茜,苏童,李春霞,陈银华,罗丽娟(2016).农杆菌介导的木薯遗传转化体系的优化.热带生物学报7,427-434.
王善平,许智宏,卫志明(1990).毛白杨叶外植体的遗传转化.植物学报32,172-177.
王运英(2016).怀山药脱毒微型块茎管内管外萌发条件及生理生化机制研究.硕士论文.新乡:河南师范大学.pp.9-19.
许云(2014).大薯遗传多样性的AFLP分析和类原球茎遗传转化体系的研究.硕士论文.海口:海南大学.pp.61-66.
杨亚萍,李永兰,梁月荣,陆建良,郑新强(2015).发根农杆菌抑菌剂的抑菌效果及对茶组培苗丛生芽的影响.茶叶科学35,437-442.
张宁,司怀军,李学才,王蒂(2004).根癌农杆菌介导的马铃薯高效遗传转化体系的研究.中国马铃薯18,132-135.
赵喜亭,蒋丽薇,王苗,朱玉婷,张文芳,李明军(2016).怀黄菊间接体胚受体再生体系的建立及CmTGA1的遗传转化.植物学报51,525-532.
Edwards K,Johnstone C,Thompson C(1991).A simple and rapid method for the preparation of plant genomic DNA for PCR analysis.Nucleic Acids Res 19,1349.
Li MJ,Li JH,Wang YP,Liu W,Guo XB,Li SJ,Han LL,Song ZH,Zhao XT,Yang QX(2015).A simple method for microtuber production in Dioscorea opposita using single nodal segments.Pakistan J Bot 47,665-668.
Mignouna HD,Abang MM,Asiedu R(2008).Genomics of yams,a common source of food and medicine in the tropics.In:Moore PH,Ming R,eds.Genomics of Tropical Crop Plants.Plant Genetics and Genomics:Crops and Models,Vol.1.Berlin:Springer.pp.549-570.
Nyaboga E,Tripathi JN,Manoharan R,Tripathi L(2014).Agrobacterium-mediated genetic transformation of yam(Dioscorea rotundata):an important tool for functional study of genes and crop improvement.Front Plant Sci 5,463.
T?r M,Ainsworth C,Mantell SH(1993).Stable transformation of the food yam Dioscorea alata L.by particle bombardment.Plant Cell Rep 12,468-473.
T?r M,Twyford CT,Funes I,Boccon-Gibod J,Ainsworth CC,Mantell SH(1998).Isolation and culture of protoplasts from immature leaves and embryogenic cell suspensions of Dioscorea yams:tools for transient gene expression studies.Plant Cell Tissue Organ Cult 53,113-126.
Zhao XT,Zhang XL,Guo XB,Li SJ,Han LL,Song ZH,Wang YY,Li JH,Li MJ(2016).Identification and validation of reference genes for qRT-PCR studies of gene expression in Dioscorea opposita.Biomed Res Int 2016,3089584.