烟草中稳定遗传转化的新技术
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摘要
农杆菌介导的植物稳定遗传转化技术已经在植物基因工程领域得到广泛应用.本文介绍了一种由农杆菌介导的新型植物遗传转化技术—发芽种子真空侵染法.应用此方法将表达载体p BI121-HC-Pro转化到烟草植株中,并优化了农杆菌的浓度及真空侵染的条件.半定量RT-PCR(Reverse transcription-PCR)方法检测结果表明这种转化方法可以使HC-Pro在烟草中表达,具有广泛的应用前景.
Agrobacterium-mediated plant stable genetic transformation technology has been widely used in plant genetic engineering. In this paper,a new technology of plant genetic transformation mediated by Agrobacterium was introduced. The vector pBI121-HC-Pro into tobacco plants was transformed by using vacuum infiltration of germinated seeds,and optimized the concentration of Agrobacterium and the condition of vacuum infiltration.The results of semi-quantitative RT-PCR showed that HC-Pro gene could be stably expressed in tobacco plants by the technology. The new technology has broad application prospects.
Agrobacterium-mediated plant stable genetic transformation technology has been widely used in plant genetic engineering. In this paper,a new technology of plant genetic transformation mediated by Agrobacterium was introduced. The vector pBI121-HC-Pro into tobacco plants was transformed by using vacuum infiltration of germinated seeds,and optimized the concentration of Agrobacterium and the condition of vacuum infiltration.The results of semi-quantitative RT-PCR showed that HC-Pro gene could be stably expressed in tobacco plants by the technology. The new technology has broad application prospects.
引文
[1]姚冉,石美丽,潘沈元,等.农杆菌介导的植物遗传转化研究进展[J].生物技术进展,2011,1(4):260-265.
[2]KATAEVA NV,ALEXANDROVA IG,BUTENKO RG,et al. Effect of applied and internal hormones on vitrification and apical necrosis of different plants cultured in vitro[J]. Plant Cell,Tissue and Organ Culture,1991,27(2):149-154.
[3]KADOTA M,NIIMI Y. Effects of cytokinin types and their concentra-tions on shoot proliferation and hyperhydricityin vitro pear cultivar shoots[J]. Plant Cell,Tissue and Organ Culture,2003,72(3):261-265.
[4]MONSALUD MJ,MATHEWS H,LITZ RE,et al. Control of hyperhydricity of mango somatic embryos[J]. Plant Cell,Tissue and Organ Culture,1995,42(2):195-206.
[5]SAHER S,PIQUERAS A,HELLIN E,et al. Prevention of hyperhydricity in micropropagated carnation shoots by bottom cooling:implications of oxidative stress[J]. Plant Cell,Tissue and Organ Culture,2005,81(2):149-158.
[6]亓建飞,李付广,张朝军,等.植物组织培养中畸形苗发生机理的研究进展[J].棉花学报,2004,16(4):243-248.
[7]ANANDALAKSHMI R,PRUSS G J,GE X,et al. A viral suppressor of gene silencing in plants[J]. Proc Natl Acad Sci USA,1998,95(22):13079-13084.
[8]BRIGNETI G,et al. Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana[J]. Embo J,1998,17(22):6739-46.
[9]李向东,李怀方,范在丰,等.马铃薯Y病毒属病毒HC-Pro研究进展[J].山东农业大学学报(自然科学版),2000,31(4):437-440.
[10]蒋琳,魏春红,李毅.病毒基因沉默抑制子及其作用机制[J].中国科学,2012,42(1):16-28.
[11]VANCE V B,ANANDALAKSHMI R,PRUSS G J,et al. A viral suppressor of gene silencing in plants[J]. Proc Natl Acad Sci USA,1998,95:13079-13084.
[12]BOUTET S,VAZQUEZ F,LIU J,et al. Arabidopsis HEN1:A genetic link between endogenous miRNA controlling development and siRNA controlling transgene silencing and virus resistance[J]. Curr Biol,2003,13(10):843-848.
[13]HAN M H,GOUD S,SONG L,et al. The Arabidopsis double-stranded RNA-binding protein HYL1 plays a role in microRNA-mediated gene regulation[J]. Proc Natl Acad Sci USA,2004,101(4):1093-1098.
[14]PARK M Y,WU G,GONZALEZ-SULSER,et al. Nuclear processing and export of microRNA in Arabidopsis[J]. Proc Natl Acad Sci USA,2005,102(10):3691-3696.
[15]GAIL J P,CHRISTOPHER B L,TROY B,et al. The potyviral suppressor of RNA silencing confers enhance dresistance to multiple pathogens[J]. Virology,2004,(320):107-120.
[16]杨丽萍,许亚男,刘雨晴,等.利用根部真空侵染法在烟草中瞬时表达外源蛋白[J].分子植物育种,2016,14(12):3385-3389.
[17]杨丽萍,金太成,徐洪伟,等.植物中瞬时表达外源基因的新型侵染技术[J].遗传,2013,35(1):111-117.
[18]YANG L P,XU Y N,LIU Y Q,et al. HC-Pro viral suppressor from tobacco vein banding mosaic virus interferes with DNA methylation and activates the salicylic acid pathway,Virology,2016,497:244-250.
[19]杨丽萍,金太成,周晓馥,利用农杆菌侵染新技术在豌豆中瞬时表达ha FGF[J].生物技术通报,2013,1(10),71-75.7
[20]YANG L P,WANG H W,LIU J Y,Et al. A simple and effective system for foreign gene expression in plants via root absorption of agrobacterial suspension,Journal of Biotechnology,2008,134:320-324.
[21]JIN T C,WANG J,ZHU X J,et al. A new transient expression system for large-scale production of recombinant proteins in plants based on airbrushing an Agrobacterium suspension,Biotechnology Report,2015,6:36-40.
[22]FAN Y J,LI W,WANG J J,et al. Efficient production of human acidic fibroblast growth factor in pea(Pisum sativum L.)plants by agroinfection of germinated seeds[J]. BMC Biotechnol,2011,11:45.
[2]KATAEVA NV,ALEXANDROVA IG,BUTENKO RG,et al. Effect of applied and internal hormones on vitrification and apical necrosis of different plants cultured in vitro[J]. Plant Cell,Tissue and Organ Culture,1991,27(2):149-154.
[3]KADOTA M,NIIMI Y. Effects of cytokinin types and their concentra-tions on shoot proliferation and hyperhydricityin vitro pear cultivar shoots[J]. Plant Cell,Tissue and Organ Culture,2003,72(3):261-265.
[4]MONSALUD MJ,MATHEWS H,LITZ RE,et al. Control of hyperhydricity of mango somatic embryos[J]. Plant Cell,Tissue and Organ Culture,1995,42(2):195-206.
[5]SAHER S,PIQUERAS A,HELLIN E,et al. Prevention of hyperhydricity in micropropagated carnation shoots by bottom cooling:implications of oxidative stress[J]. Plant Cell,Tissue and Organ Culture,2005,81(2):149-158.
[6]亓建飞,李付广,张朝军,等.植物组织培养中畸形苗发生机理的研究进展[J].棉花学报,2004,16(4):243-248.
[7]ANANDALAKSHMI R,PRUSS G J,GE X,et al. A viral suppressor of gene silencing in plants[J]. Proc Natl Acad Sci USA,1998,95(22):13079-13084.
[8]BRIGNETI G,et al. Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana[J]. Embo J,1998,17(22):6739-46.
[9]李向东,李怀方,范在丰,等.马铃薯Y病毒属病毒HC-Pro研究进展[J].山东农业大学学报(自然科学版),2000,31(4):437-440.
[10]蒋琳,魏春红,李毅.病毒基因沉默抑制子及其作用机制[J].中国科学,2012,42(1):16-28.
[11]VANCE V B,ANANDALAKSHMI R,PRUSS G J,et al. A viral suppressor of gene silencing in plants[J]. Proc Natl Acad Sci USA,1998,95:13079-13084.
[12]BOUTET S,VAZQUEZ F,LIU J,et al. Arabidopsis HEN1:A genetic link between endogenous miRNA controlling development and siRNA controlling transgene silencing and virus resistance[J]. Curr Biol,2003,13(10):843-848.
[13]HAN M H,GOUD S,SONG L,et al. The Arabidopsis double-stranded RNA-binding protein HYL1 plays a role in microRNA-mediated gene regulation[J]. Proc Natl Acad Sci USA,2004,101(4):1093-1098.
[14]PARK M Y,WU G,GONZALEZ-SULSER,et al. Nuclear processing and export of microRNA in Arabidopsis[J]. Proc Natl Acad Sci USA,2005,102(10):3691-3696.
[15]GAIL J P,CHRISTOPHER B L,TROY B,et al. The potyviral suppressor of RNA silencing confers enhance dresistance to multiple pathogens[J]. Virology,2004,(320):107-120.
[16]杨丽萍,许亚男,刘雨晴,等.利用根部真空侵染法在烟草中瞬时表达外源蛋白[J].分子植物育种,2016,14(12):3385-3389.
[17]杨丽萍,金太成,徐洪伟,等.植物中瞬时表达外源基因的新型侵染技术[J].遗传,2013,35(1):111-117.
[18]YANG L P,XU Y N,LIU Y Q,et al. HC-Pro viral suppressor from tobacco vein banding mosaic virus interferes with DNA methylation and activates the salicylic acid pathway,Virology,2016,497:244-250.
[19]杨丽萍,金太成,周晓馥,利用农杆菌侵染新技术在豌豆中瞬时表达ha FGF[J].生物技术通报,2013,1(10),71-75.7
[20]YANG L P,WANG H W,LIU J Y,Et al. A simple and effective system for foreign gene expression in plants via root absorption of agrobacterial suspension,Journal of Biotechnology,2008,134:320-324.
[21]JIN T C,WANG J,ZHU X J,et al. A new transient expression system for large-scale production of recombinant proteins in plants based on airbrushing an Agrobacterium suspension,Biotechnology Report,2015,6:36-40.
[22]FAN Y J,LI W,WANG J J,et al. Efficient production of human acidic fibroblast growth factor in pea(Pisum sativum L.)plants by agroinfection of germinated seeds[J]. BMC Biotechnol,2011,11:45.
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