VIGS实验技术体系在月季中的应用及优化
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摘要
旨在月季中建立高效、快速的VIGS实验技术体系,以期能够改进VIGS技术体系在月季等木本植物表达效率低等缺陷。采用RT-PCR方法进行月季RhPDS基因片段的克隆,在以pTRV2为原载体,采用双酶切的方法构建了pTRV2-RhPDS重组载体。经双酶切及测序验证后,通过电击法转化农杆菌GV3101,并分别采用高压喷枪法对月季植株进行侵染、采用真空渗透法对扦插苗以及组培苗进行侵染。采用qRT-PCR方法分析RhPDS基因的表达。结果显示:构建的重组载体pTRV2-RhPDS,经双酶切验证,显示条带单一清晰,符合预期目标。在侵染方法和材料上,采用真空渗透法侵染组培苗是最为快速、高效的方式。该试验获得了较为高效、快速VIGS技术体系,提高了VIGS技术体系在月季上表达效率。
This study established an efficient and fast VIGS experimental system in rosa hybrida to improve the expression efficiency in rosa hybrida and other woody plants by VIGS.The coding sequence of RhPDS was amplified using RT-PCR method.pTRV2 was used as the original vector,and pTRV2-RhPDS vectors were constructed by double digestion.The test result was validated by double digestion and sequencing,and then the VIGS expression vectors were transformed into Agrobacterium strain GV3101 by eletroporation.The whole plant was sprayed by a portable air compressor.The vacuum infiltration method was used for rose cuttings and tissue culture.The expression level of RhPDS was detected by RT-qPCR.The results showed that the recombinant vector pTRV2-RhPDS,verified by double enzyme digestion,had a single and clear band,which met the target.Considering the methods and materials,vacuum osmosis was the most rapid and efficient way to infect tissue culture seedling.In this research,we obtained an optimized VIGS technical system and improved the expression efficiency of VIGS in rosa hybrida.
This study established an efficient and fast VIGS experimental system in rosa hybrida to improve the expression efficiency in rosa hybrida and other woody plants by VIGS.The coding sequence of RhPDS was amplified using RT-PCR method.pTRV2 was used as the original vector,and pTRV2-RhPDS vectors were constructed by double digestion.The test result was validated by double digestion and sequencing,and then the VIGS expression vectors were transformed into Agrobacterium strain GV3101 by eletroporation.The whole plant was sprayed by a portable air compressor.The vacuum infiltration method was used for rose cuttings and tissue culture.The expression level of RhPDS was detected by RT-qPCR.The results showed that the recombinant vector pTRV2-RhPDS,verified by double enzyme digestion,had a single and clear band,which met the target.Considering the methods and materials,vacuum osmosis was the most rapid and efficient way to infect tissue culture seedling.In this research,we obtained an optimized VIGS technical system and improved the expression efficiency of VIGS in rosa hybrida.
引文
[1]Senthilkumar M,Mysore K S.Tobacco rattle virus-based virusinduced gene silencing in Nicotiana benthamiana.[J].Nature Protocols,2014,9(7):1549-62.
[2]姚丙晨,孙玥,孙林静,等.病毒诱导的基因沉默的发展及在植物生物逆境上的应用[J].中国农学通报,2016,32(9):131-136.
[3]Hsieh M H,Pan Z J,Lai P H,et al.Virus-induced gene silencing unravels multiple transcription factors involved in floral growth and development in Phalaenopsis orchids[J].Journal of Experimental Botany,2013,64(12):3869.
[4]Amap M,O’Rourke J A,Van d M M,et al.Transcriptome analyses and virus induced gene silencing identify genes in the Rpp4-mediated Asian soybean rust resistance pathway[J].Functional Plant Biology Fpb,2013,40(10):1029-1047.
[5]Liscombe D K,O’Connor S E.A virus-induced gene silencing approach to understanding alkaloid metabolism in Catharanthus roseus[J].Phytochemistry,2011,72(16):1969.
[6]Chang X,Donnelly L,Sun D,et al.A Petunia HomeodomainLeucine Zipper Protein,Ph HD-Zip,Plays an Important Role in Flower Senescence[J].Plos One,2014,9(2):e88320.
[7]Eybishtz A,Peretz Y,Sade D,et al.Tomato yellow leaf curl virus infection of a resistant tomato line with a silenced sucrose transporter gene Le HT1 results in inhibition of growth,enhanced virus spread,and necrosis[J].Planta,2010,231(3):537-548.
[8]Kang Y,Kim R,Cho H,et al.Silencing of a BYPASS1 homolog results in root-independent pleiotrophic developmental defects in Nicotiana benthamiana.[J].Plant Molecular Biology,2008,68(4-5):423-437.
[9]Bouvier F,Linka N,Isner J C,et al.Arabidopsis SAMT1 Defines a Plastid Transporter Regulating Plastid Biogenesis and Plant Development[J].Plant Cell,2006,18(11):3088-105.
[10]Jiang C Z,Chen J C,Reid M S,et al.Functional analysis of genes associated with flower senescence[J].Acta Horticulturae,2005,682(682).
[11]Quadrana L,Rodriguez M C,López M,et al.Coupling VirusInduced Gene Silencing to Exogenous Green Fluorescence Protein Expression Provides a Highly Efficient System for Functional Genomics in Arabidopsis and across All Stages of Tomato Fruit Development[J].Plant Physiology,2011,156(3):1278-1291.
[12]Liu B,Watanabe S,Uchiyama T,et al.The Soybean Stem Growth Habit Gene Dt1 Is an Ortholog of Arabidopsis TERMINAL FLOWER1[J].Plant Physiology,2010,153(1):198-210.
[13]Bruun-Rasmussen M,Madsen C T,Jessing S,et al.Stability of Barley stripe mosaic virus-induced gene silencing in barley[J].Molecular plant-microbe interactions:MPMI,2007,20(11):1323.
[14]Jia H F,Chai Y M,Li C L,et al.Abscisic Acid Plays an Important Role in the Regulation of Strawberry Fruit Ripening[J].Plant Physiology,2011,157(1):188.
[15]张新华,李富军.病毒诱导的基因沉默技术及其在植物中的研究进展[J].西北植物学报,2012,32(2):419-424.
[16]Liu Y L,Schiff M,Dinesh-Kumar S P.Virus-induced gene silencing in tomato[J].Plant Journal,2002,31(6):777-786.
[17]Yan H X,Fu D Q,Zhu B Z,et al.Sprout vacuum-infiltration:a simple and efficient agroinoculation method for virus-induced gene silencing in diverse solanaceous species[J].Plant Cell Reports,2012,31(9):1713-1722.
[18]Livak K J,Schmittgen T D.Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2-ΔΔC T,Method[J].Methods,2012,25(4):402-408.
[19]Bennypaul H S,Mutti J S,Rustgi S,et al.Virus-induced gene silencing(VIGS)of genes expressed in root,leaf,and meiotic tissues of wheat[J].Functional&Integrative Genomics,2012,12(1):143.
[20]傅达奇.番茄中病毒诱导基因沉默体系的建立及Le EIN2基因功能分析[D].北京:中国农业大学,2005.
[21]Zeng S,Liang S,Zhang Y Y,et al.In vitro flowering red miniature rose[J].Biologia Plantarum,2013,57(3):401-409.
[22]Sun D,Li S,Niu L,et al.Ph OBF1,a petunia ocs element binding factor,plays an important role in antiviral RNA silencing.[J].Journal of Experimental Botany,2017,68(5).
[23]Senthil-Kumar M,Mysore K S.Virus-induced gene silencing can persist for more than 2 years and also be transmitted to progeny seedlings in Nicotiana benthamiana and tomato[J].Plant Biotechnology Journal,2011,9(7):797-806.
[2]姚丙晨,孙玥,孙林静,等.病毒诱导的基因沉默的发展及在植物生物逆境上的应用[J].中国农学通报,2016,32(9):131-136.
[3]Hsieh M H,Pan Z J,Lai P H,et al.Virus-induced gene silencing unravels multiple transcription factors involved in floral growth and development in Phalaenopsis orchids[J].Journal of Experimental Botany,2013,64(12):3869.
[4]Amap M,O’Rourke J A,Van d M M,et al.Transcriptome analyses and virus induced gene silencing identify genes in the Rpp4-mediated Asian soybean rust resistance pathway[J].Functional Plant Biology Fpb,2013,40(10):1029-1047.
[5]Liscombe D K,O’Connor S E.A virus-induced gene silencing approach to understanding alkaloid metabolism in Catharanthus roseus[J].Phytochemistry,2011,72(16):1969.
[6]Chang X,Donnelly L,Sun D,et al.A Petunia HomeodomainLeucine Zipper Protein,Ph HD-Zip,Plays an Important Role in Flower Senescence[J].Plos One,2014,9(2):e88320.
[7]Eybishtz A,Peretz Y,Sade D,et al.Tomato yellow leaf curl virus infection of a resistant tomato line with a silenced sucrose transporter gene Le HT1 results in inhibition of growth,enhanced virus spread,and necrosis[J].Planta,2010,231(3):537-548.
[8]Kang Y,Kim R,Cho H,et al.Silencing of a BYPASS1 homolog results in root-independent pleiotrophic developmental defects in Nicotiana benthamiana.[J].Plant Molecular Biology,2008,68(4-5):423-437.
[9]Bouvier F,Linka N,Isner J C,et al.Arabidopsis SAMT1 Defines a Plastid Transporter Regulating Plastid Biogenesis and Plant Development[J].Plant Cell,2006,18(11):3088-105.
[10]Jiang C Z,Chen J C,Reid M S,et al.Functional analysis of genes associated with flower senescence[J].Acta Horticulturae,2005,682(682).
[11]Quadrana L,Rodriguez M C,López M,et al.Coupling VirusInduced Gene Silencing to Exogenous Green Fluorescence Protein Expression Provides a Highly Efficient System for Functional Genomics in Arabidopsis and across All Stages of Tomato Fruit Development[J].Plant Physiology,2011,156(3):1278-1291.
[12]Liu B,Watanabe S,Uchiyama T,et al.The Soybean Stem Growth Habit Gene Dt1 Is an Ortholog of Arabidopsis TERMINAL FLOWER1[J].Plant Physiology,2010,153(1):198-210.
[13]Bruun-Rasmussen M,Madsen C T,Jessing S,et al.Stability of Barley stripe mosaic virus-induced gene silencing in barley[J].Molecular plant-microbe interactions:MPMI,2007,20(11):1323.
[14]Jia H F,Chai Y M,Li C L,et al.Abscisic Acid Plays an Important Role in the Regulation of Strawberry Fruit Ripening[J].Plant Physiology,2011,157(1):188.
[15]张新华,李富军.病毒诱导的基因沉默技术及其在植物中的研究进展[J].西北植物学报,2012,32(2):419-424.
[16]Liu Y L,Schiff M,Dinesh-Kumar S P.Virus-induced gene silencing in tomato[J].Plant Journal,2002,31(6):777-786.
[17]Yan H X,Fu D Q,Zhu B Z,et al.Sprout vacuum-infiltration:a simple and efficient agroinoculation method for virus-induced gene silencing in diverse solanaceous species[J].Plant Cell Reports,2012,31(9):1713-1722.
[18]Livak K J,Schmittgen T D.Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2-ΔΔC T,Method[J].Methods,2012,25(4):402-408.
[19]Bennypaul H S,Mutti J S,Rustgi S,et al.Virus-induced gene silencing(VIGS)of genes expressed in root,leaf,and meiotic tissues of wheat[J].Functional&Integrative Genomics,2012,12(1):143.
[20]傅达奇.番茄中病毒诱导基因沉默体系的建立及Le EIN2基因功能分析[D].北京:中国农业大学,2005.
[21]Zeng S,Liang S,Zhang Y Y,et al.In vitro flowering red miniature rose[J].Biologia Plantarum,2013,57(3):401-409.
[22]Sun D,Li S,Niu L,et al.Ph OBF1,a petunia ocs element binding factor,plays an important role in antiviral RNA silencing.[J].Journal of Experimental Botany,2017,68(5).
[23]Senthil-Kumar M,Mysore K S.Virus-induced gene silencing can persist for more than 2 years and also be transmitted to progeny seedlings in Nicotiana benthamiana and tomato[J].Plant Biotechnology Journal,2011,9(7):797-806.