利用CRISPR/Cas9技术敲除拟南芥转录因子MYB40的两种可变剪接体
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摘要
MYB家族转录因子在植物抵抗非生物胁迫过程中发挥着重要功能。前期研究发现强旱生植物霸王转录因子ZxMYB315参与到植物抗逆过程中,其在拟南芥中的同源基因MYB40的两种不同可变剪接体MYB40.1和MYB40.2对盐胁迫均有明显响应,但其在植物抗逆过程中的功能还未见报道。分析了在盐处理条件下MYB40.1及MYB40.2的表达模式,并利用CRISPR/Cas9技术,获得了2个MYB40.1被编辑株系,及3个MYB40.1和MYB40.2被同时编辑株系,为揭示MYB40这两种可变剪接体在拟南芥响应逆境胁迫过程中的功能和分子机理奠定了基础。
MYB transcription factors play important roles in various abiotic stress signaling pathways.In a previous study,we found that ZxMYB315 fromZygophyllum xanthoxylumis involved in plant responses to salt and drought stresses.In addition,the ortholog of ZxMYB315 in Arabidopsis,MYB40,has two splice variants,MYB40.1and MYB40.2.Expression levels of both MYB40.1and MYB40.2are affected by salt treatment.However,the functions of these changes in response to abiotic stress are still unclear.In this study,we further analyzed the expression patterns of MYB40.1and MYB40.2under salt treatment.In particular,two heterozygous mutations for MYB40.1and three heterozygous mutations for both MYB40.1and MYB40.2were generated using the gene-editing technique CRISPR/Cas9.These mutants now provide a foundation for studying functions and molecular mechanisms of MYB40 in various abiotic stress signaling pathways.
MYB transcription factors play important roles in various abiotic stress signaling pathways.In a previous study,we found that ZxMYB315 fromZygophyllum xanthoxylumis involved in plant responses to salt and drought stresses.In addition,the ortholog of ZxMYB315 in Arabidopsis,MYB40,has two splice variants,MYB40.1and MYB40.2.Expression levels of both MYB40.1and MYB40.2are affected by salt treatment.However,the functions of these changes in response to abiotic stress are still unclear.In this study,we further analyzed the expression patterns of MYB40.1and MYB40.2under salt treatment.In particular,two heterozygous mutations for MYB40.1and three heterozygous mutations for both MYB40.1and MYB40.2were generated using the gene-editing technique CRISPR/Cas9.These mutants now provide a foundation for studying functions and molecular mechanisms of MYB40 in various abiotic stress signaling pathways.
引文
[1] Golldack D,Luking I,Yang O.Plant tolerance to drought and salinity:Stress regulating transcription factors and their functional significance in the cellular transcriptional network.Plant Cell Reports,2011,30(8):1383-1391.
[2] Roy S.Function of MYB domain transcription factors in abiotic stress and epigenetic control of stress response in plant genome.Plant Signaling&Behavior,2016,11(1):1559-2324.
[3] Urao T,Yamaguchi-Shinozaki K,Urao S,et al.An Arabidopsis myb homolog is induced by dehydration stress and its gene product binds to the conserved MYB recognition sequence.Plant Cell,1993,5(11):1529-1539.
[4] Dai X,Xu Y,Ma Q,et al.Over-expression of an R1R2R3 MYB gene,OsMYB3R-2,increases tolerance to freezing,drought,and salt stress in transgenic Arabidopsis.Plant Physiology,2007,143(4):1739-1751.
[5] Shin D,Moon S J,Han S,et al.Expression of StMYB1R-1,a novel potato single MYB-like domain transcription factor,increases drought tolerance.Plant Physiology,2011,155(1):421-432.
[6] Meng X,Wang J R,Wang G D,et al.An R2R3-MYB gene,LeAN2,positively regulated the thermo-tolerance in transgenic tomato.Journal of Plant Physiology,2015,175:1-8.
[7] Rivero L,Scholl R,Holomuzki N,et al.Handling Arabidopsis plants:Growth,preservation of seeds,transformation,and genetic crosses//Sanchez-Serrano J J,Salinas J.Arabidopsis protocol.London:Human Press,2013:9-11.
[8] Ma X,Zhang Q,Zhu Q,et al.A robust CRISPR/Cas9system for convenient,high-efficiency multiplex genome editing in monocot and dicot plants.Molecular Plant,2015,8(8):1274-1284.
[9] Yan L,Wei S,Wu Y,et al.High-efficiency genome editing in Arabidopsis using yao promoter-driven CRISPR/Cas9system.Molecular Plant,2015,8(12):1820-1823.
[10] Koevoets I T,Venema J H,Elzenga J T,et al.Roots withstanding their environment:Exploiting root system architecture responses to abiotic stress to improve crop tolerance.Frontiers in Plant Science,2016,7:1335-1353.
[11] Rosegrant M W,Ringler C,Zhu T.Water for agriculture:Maintaining food security under growing scarcity.Annual Review of Environment&Resources,2009,34:205-222.
[12] Smedema L K,Shiati K.Irrigation and salinity:A perspective review of the salinity hazards of irrigation development in the arid zone.Irrigation&Drainage Systems,2002,16(2):161-174.
[13] Joshi R,Wani S H,Singh B,et al.Transcription factors and plants response to drought stress:Current understanding and future directions.Frontiers in Plant Science,2016,7:1029-1043.
[14] Seo P J,Kim M J,Ryu J,et al.Two splice variants of the IDD14transcription factor competitively form nonfunctional heterodimers which may regulate starch metabolism.Nature Communications,2011,2:doi:10.1038/ncomms1303.
[15] Li J,Li X,Guo L,et al.A subgroup of MYBtranscription factor genes undergoes highly conserved alternative splicing in Arabidopsis and rice.Journal of Experimental Botany,2006,57(6):1263-1273.
[16] Cheng Q,Zhou Y,Liu Z,et al.An alternatively spliced heat shock transcription factor,OsHSFA2dI,functions in the heat stress-induced unfolded protein response in rice.Plant Biology,2015,17(2):419-429.
[17] Liu J,Sun N,Liu M,et al.An autoregulatory loop controlling Arabidopsis HsfA2expression:Role of heat shock-induced alternative splicing.Plant Physiology,2013,162:512-521.
[18] Bao A K,Bai T H,Zhao T X,et al.CRISPR/Cas9:A gene targeting technology and its application in the study of plant genetic function.Acta Prataculturae Sinica,2017,26(7):190-200.包爱科,白天惠,赵天璇,等.CRISPR/Cas9系统:基因组定点编辑技术及其在植物基因功能研究中的应用.草业学报,2017,26(7):190-200.
[19] Bortesi L,Fischer R.The CRISPR/Cas9system for plant genome editing and beyond.Biotechnology Advances,2015,33:41
[20] Ye G N,Stone D,Pang S Z,et al.Arabidopsis ovule is the target for Agrobacteriumin planta vacuum infiltration transformation.Plant Journal,1999,19:249-257.
[21] Li H J,Liu N Y,Shi D Q,et al.YAO is a nucleolar WD40-repeat protein critical for embryogenesis and gametogenesis in Arabidopsis.BMC Plant Biology,2010,10:169-180.
[2] Roy S.Function of MYB domain transcription factors in abiotic stress and epigenetic control of stress response in plant genome.Plant Signaling&Behavior,2016,11(1):1559-2324.
[3] Urao T,Yamaguchi-Shinozaki K,Urao S,et al.An Arabidopsis myb homolog is induced by dehydration stress and its gene product binds to the conserved MYB recognition sequence.Plant Cell,1993,5(11):1529-1539.
[4] Dai X,Xu Y,Ma Q,et al.Over-expression of an R1R2R3 MYB gene,OsMYB3R-2,increases tolerance to freezing,drought,and salt stress in transgenic Arabidopsis.Plant Physiology,2007,143(4):1739-1751.
[5] Shin D,Moon S J,Han S,et al.Expression of StMYB1R-1,a novel potato single MYB-like domain transcription factor,increases drought tolerance.Plant Physiology,2011,155(1):421-432.
[6] Meng X,Wang J R,Wang G D,et al.An R2R3-MYB gene,LeAN2,positively regulated the thermo-tolerance in transgenic tomato.Journal of Plant Physiology,2015,175:1-8.
[7] Rivero L,Scholl R,Holomuzki N,et al.Handling Arabidopsis plants:Growth,preservation of seeds,transformation,and genetic crosses//Sanchez-Serrano J J,Salinas J.Arabidopsis protocol.London:Human Press,2013:9-11.
[8] Ma X,Zhang Q,Zhu Q,et al.A robust CRISPR/Cas9system for convenient,high-efficiency multiplex genome editing in monocot and dicot plants.Molecular Plant,2015,8(8):1274-1284.
[9] Yan L,Wei S,Wu Y,et al.High-efficiency genome editing in Arabidopsis using yao promoter-driven CRISPR/Cas9system.Molecular Plant,2015,8(12):1820-1823.
[10] Koevoets I T,Venema J H,Elzenga J T,et al.Roots withstanding their environment:Exploiting root system architecture responses to abiotic stress to improve crop tolerance.Frontiers in Plant Science,2016,7:1335-1353.
[11] Rosegrant M W,Ringler C,Zhu T.Water for agriculture:Maintaining food security under growing scarcity.Annual Review of Environment&Resources,2009,34:205-222.
[12] Smedema L K,Shiati K.Irrigation and salinity:A perspective review of the salinity hazards of irrigation development in the arid zone.Irrigation&Drainage Systems,2002,16(2):161-174.
[13] Joshi R,Wani S H,Singh B,et al.Transcription factors and plants response to drought stress:Current understanding and future directions.Frontiers in Plant Science,2016,7:1029-1043.
[14] Seo P J,Kim M J,Ryu J,et al.Two splice variants of the IDD14transcription factor competitively form nonfunctional heterodimers which may regulate starch metabolism.Nature Communications,2011,2:doi:10.1038/ncomms1303.
[15] Li J,Li X,Guo L,et al.A subgroup of MYBtranscription factor genes undergoes highly conserved alternative splicing in Arabidopsis and rice.Journal of Experimental Botany,2006,57(6):1263-1273.
[16] Cheng Q,Zhou Y,Liu Z,et al.An alternatively spliced heat shock transcription factor,OsHSFA2dI,functions in the heat stress-induced unfolded protein response in rice.Plant Biology,2015,17(2):419-429.
[17] Liu J,Sun N,Liu M,et al.An autoregulatory loop controlling Arabidopsis HsfA2expression:Role of heat shock-induced alternative splicing.Plant Physiology,2013,162:512-521.
[18] Bao A K,Bai T H,Zhao T X,et al.CRISPR/Cas9:A gene targeting technology and its application in the study of plant genetic function.Acta Prataculturae Sinica,2017,26(7):190-200.包爱科,白天惠,赵天璇,等.CRISPR/Cas9系统:基因组定点编辑技术及其在植物基因功能研究中的应用.草业学报,2017,26(7):190-200.
[19] Bortesi L,Fischer R.The CRISPR/Cas9system for plant genome editing and beyond.Biotechnology Advances,2015,33:41
[20] Ye G N,Stone D,Pang S Z,et al.Arabidopsis ovule is the target for Agrobacteriumin planta vacuum infiltration transformation.Plant Journal,1999,19:249-257.
[21] Li H J,Liu N Y,Shi D Q,et al.YAO is a nucleolar WD40-repeat protein critical for embryogenesis and gametogenesis in Arabidopsis.BMC Plant Biology,2010,10:169-180.