沙冬青子叶原生质体瞬时表达体系的建立及其AmDREB1蛋白的亚细胞定位
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摘要
以沙冬青(Ammopiptanthus mongolicus(Maxim.ex Kom.)Cheng f.)幼苗的子叶为材料,对其原生质体的分离、纯化和瞬时表达体系进行了研究。结果表明,子叶原生质体分离的最佳酶解液组成为CPW溶液+3.0%纤维素酶R-10+0.5%离析酶R-10+0.3%半纤维素酶+9.0%甘露醇(p H5.8);最佳酶解条件为室温、避光、40 r/min轻摇14 h。采用W5溶液作为漂洗液将酶解物稀释后进行过滤,将过滤液在4℃、700 r/min条件下离心5 min,所得纯化原生质体的产量约为2.50×106cells/g,活力达到90%;以纯化的原生质体作为受体,利用聚乙二醇(PEG)介导法成功将植物瞬时表达载体p BI-GFP导入其中,转化效率达到50.8%。利用本研究建立的原生质体瞬时表达体系,检测到沙冬青脱水应答转录因子Am DREB1定位于细胞核内。
Using young cotyledons of Ammopiptanthus mongolicus(Maxim. ex Kom.) Cheng f.seedlings as donor material,the technical system for the isolation,purification,and transient expression of the cotyledon protoplasts was studied. Results showed that the optimal enzyme solution for the protoplast isolation was CPW solution + 3.0% cellulase R-10 + 0.5%macerozyme R-10 + 0.3% hemicellulose + 9.0% mannitol(p H5.8), and the optimal enzymolysis conditions were gentle shaking of the enzyme solution containing cotyledon tissues at 40 r/min in the dark for 14 h at room temperature. Using the W5 solution as a washing solution,the enzymatic hydrolysate was diluted and filtered,with the filtrate then centrifuged at 4℃ and 700 r/min for 5 min. The purified protoplast yield was approximately2.50 × 106 cells/g and the protoplast viability reached 90%. Using the PEG-mediated method,the protoplasts were successfully transformed with plant transient expression vector p BI-GFPand the transformation efficiency was 50.8%. Moreover, using the established protoplast transient expression system,a dehydration-responsive transcription factor of A. mongolicus,namely Am DREB1,was found in the nucleus.
Using young cotyledons of Ammopiptanthus mongolicus(Maxim. ex Kom.) Cheng f.seedlings as donor material,the technical system for the isolation,purification,and transient expression of the cotyledon protoplasts was studied. Results showed that the optimal enzyme solution for the protoplast isolation was CPW solution + 3.0% cellulase R-10 + 0.5%macerozyme R-10 + 0.3% hemicellulose + 9.0% mannitol(p H5.8), and the optimal enzymolysis conditions were gentle shaking of the enzyme solution containing cotyledon tissues at 40 r/min in the dark for 14 h at room temperature. Using the W5 solution as a washing solution,the enzymatic hydrolysate was diluted and filtered,with the filtrate then centrifuged at 4℃ and 700 r/min for 5 min. The purified protoplast yield was approximately2.50 × 106 cells/g and the protoplast viability reached 90%. Using the PEG-mediated method,the protoplasts were successfully transformed with plant transient expression vector p BI-GFPand the transformation efficiency was 50.8%. Moreover, using the established protoplast transient expression system,a dehydration-responsive transcription factor of A. mongolicus,namely Am DREB1,was found in the nucleus.
引文
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[19]景艳春,康向阳,王君,李代丽.新疆杨叶肉原生质体游离和纯化的研究[J].西北植物学报,2007,27(3):509-514.Jing YC,Kang XY,Wang J,Li DL.Isolation and purification of mesophyll protoplasts of Populus alba L.var.pyramidalis[J].Acta Botanica Boreali-Occidentalia Sinica,2007,27(3):509-514.
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[21]刘艳丽,金孝芳,马林龙,曹丹,龚自明,韦朝领.茶树叶肉原生质体的分离与纯化[J].植物科学学报,2017,35(6):908-911.Liu YL,Jin XF,Ma LL,Cao D,Gong ZM,Wei CL.Isolation and purification of mesophyll protoplasts from the leaves of Camellia sinensis[J].Plant Science Journal,2017,35(6):908-911.
[22]廖嘉明,王伯初,王益川,田继权.拟南芥叶肉原生质体分离条件的优化研究[J].西北植物学报,2010,30(6):1271-1276.Liao JM,Wang BC,Wang YC,Tian JQ.Optimization conditions of Arabidopsis mesophyll protoplast isolation[J].Acta Botanica Boreali-Occidentalia Sinica,2010,30(6):1271-1276.
[2]Yoo SD,Cho YH,Sheen J.Arabidopsis mesophyll protoplasts:a versatile cell system for transient gene expression analysis[J].Nat Protoc,2007,2:1565-1572.
[3]Zhai Z,Sooksa-nguan T,Vatamaniuk OK.Establishing RNA interference as a reverse-genetic approach for gene functional analysis in protoplasts[J].Plant Physiol,2009,149:642-652.
[4]Zhang Y,Su J,Duan S,Ao Y,Dai J,et al.A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes[J].Plant Methods,2011,7:30-43.
[5]赵文婷,魏建和,刘晓东,高志晖.植物瞬时表达技术的主要方法与应用进展[J].生物技术通讯,2013,24(2):294-300.Zhao WT,Wei JH,Liu XD,Gao ZH.Advance of the main methods and applications of plant transient expression system[J].Letters in Biotechnology,2013,24(2):294-300.
[6]Ficher R,Hain R.Tobacco protoplast transformation and use for functional analysis of newly isolated genes and gene constructs[J].Method Cell Biol,1995,50:401-410.
[7]Jiang L,Wang J,Liu Z,Wang L,Zhang F,et al.Silencing induced by inverted repeat constructs in protoplasts of Nicotiana benthamiana[J].Plant Cell Tiss Organ Cult,2010,100:139-148.
[8]Bart R,Chern M,Park CJ,Bartley L,Ronald PC.A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts[J].Plant Methods,2006,2:13-21.
[9]Guo JJ,Morrell-Falvey JL,LabbéJL,Muchero W,Kalluri UC,et al.Highly efficient isolation of Populus mesophyll protoplasts and its application in transient expression assays[J].PLo S One,2012,7(9):e44908.
[10]舒英杰,黄丽燕,陈明,陶源,王占奎,麻浩.基于亚细胞定位的大豆和鹰嘴豆原生质体分离体系的建立与优化[J].生物工程学报,2017,33(6):976-985.Shu YJ,Huang LY,Chen M,Yuan T,Wang ZK,Ma H.Establishment and optimization of systems for protoplasts isolation of soybean and chickpea that used in subcellular location[J]Chinese Journal of Biotechnology,2017,33(6):976-985.
[11]Pitzschke A,Persak H.Poinsettia protoplasts-a simple,robust and efficient system for transient gene expression studies[J].Plant Methods,2012,8:14.
[12]Masani MYA,Noll GA,Parveez GKA,Sambanthamurthi R,Prüfer D.Efficient transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection[J].PLo S One,2014,9(5):e96831.
[13]李妮娜,丁林云,张志远,郭旺珍.棉花叶肉原生质体分离及目标基因瞬时表达体系的建立[J].作物学报,2014,40(2):231-239.Li NN,Ding LY,Zhang ZY,Guo WZ.Isolation of mesophyll protoplast and establishment of gene transient expression system in cotton[J].Acta Agronomica Sinica,2014,40(2):231-239.
[14]Nanjareddy K,Arthikala MK,Blanco L,Arellano ES,Lara M.Protoplast isolation,transient transformation of leaf mesophyll protoplasts and improved Agrobacteriummediated leaf disc infiltration of Phaseolus vulgaris:tools for rapid gene expression analysis[J].BMC Biotechnol,2016,16:53.
[15]周宜君,刘春兰,冯金朝,贾晓红.沙冬青抗旱、抗寒机理的研究进展[J].中国沙漠,2001,21(3):312-315.Zhou YJ,Liu CL,Feng JC,Jia XH.Advances of droughtresistance and frigid-resistance mechanism research on Ammopiptanthus mongolicus[J].Journal of Desert Research,2001,21(3):312-315.
[16]Zhou Y,Gao F,Liu R,Feng J,Li H.De novo sequencing and analysis of root transcriptome using 454 pyrosequencing to discover putative genes associated with drought tolerance in Ammopiptanthus mongolicus[J].BMC Genomics,2012,13:266.
[17]Wu YQ,Wei W,Pang XY,Wang XF,Zhang HL,et al.Comparative transcriptome profiling of a desert evergreen shrub,Ammopiptanthus mongolicus,in response to drought and cold stresses[J].BMC Genomics,2014,15:671.
[18]Pang T,Guo L,Shim D,Cannon N,Tang S,Chen J.Characterization of the transcriptome of the xerophyte Ammopiptanthus mongolicus leaves under drought stress by454 pyrosequencing[J].PLo S One,2015,10(8):e0136495.
[19]景艳春,康向阳,王君,李代丽.新疆杨叶肉原生质体游离和纯化的研究[J].西北植物学报,2007,27(3):509-514.Jing YC,Kang XY,Wang J,Li DL.Isolation and purification of mesophyll protoplasts of Populus alba L.var.pyramidalis[J].Acta Botanica Boreali-Occidentalia Sinica,2007,27(3):509-514.
[20]Lata C,Prasd M.Role of DREBs in regulation of abiotic stress responses in plants[J].J Exp Bot,2011,62:4731-4748.
[21]刘艳丽,金孝芳,马林龙,曹丹,龚自明,韦朝领.茶树叶肉原生质体的分离与纯化[J].植物科学学报,2017,35(6):908-911.Liu YL,Jin XF,Ma LL,Cao D,Gong ZM,Wei CL.Isolation and purification of mesophyll protoplasts from the leaves of Camellia sinensis[J].Plant Science Journal,2017,35(6):908-911.
[22]廖嘉明,王伯初,王益川,田继权.拟南芥叶肉原生质体分离条件的优化研究[J].西北植物学报,2010,30(6):1271-1276.Liao JM,Wang BC,Wang YC,Tian JQ.Optimization conditions of Arabidopsis mesophyll protoplast isolation[J].Acta Botanica Boreali-Occidentalia Sinica,2010,30(6):1271-1276.