作物学报 ›› 2014, Vol. 40 ›› Issue (02): 231-239.doi: 10.3724/SP.J.1006.2014.00231
李妮娜,丁林云,张志远,郭旺珍*
LI Ni-Na,DING Lin-Yun,ZHANG Zhi-Yuan,GUO Wang-Zhen*
摘要:
以棉花幼嫩子叶为外植体材料,分析影响棉花叶肉原生质体分离及目标基因转化的主要因素,以棉花叶肉原生质体为受体,建立稳定、高效的目标基因瞬时表达与鉴定体系。技术体系包括,选择自然生长12 d的棉花幼嫩子叶为外植体材料,混合1.5%纤维素酶、0.4%离析酶、0.5 mol L–1甘露醇、20 mmol L–1 KCl、20 mmol L–1 MES、0.1 mol L–1 CaCl2和1.0 g L–1 BSA等酶液,在28℃黑暗条件下振荡酶解8 h,可游离出浓度达1.0×106 m L–1以上的纯净棉花叶肉原生质体。利用该方法将棉花锌指蛋白基因GhZFP2整合到pJIT166-GFP质粒载体,构建了GhZFP2:GFP融合载体,采用40% PEG(4000)介导转化,获得高转化率的棉花叶肉原生质体。对目标基因瞬时表达产物检测表明,GhZFP2蛋白清晰定位在细胞核上。
[1]Werr W, Lörz H. Transient gene expression in a Gramineae cell line. Mol Gen Genet, 1986, 202: 471–475[2]Marion J, Bach L, Bellec Y, Meyer C, Gissot L, Faure J D. Systematic analysis of protein subcellular localization and interaction using high-throughput transient transformation of Arabidopsis seedlings. Plant J, 2008, 56: 169–179[3]王华忠, 陈雅平, 陈佩度. 植物瞬间表达系统与功能基因组学研究. 生物工程学报, 2007, 33: 367–374Wang H Z, Chen Y P, Chen P D. Plant transient expression system in functional genomics. Chin J Biotechnol, 2007, 33: 367–374 (in Chinese with English abstract)[4]杨捷, Showalter A M. 拟南芥中一个经典阿拉伯半乳糖蛋白的亚细胞定位. 福州大学学报(自然科学版), 2010, 38: 753–757 Yang J, Showalter A M. Subcellular localization of a classical Arabinogalactan - protein from Arabidopsis. J Fuzhou Univ (Nat Sci Edn), 2010, 38: 753–757 (in Chinese with English abstract)[5]刘肖飞, 梁卫红. 根癌农杆菌介导的GFP在洋葱表皮细胞定位研究. 河南师范大学学报(自然科学版), 2009, 37(1): 123–125 Liu X F, Liang W H. Localization analysis of GFP in onion epidermal cell via Agrobacterium tumefaciens-Mediated transformation. J Henan Normal Univ (Nat Sci Edn), 2009, 37(1): 123–125 (in Chinese with English abstract)[6]杨光, 曹雪, 房经贵, 黄振喜, 陶建敏, 王晨. 葡萄花发育基因的亚细胞定位和表达分析. 中国农业科学, 2011, 44: 641–650 Yang G, Zao X, Fang J G, Huang Z X, Tao J M, Wang C. Sub-Cellular localization and expression analysis of genes involved in grapevine floral development. Sci Agri Sin, 2011, 44: 641–650 (in Chinese with English abstract)[7]Zou J J,Wei F J, Wang C ,Wu J J, Ratnasekera D, Liu W X, Wu W H. Arabidopsis calcium dependent protein kinase CPK10 functions in abscisic acid and Ca2+ mediated stomata regulation in response to drought stress. Plant Physiol, 2010, 154: 1232–1243[8]Jiang L, Wang J, Liu Z, Wang L, Zhang F, Liu G C, Zhong Q. Silencing induced by inverted repeat constructs in protoplasts of Nicotiana benthamiana. Plant Cell Tiss Organ Cult, 2010, 100: 139–148[9]孙鹤, 郎志宏, 朱莉, 黄大昉. 玉米、小麦、水稻原生质体制备条件优化. 生物工程学报, 2013, 29: 224–234 Sun H, Lang Z H, Zhu L, Huang D F. Optimized condition for protoplast isolation from maize, wheat and rice leaves. Chin J Biotech, 2013, 29: 224–234 (in Chinese with English abstract)[10]Zhang H, Ni L, Liu Y P, Wang Y F, Zhang A Y, Tan M P, Jiang M Y. The C2H2-type zinc finger protein ZFP182 is involved in abscisic acid-induced antioxidant defense in rice. J Integr Plant Biol, 2012, 54: 500–510[11]Zhang Y, Xiao W K, Luo L J, Pang J H, Rong W, He C Z. Down regulation of OsPK1, a cytosolic pyruvate kinase, by T-DNA in sertion causes dwar?sm and panicle enclosure in rice. Planta, 2012, 235: 25–38[12]Paterson A H, Wendel J F, Gundlach H, Guo H, Jenkins J, Jin D, Llewellyn D, Showmaker K C, Shu S, Udall J, Yoo M, Byers R, Chen W, Doron-Faigenboim A, Duke M V, Gong L, Grimwood J, Grover C, Grupp K, Hu G, Lee T, Li J, Lin L, Liu T, Marler B S, Page J T, Roberts A W, Romanel E, Sanders W S, Szadkowski E, Tan X, Tang H, Xu C, Wang J, Wang Z, Zhang D, Zhang L, Ashfari H, Bedon F, Bowers J E, Brubaker C L, Chee P W, Das S, Gingle A R, Haigler C H, Harker D, Hoffmann L V, Hovav R, Jones D C, Lemke C, Mansoor S, Rahman M, Rainville L N, Rambani A, Reddy U K, Rong J, Saranga Y, Scheffler B E, Scheffler J A, Stelly D M, Triplett B A, Deynze A V, Vaslin M F S, Waghmare V N, Walford S, Wright R J, Zaki E A, Zhang T, Dennis E S, Mayer K F X, Peterson D G, Rokhsar D S, Wang X, Schmutz J. Repeated polyploidization of Gossypium genomes and the evolution of spinnable cotton fibres. Nature, 2012, 492: 423–427[13]Wang K B, Wang Z W, Li F G, Ye W W, Wang J Y, Song G L, Yue Z, Cong L, Shang H H, Zhu S L, Zou C S, LI Q, Yuan Y L, Lu C R, Wei H L, Gou C Y, Zheng Z Q, Yin Y, Zhang X Y, Liu K, Wang B, Song C, Shi N, Kohel R J, Percy R G, Yu J Z, Zhu Y X, Wang J, Yu S X . The draft genome of a diploid cotton Gossypium raimondii. Nat Genet, 2012, 44: 1098–1103[14]王晋成. 五个棉花逆境相关锌指蛋白基因的克隆与功能研究. 南京农业大学硕士学位论文, 2010Wang J C. Cloning and characterization of five zinc-finger proteins (ZFPs) related with stress in cotton. MS Thesis of Nanjing Agricultural University, 2010 (in Chinese with English abstract)[15]Yoo S D, Cho Y H, Sheen J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocols, 2007, 2: 1565–1572[16]于晓玲, 李春强, 彭明. 植物原生质体技术及其应用. 中国农学通报, 2009, 25(8): 22–26 Yu X L, Li C Q, Peng M. Advances on the research of protoplast technology. Chin Agri Sci Bull, 2009, 25(8): 2–26 (in Chinese with English abstract)[17]Cooking E. A method for the isolation of plant protoplasts and vacuoles. Nature, 1960, 187: 962–963[18]Nagata T, Takebe I. Cell wall regeneration and cell division in isolated tobacco mesophyll protoplasts. Planta, 1970, 92: 301–308[19]Kitajima A, Asatsuma S, Okada H, Hamada Y, Kaneko K, Nanjo Y, Kawagoe Y, Toyooka K, Matsuoka K, Takeuchi M, Nakano A, Mitsui T. The rice alpha-amylase glycoprotein is targeted from the Golgi apparatus through the secretory pathway to the plastids. Plant Cell, 2009, 21: 2844–2858[20]周颖, 姜国勇. 玉米核糖体失活蛋白基因z108在烟草原生质体中的转化及其表达. 生物技术通报, 2010, (4): 99–102 Zhou Y, Jiang G Y. Transformation and expression of maize ribosome-inactivating protein gene z108 in Tobacco protoplasts. Biotechnol Bull, 2010, (4): 99–102 (in Chinese with English abstract)[21]Zhang Y, Su J B, Duan S, Ao Y, Dai J R, Liu J, Wang P, Li Y G, Liu B, Feng D R, Wang J F,Wang H B. A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes. Plant Methods , 2011, 7: 30[22]秦永华, 刘进元. 棉花组织培养与植株再生. 分子植物育种, 2006, 4: 583–592 Qin Y H, Liu J Y. Cotton tissue culture and plant regeneration. Mol Plant Breed, 2006, 4: 583–592 (in Chinese with English abstract)[23]汪静儿, 孙玉强, 燕树锋, 道德, 沈晓佳, 祝水金. 陆地棉原生质体培养与植株再生技术研究. 棉花学报, 2008, 20: 403–407 Wang J E, Sun Y Q, Yan S F, Dao D, Shen X J, Zhu S J. Study on the plant regeneration from protoplasts of upland cotton (Gossypium hirsutum L.) via somatic embryogenesis. Cotton Sci, 2008, 20: 403–407 (in Chinese with English abstract)[24]Fu L L, Yang X Y, Zhang X L, Wang Z W, Feng C H, Liu Z X, Jiang P Y, Zhang J L. Regeneration and identification of interspecific asymmetric somatic hybrids obtained by donor-recipient fusion in cotton. Chin Sci Bull, 2009, 54: 2219–2227[25]王喆之, 张苏锋, 胡正海. 陆地棉胚性愈伤组织原生质体的制备、培养及植株再生. 植物学报, 1998, 40: 234–240 Wang Z Z, Zhang S F, Hu Z H. Isolation, culture and plant regeneration of protoplasts from an embryogenic callus tissue of Gossypium hirsutum. Acta Bot Sin, 1998, 40: 234–240 (in Chinese with English abstract)[26]李仁敬, 张忠新, 石玉瑚. 棉花叶肉原生质体的分离初报. 遗传, 1980, 2(4): 36 Li R J, Zhang Z X, Shi Y H. A preliminary report on the separation of cotton mesphyll protoplast. Hereditas, 1980, 2(4): 36 (in Chinese with English abstract)[27]Yang X Y, Zhang X L, Jin S X, Fu L L, Wang L G. Production and characterization of asymmetric hybrids between upland cotton Coker 201 (Gossypium hirsutum) and wild cotton (G. klozschianum Anderss). Plant Cell Tiss Organ Cult, 2007, 89: 225–235[28]汪静儿, 孙玉强, 祝水金. 棉花原生质体培养与体细胞杂交研究进展. 棉花学报, 2007, 19: 139–144 Wang J E, Sun Y Q, Zhu S J. Advances in cotton protoplast culture and somatic hybridization. Cotton Sci, 2007, 19: 139–144 (in Chinese with English abstract)[29]白珊珊, 尹敏娟, 张磊, 康向阳. 新疆杨愈伤组织原生质体的游离与纯化. 生物技术通讯, 2011, 22: 49–52 Bai S S, Yin M J, Zhang L, Kang X Y. Isolation and purification of callus-derived protoplasts of Populus Alba L var pyramidalis. Lett Biotechnol, 2011, 22: 49–52 (in Chinese with English abstract)[30]张红梅, 王俊丽. 植物原生质体游离及应用. 河北林果研究, 2002, 17: 376–382 Zhang H M, Wang J L. Plant protoplast isolation, culture and application. Hebei J For Orchard Res, 2002, 17: 376–382 (in Chinese with English abstract)[31]陈名红, 熊立, 陈学军. 烟草叶肉原生质体分离和纯化研究. 云南民族大学学报(自然科学版), 2005, 14: 326–329 Chen M H, Xiong L, Chen X J. Isolation and purification of mesophyll protoplast in Tabacco. J Yunnan National Univ (Nat Sci Edn), 2005, 14: 326–329 (in Chinese with English abstract)[32]He P, Shan L, Sheen J. The use of protoplasts to study innate immune responses. Methods Mol Biol, 2007, 354: 1–9[33]Sun Y Q, Zhang X L, Huang C, Nie Y C, Guo X P. Plant regeneration via somatic embryogenesis from protoplast s of six explants in Coker 201 (Gossypium hirsutum). Plant Cell Tiss Organ Cult, 2005, 82: 309–315[34]Gao X Q, Wheeler T, Li Z H, Kenerley C M, He P, Shan L B. Silencing GhNDR1 and GhMKK2 compromised cotton resistance to Verticillium wilt. Plant J, 2011, 66: 293–305[35]梁大伟. 拟南芥叶肉原生质体分离及瞬时表达体系的建立. 兰州大学硕士学位论文, 2009Liang D W. Isolation of mesphyll protoplast from Arabidopsis thaliana and the setup of transient expression assay system. MS Thesis of Lanzhou University, 2009 (in Chinese with English abstract)[36]Maas C, Werr W. Mechanism and optimized condition for PEG mediated DNA transfection into plant protoplast. Plant Cell Reps, 1989, 8: 148–151[37]Lazzeri P A, Brettschneider R, Luhrs R, Lorz H. Stable transformation of barley via PEG-induced direct DNA uptake into protoplasts. Theor Appl Genet, 1991, 81: 437–444[38]Kirschner M, Winkelhaus S, Thierfelder J M, Nover L. Transient expression and heat-induced co-aggregation of endogenous and heterologous small heat-stress proteins in tobacco protoplasts. Plant J, 2000, 24: 397–411[39]黄瑾, 李燕, 张薇, 李旭锋, 杨毅. 甘蓝型油菜瞬时表达系统研究. 四川大学学报(自然科学版), 2008, 45(增刊): 19–24 Huang J, Li Y, Zhang W, Li X F, Yang Y. Transient expression system in protoplasts of Brassica napus L. J Sichuan Univ (Nat Sci Edn), 2008, 45(suppl): 19–24 (in Chinese with English abstract)[40]李文彬, 王革娇, Stanchina E, Castiglione S, 孙勇如, Sala F. PEG介导原生质体转化获得水稻转基因植株. 植物学报, 1995, 37: 409–412Li W S, Wang G J, Stanchina E, Castiglione S, Sun Y R, Sala F. Transgenic rice plants produced by PEG-mediated plasmid uptake into protoplasts. Acta Bot Sin, 1995, 37: 409–412 (in Chinese with English abstract) |
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