Protoplast Isolation from Elephant Grass(Pennisetum purpureum ‘Huanan’)for Transient Gene Expression

doi:10.11733/j.issn.1007-0435.2015.03.020

Acta Agrestia Sinica ›› 2015, Vol. 23 ›› Issue (3): 571-579.DOI: 10.11733/j.issn.1007-0435.2015.03.020

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Protoplast Isolation from Elephant Grass(Pennisetum purpureum ‘Huanan’)for Transient Gene Expression

TANG Ran, PENG Xiao-qun, XIE Xin-ming

College of Agriculture, South China Agricultural University, Guangzhou, Guangdong Province 510642, China

Received:2014-06-06 Revised:2014-08-27 Online:2015-06-15 Published:2015-07-02

华南象草原生质体分离及基因瞬时表达研究

唐然, 彭小群, 解新明

华南农业大学农学院, 广东广州 510642

通讯作者: 解新明
作者简介:唐然(1988-),男,云南昆明人,博士研究生,主要从事牧草生物技术研究,E-mail:tangran@stu.scau.edu.cn
基金资助:
国家自然科学基金(31272491) (30972138);教育部高等学校博士学科点专项科研基金(20124404110011)资助

Abstract

Abstract:

Elephant grass (Pennisetum purpureum) is a kind of typical C4 clonal plant, which grows well in south China. It is commonly used as forage grass, green energy grass as well as nonwood fiber. Young leaf sheaths of elephant grass were used for mesophyll protoplasts isolation in this study. 5 factors of cellulase concentration, macerozyme concentration, pH, mannitol concentration, enzymolysis time were studied to optimize isolation conditions based on L₁₆(4⁵) orthogonal test. And the isolated elephant grass protoplasts were used for gene transient expression assays, aiming to lay the foundation for gene functional analysis in elephant grass. The results showed that the optimum conditions for elephant grass mesophyll protoplasts isolation were in the enzyme combinations containing 1.5% cellulase, 0.75% macerozyme, 0.5 mol·L^-1 mannitol, pH 5.8. The digestion was conducted in the dark under 28℃ for 4 h with gentle shaking. Under these conditions, the protoplasts yield was 5.11×10⁶ cells·g FW^-1 (fresh weight), and the vitality was 91.08%. Variance analysis of orthogonal test indicated that the enzymolysis time is the most important factor influencing both protoplast yield and vitality. Two plasmid vectors, carried green and red fluorescent protein respectively, were then transformed into purified mesophyll protoplasts by PEG-mediated method. The green and red fluorescent signal were detected in the transient transformed protoplasts, obtained the highest transformation efficiency of 77.47%, and co-transformation efficiency of 8.79%. The isolated protoplasts could be used for gene transient assay in elephant grass.

Key words: Pennisetum purpureum, Protoplast, PEG-mediated, Transient expression

摘要：

以华南象草(Pennisetum purpureum)幼叶鞘为材料,采用L₁₆(4⁵)正交试验设计,对分离原生质体过程中的纤维素酶浓度、离析酶浓度、酶解液pH、甘露醇浓度、酶解时间5个因素进行筛选,建立了高效的象草叶鞘原生质体分离体系,并进行目标基因的瞬时转化,以期为象草基因功能研究奠定基础。结果表明:叶鞘在含有1.5%纤维素酶R-10,0.75%离析酶R-10,0.5 mol·L^-1 甘露醇,pH为5.8的酶解液中酶解4 h,原生质体产量达5.11×10⁶个·g FW^-1,活力达91.08%,酶解时间对象草原生质体产量和活力的影响最大。用PEG介导法将2个分别带有绿色荧光蛋白和红色荧光蛋白的载体转入原生质体中,转化效率最高可达77.47%,共转化效率为8.79%。所分离的原生质体可用于基因的瞬时表达研究。

关键词: 象草, 原生质体, PEG介导, 瞬时表达

CLC Number:

TANG Ran, PENG Xiao-qun, XIE Xin-ming. Protoplast Isolation from Elephant Grass(Pennisetum purpureum ‘Huanan’)for Transient Gene Expression[J]. Acta Agrestia Sinica, 2015, 23(3): 571-579.

唐然, 彭小群, 解新明. 华南象草原生质体分离及基因瞬时表达研究[J]. 草地学报, 2015, 23(3): 571-579.

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References

[1] Hisano H, Nandakumar R, Wang Z Y. Genetic modification of lignin biosynthesis for improved biofuel production [J]. In Vitro & Developmental Biology-Plant,2009,45(3):306-313
[2] Cao J, Yao D, Lin F, et al. PEG-mediated transient gene expression and silencing system in maize mesophyll protoplasts: A valuable tool for signal transduction study in maize [J]. Acta Physiologiae Plantarum,2014,36(5):1271-1281
[3] Marion J, Bach L, Bellec Y, et al. Systematic analysis of protein subcellular localization and interaction using high-throughput transient transformation of Arabidopsis seedlings [J]. The Plant Journal,2008,56(1):169-179
[4] 柏锡,贾蓓,吕德康,等. 水稻Os03g19020基因的克隆及亚细胞定位分析[J]. 东北农业大学学报,2014,45(1):29-33
[5] Yang J W, Fu J X, Li J, et al. A novel co-immunoprecipitation protocol based on protoplast transient gene expression for studying protein-protein interactions in rice [J]. Plant Molecular Bology Reporter,2014,32(1):153-161
[6] Sheen J. Signal transduction in maize and Arabidopsis mesophyll protoplasts [J]. Plant Physiology,2001,127(4):1466-1475
[7] Davey M R, Anthony P, Power J B, et al. Plant protoplasts: Status and biotechnological perspectives [J]. Biotechnology Advances,2005,23(2):131-171
[8] 王莉,姚占军. 植物原生质体的制备与活力检测研究进展[J]. 生物技术通报,2009(S1):46-50
[9] 赵严伟. 拟南芥原生质体分离及水稻植酸酶基因OsMINPP瞬时表达研究[D]. 长沙:湖南农业大学,2011
[10] Zhang Y, Su J, Duan S, 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(1):30
[11] Cocking E C. A method for the isolation of plant protoplasts and vacuoles [J]. Nature Protocols,1960(187):962-963
[12] Yoo S D, Cho Y H, Sheen J. Arabidopsis mesophyll protoplasts: A versatile cell system for transient gene expression analysis [J]. Nature Protocols,2007,2(7):1565-1572
[13] Ueki S, Lacroix B, Krichevsky A, et al. Functional transient genetic transformation of Arabidopsis leaves by biolistic bombardment [J]. Nature Protocols,2009,4(1):71-77
[14] Reich T J, Iyer V N, Miki B L. Efficient transformation of alfalfa protoplasts by the intranuclear microinjection of Ti plasmids [J]. Nature Biotechnology,1986,4(11):1001-1004
[15] 陈蓁蓁,张宁,张文申,等. 蛋白质分子荧光探针研究及其应用新进展[J]. 分析化学,2006,34(9):1341-1347
[16] 夏玉凤. 以gfp为报告基因研究蛋白亚细胞定位[J]. 生物技术通报,2006(2):11-13
[17] Vermeer J E M, Van Munster E B, Vischer N O, et al. Probing plasma membrane microdomains in cowpea protoplasts using lapidated GFP-fusion proteins and multimode FRET microscopy [J]. Journal of Microscopy,2004,214(2):190-200
[18] Ali G S, Prasad K V S K, Hanumappa M, et al. Analyses of in vivo interaction and mobility of two spliceosomal proteins using FRAP and BiFC [J]. PLoS One,2008,3(4):e1953
[19] 王娟,李玉珠,师尚礼. 苜蓿愈伤组织原生质体游离与培养[J]. 草地学报,2010,18(2):258-262
[20] 陶茸,李玉珠,王娟,等. 扁蓿豆愈伤组织原生质体分离条件的研究[J]. 草地学报,2011,19(2):288-293
[21] 解新明,周峰,赵燕慧,等. 多年生能源和草的产能和生态效益[J]. 生态学报,2008,28(5):2329-2342
[22] 李有涵,唐然,解新明. 华南象草分株构件生长极其生物量分配的相关性[J]. 生态学杂志,2011,30(9):1875-1880
[23] Tang R, Zhang X Q, Li Y H, et al. Cloning and in silico analysis of a cinnamyl alcohol dehydrogenase gene in Pennisetum purpureum [J]. Journal of Genetics,2014,93(1):145-158
[24] 孙鹤,郎志宏,朱莉,等. 玉米、小麦、水稻原生质体制备条件优化[J]. 生物工程学报,2013,29(2):224-234
[25] 刘晓光,刘孟军,宁强,等. 冬枣花药愈伤组织的诱导及原生质体分离[J]. 中国农学通报,2009,25(2):100-104
[26] 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[J]. 生物工程学报,2007,23(3):367-374
[27] 李妮娜,丁林云,张志远,等. 棉花椰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
[28] 赵红娟,张博,陈爱萍,等. 酶解对苜蓿子叶原生质体体分离效果的影响[J]. 草地学报,2008,16(1):50-53
[29] 马晖玲,赵小强,白小明,等. 草地早熟禾午夜Ⅱ号原生质体培养及植株再生[J]. 草地学报,2010,18(1):103-107
[30] 袁彬,潘学军. 毛葡萄原生质体分离与纯化研究[J]. 西南大学学报,2010,32(12):97-101
[31] 王鹏凯,吕伟光,胡雪怡,等. 北美鹅掌楸原生质体的分离与培养[J]. 西北植物学报,2013,33(2):254-260
[32] 李玉珠,师尚礼. 杂花和紫花苜蓿原生质体分离培养条件的筛选[J]. 西北植物学报,2014,34(1):184-192.
[33] 羿德磊. 桑树原生质体分离及融合的研究[D]. 泰安:山东农业大学,2012
[34] 李贵,李必元,王五宏,等. 结球甘蓝下胚轴原生质体培养再生植株体系的优化研究[J]. 西北植物学报,2012,32(12):2438-2443
[35] 张凌云,师尚礼. 俄罗斯杂花苜蓿愈伤组织原生质体游离与培养条件的优化[J]. 中国草地学报,2013, 35(3):12-17
[36] Bart R, Chern M, Park C J, et al. A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts [J]. Plant Methods,2006,2(1):13-21

Protoplast Isolation from Elephant Grass(Pennisetum purpureum ‘Huanan’)for Transient Gene Expression

华南象草原生质体分离及基因瞬时表达研究

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[3]	ZHANG Ling-yun, SHI Shang-li. Selecting the Electrofusion Condition of Alfalfa Protoplast [J]. , 2013, 21(4): 769-775.
[4]	LI Yu-zhu, SHI Shang-li, HE Yan-yu. Effects of Different Fluorescent Dyes on Staining Alfalfa Protoplast [J]. , 2012, 20(2): 348-351.
[5]	TAO Rong, LI Yu-zhu, WANG Juan, AN Hui-hui, GUO Jing, SHI Shang-li. Protoplasts Isolation Conditions of Melilotoides ruthenica Callus [J]. , 2011, 19(2): 288-293.
[6]	LIU Xin, YU Bing, CHEN Xiao-yan, WANG Qing. Improving Protoplast Dissociation Yield and Division Frequency of Glycyrrhizia inflata Batal. [J]. , 2011, 19(2): 294-298.
[7]	LI Yu-zhu, TAO Rong, WANG Juan, SHI Shang-li, MA Hui-ling, ZHAO Xiao-qiang. Enzymolysis and Isolation Conditions of Protoplasts from Cotyledons and Calli in Lotus corniculatus [J]. , 2010, 18(6): 798-804.
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[9]	MA Hui-ling, ZHAO Xiao-qiang, BAI Xiao-ming. Protoplast Culture and Plant Regeneration of Midnight Ⅱ(Poa Pratensis L.) [J]. , 2010, 18(1): 103-107.
[10]	ZHAO Hong-juan, ZHANG Bo, CHEN Ai-ping, LI Pei-ying, XIAO Yan. Effects of Different Enzymolysis Conditions on the Cotyledons Protoplast Isolation of Alfalfa [J]. Acta Agrestia Sinica, 2008, 16(1): 50-53.
[11]	Shu Wenhua, Geng Huazhu, Sun Yongru. PROTOPLAST CULTURE AND PLANT REGENERATION FROM MESOPHYLL OF ALFALFA (MEDICAGO SATIVA L.) [J]. , 1994, 2(1): 40-44.
[12]	Bai Jingren, He Maotai, Yuan Qing, Sha ren, Li Yonggan . PLANT REGENERATION FROM MESOPHYLL PROTOPLASTS OF WILD MEDICAGO FALCATA [J]. , 1994, 2(1): 59-63.