梨叶片原生质体制备方法的建立及其基因瞬时转化试验

doi:10.16420/j.issn.0513-353x.2022-0264

园艺学报 ›› 2023, Vol. 50 ›› Issue (5): 1141-1150.doi: 10.16420/j.issn.0513-353x.2022-0264

梨叶片原生质体制备方法的建立及其基因瞬时转化试验

高成昱¹^,^*, 王艺衡¹^,^*, 靳江周¹, 李涛¹, 李金斗¹, 周梦瑶¹, 张海霞¹^,², 马辉¹^,², 张玉星¹^,²^,^**(), 亓宝秀¹^,³^,^**, 许建锋¹^,²^,^**

¹河北农业大学园艺学院，河北保定 071051
²河北省梨技术创新中心，河北保定 071051
³英国利物浦约翰摩尔大学药学和分子生物学学院，英国利物浦 L3 3AF

收稿日期:2022-11-07 修回日期:2023-03-11 出版日期:2023-05-25 发布日期:2023-05-31
通讯作者: **（E-mail：xjf@hebau.edu.cn）
（E-mail：b.qi@ljmu.ac.uk）
（E-mail：zhyx@hebau.edu.cn）
作者简介:第一联系人：
^*共同第一作者
基金资助:
广东省重点领域研发计划项目(2020B0202010003);国家重点研发计划项目(2019YFD1001404-5);河北省重点研发计划项目(21326308D-1-4);河北省现代农业产业技术体系梨创新团队项目(HBCT2018100202);大学生创新创业项目(201810086040)

Method for High Efficiency Isolation of Protoplasts from Pear Leaves and Its Utility for Gene Transient Expression

GAO Chengyu¹^,^*, WANG Yiheng¹^,^*, JIN Jiangzhou¹, LI Tao¹, LI Jindou¹, ZHOU Mengyao¹, ZHANG Haixia¹^,², MA Hui¹^,², ZHANG Yuxing¹^,²^,^**(), QI Baoxiu¹^,³^,^**, XU Jianfeng¹^,²^,^**

¹College of Horticulture，Hebei Agricultural University，Baoding，Hebei 071051，China
²Hebei Pear Technology and Innovation Center，Baoding，Hebei 071051，China
³School of Pharmacy and Biomolecular Sciences，Liverpool John Moores University，Byrom Street，Liverpool L3 3AF，UK

Received:2022-11-07 Revised:2023-03-11 Published:2023-05-25 Online:2023-05-31

摘要/Abstract

摘要：

以‘新梨7号’梨叶片为材料，探究影响原生质体分离的主要因素，制备高产优质的叶肉原生质体，并进行基因瞬时转化和表达。将继代30 d的组培苗嫩叶在最佳酶解液（1.0%纤维素酶，0.1%果胶酶，0.4%离析酶，0.5 mol · L^-1甘露醇，20 mmol · L^-1 KCl，20 mmol · L^-1 MES，10 mmol · L^-1CaCl₂，1.0 g · L^-1 BSA）中酶解12 h，可制备出产量为1.09 × 10⁶protopl · mL^-1、活力为90%的原生质体。将1 ~ 2 µg · mL^-1 pROK2-GFP质粒DNA通过40% PEG-4000介导转化至上述原生质体中并培养12 h，可以观察到GFP绿色荧光信号，转化率可达40%。

关键词: 梨, 叶片, 原生质体制备, 瞬时转化

Abstract:

High quality and quantity of protoplasts were succefully isolated from the mesophyll cells of leaves of tissue cultured‘Xinli 7’pear seedlings. This was achieved by optimizing the composition of the cell wall degrading enzymes and the reaction solution where the best results were obtained when the leaves of 30-days old tissue cultured pear seedlings were treated with 1.0% cellulase，0.1% pectinase and 0.4% macerozyme in a buffer containing 20 mmol · L^-1 MES，0.5 mol · L^-1 of D-mannitol，20 mmol · L^-1 of potassium chloride，10 mmol · L^-1of calcium chloride and 1.0 g · L^-1 of bovine serum albumin（pH 5.7）. Approximately 1.09 × 10⁶ protopl · mL^-1 were recovered with their viability being above 90%. To test their usefulness in genetic transformation in transient expression of proteins 1-2 µg · mL^-1 of pROK2- GFP plasmid DNA was transformed into the prepared protoplasts with 40% PEG-4000. After being cultured for 12 hours GFP was clearly observed in about 40% of the protoplasts，the transformation efficiency reached 40%.

Key words: pear, leaf, protoplast isolation, protoplast transient transformation

中图分类号:

S661.2

高成昱, 王艺衡, 靳江周, 李涛, 李金斗, 周梦瑶, 张海霞, 马辉, 张玉星, 亓宝秀, 许建锋. 梨叶片原生质体制备方法的建立及其基因瞬时转化试验[J]. 园艺学报, 2023, 50(5): 1141-1150.

GAO Chengyu, WANG Yiheng, JIN Jiangzhou, LI Tao, LI Jindou, ZHOU Mengyao, ZHANG Haixia, MA Hui, ZHANG Yuxing, QI Baoxiu, XU Jianfeng. Method for High Efficiency Isolation of Protoplasts from Pear Leaves and Its Utility for Gene Transient Expression[J]. Acta Horticulturae Sinica, 2023, 50(5): 1141-1150.

图/表 4

表1 梨叶片原生质体最佳分离条件筛选

Table 1 The experimental design for screening the best conditions for protoplast isolation from pear leaves

筛选对象 Screening object	纤维素酶/% Cellulase	果胶酶/% Pectinase	离析酶/% Macerozyme	甘露醇/ （mol · L^-1） Mannitol	酶解时间/h Enzymatic hydrolysis time	原生质体产量/ （× 10⁵ protopl · mL^-1） Protoplast yield
纤维素酶浓度 Cellulase concentration	0.5	0.1	0.5	0.5	12	3.50 b
	1.0	0.1	0.5	0.5	12	5.00 a
	1.5	0.1	0.5	0.5	12	2.88 b
	2.0	0.1	0.5	0.5	12	2.03 b
	2.5	0.1	0.5	0.5	12	2.00 b
果胶酶浓度 Pectinase concentration	1.0	0.0	0.5	0.5	12	4.85 b
	1.0	0.1	0.5	0.5	12	10.67 a
	1.0	0.3	0.5	0.5	12	5.86 b
	1.0	0.5	0.5	0.5	12	5.32 b
	1.0	0.7	0.5	0.5	12	3.81 c
离析酶浓度 Macerozyme concentration	1.0	0.1	0.2	0.5	12	2.55 c
	1.0	0.1	0.3	0.5	12	3.33 bc
	1.0	0.1	0.4	0.5	12	6.92 a
	1.0	0.1	0.5	0.5	12	4.84 b
	1.0	0.1	0.6	0.5	12	3.45 bc
甘露醇浓度 Mannitol concentration	1.0	0.1	0.4	0.3	12	6.17 cd
	1.0	0.1	0.4	0.4	12	7.17 bc
	1.0	0.1	0.4	0.5	12	10.93 a
	1.0	0.1	0.4	0.6	12	8.50 b
	1.0	0.1	0.4	0.7	12	5.37 d
酶解时间 Reaction time	1.0	0.1	0.4	0.5	6	1.83 c
	1.0	0.1	0.4	0.5	8	2.38 bc
	1.0	0.1	0.4	0.5	10	2.86 b
	1.0	0.1	0.4	0.5	12	3.63 a
	1.0	0.1	0.4	0.5	14	2.17 c
	1.0	0.1	0.4	0.5	16	0.16 d

图1 梨田间植株叶片和组培苗叶片的原生质体分离 A：田间生长的叶片；B：酶解后的田间叶片叶丝；C：从田间苗叶片提取的原生质体；D：组培苗的叶片；E：酶解后的组培苗叶片叶丝；F：从组培苗叶片提取的原生质体。

Fig. 1 Protoplast isolation from leaves of pear field plants and tissue culture seedlings A：Leaves of field grown seedlings；B：Leaf stripes of field grown seedlings after enzymatic hydrolysis；C：Protoplasts extracted from leaves of field grown seedlings；D：Leaves of tissue cultured seedlings；E：Leaf stripes of tissue cultured seedlings after enzymatic hydrolysis；F：Protoplasts extracted from leaves of tissue cultured seedlings.

图2 梨组培苗叶片叶龄对原生质体提取及其活性的影响

Fig. 2 Effects of leaf age of pear tissue culture seedlings on protoplast extraction and viability

图3 梨叶肉原生质体的瞬时转化结果 A：明场下细胞状态；B：GFP绿色荧光；C：明场与GFP荧光叠加；D：单细胞明场下状态；E：黑白背景的明场；F：叶绿体红色荧光；G：GFP绿色荧光；H：黑白背景的明场与GFP荧光叠加。

Fig. 3 Transient transformation and GFP expression of the pear mesophyll protoplasts A：Cell morphology observed under bright field microscopy；B：GFP fluorescence observed under UV microcopy；C：Merge of A and B；D：Single cell observed under bright field at high magnification；E：Same as in D，but in black and white background；F：Autofluorescence of the chloroplast；G：GFP green fluorescence；H：Overlay of E & G.

参考文献 32

[1]	Cai X D, Fu J, Guo W W. 2017. Mitochondrial genome of callus protoplast has a role in mesophyll protoplast regeneration in Citrus:evidence from transgenic GFP somatic homo-fusion. Horticultural Plant Journal, 3 (5):177-182. doi: 10.1016/j.hpj.2017.10.001 URL
[2]	Cao J M, Yao D M, Lin F, Jiang M Y. 2014. PEG-mediated transient gene expression and silencing system in maize mesophyll protoplasts:a valuable tool for signal transduction study in maize. Acta Physiologiae Plantarum, 36 (5):1271-1281. doi: 10.1007/s11738-014-1508-x URL
[3]	Davey M R, Anthony P, Power J B, Lowe K C. 2005. Plant protoplasts:status and biotechnological perspectives. Biotechnology Advances, 23 (2):131-171. doi: 10.1016/j.biotechadv.2004.09.008 URL
[4]	Fan Zhi-qiang, Du Xi-hua. 1999. Study on factors affecting grape protoplast separation. Shandong Forestry Science and Technology,(5):1-4. (in Chinese)
	范志强, 杜希华. 1999. 影响葡萄原生质体分离因素的研究. 山东林业科技,(5):1-4.
[5]	Faraco M, Di Sansebastiano G P, Spelt K, Koes R E, Quattrocchio F M. 2011. One protoplast is not the other! Plant Physiology, 156 (2):474-478. doi: 10.1104/pp.111.173708 pmid: 21454800
[6]	Gu Xiao-feng, Luo Zheng-rong. 2002. Studies on the isolation,culture of protoplast and plant regeration in Luotiantianshi persimmon. Acta Horticulturae Sinica,(4):369-371. (in Chinese)
	谷晓峰, 罗正荣. 2002. 罗田甜柿原生质体分离、培养及植株再生. 园艺学报,(4):369-371.
[7]	Guo J J, Morrell-Falvey J L, Labbé J L, Muchero W, Kalluri U C, Tuskan G A, Chen J G. 2012. Highly efficient isolation of Populus mesophyll protoplasts and its application in transient expression assays. PLoS ONE, 7 (9):e44908. doi: 10.1371/journal.pone.0044908 URL
[8]	Hong S Y, Seo P J, Cho S H, Park C M. 2012. Preparation of leaf mesophyll protoplasts for transient gene expression in Brachypodium distachyon. Journal of Plant Biology, 55 (5):390-397. doi: 10.1007/s12374-012-0159-y URL
[9]	He P, Shan L, Sheen J. 2007. The use of protoplasts to study innate immune responses. Methods Mol Biol, 354:1-9. pmid: 17172739
[10]	Jia X Y, Zhang X H, Qu J M, Han R. 2016. Optimization conditions of wheat mesophyll protoplast isolation. Agricultural Sciences, 7 (12):7.
[11]	Lai Q, Wang Y L, Zhou Q Y, Zhao Z. 2020. Isolation and purification of mesophyll protoplasts from Ginkgo biloba L. Cytologia, 85 (1):27-32. doi: 10.1508/cytologia.85.27 URL
[12]	Li Ni-na, Ding Lin-Yun, Zhang Zhi-yuan, Guo Wang-zhen. 2014. Isolation of mesophyll protoplast and establishment of gene transient ex-pression system in cotton. Acta Agronomica Sinica, 40 (2):231-239. (in Chinese)
	李妮娜, 丁林云, 张志远, 郭旺珍. 2014. 棉花叶肉原生质体分离及目标基因瞬时表达体系的建立. 作物学报, 40 (2):231-239.
[13]	Li X Y. 2011. A transient expression assay using Arabidopsis mesophyll protoplasts. Bio-Protocol, 1 (10):1-6.
[14]	Lu Rong-sheng, Han Mei-li. 1998. Advances in protoplast culture of woody plants. Guangxi Forestry Science,(4):197-201. (in Chinese)
	陆荣生, 韩美丽. 1998. 木本植物原生质体培养研究进展. 广西林业科学,(4):197-201.
[15]	Li Qing, Yu Hai-peng, Zhang Zi-hao, Sun Zheng-wen, Zhang Yan, Zhang Dong-mei, Wang Xing-fen, Ma Zhi-ying, Yan Yuan-yuan. 2021. Optimization of cotton mesophyll protoplast transient expression system. Scientia Agricultura Sinica, 54 (21):4514-4524. (in Chinese) doi: 10.3864/j.issn.0578-1752.2021.21.003
	李青, 鱼海鹏, 张子豪, 孙正文, 张艳, 张冬梅, 王省芬, 马峙英, 阎媛媛. 2021. 棉花真叶原生质体分离及瞬时表达体系的优化. 中国农业科学, 54 (21):4514-4524. doi: 10.3864/j.issn.0578-1752.2021.21.003
[16]	Liao Jingjing, Niu Congcong, Xie Qunjie, Xing Qiaojuan, Qi Hongyan. 2017. The recent advances of transient expression system in horticultural plants. Acta Horticulturae Sinica, 44 (9):1796-1810. (in Chinese)
	廖晶晶, 牛聪聪, 解群杰, 邢巧娟, 齐红岩. 2017. 基因瞬时表达技术在园艺植物上的应用研究进展. 园艺学报 44 (9):1796-1810.
[17]	Liu Xiao-guang, Zhao Mian, Liu Meng-jun, Ning Qiang. 2005. Research progress of fruit tree protoplast culture. Molecular Plant Breeding,(6):143-148. (in Chinese)
	刘晓光, 赵绵, 刘孟军, 宁强. 2005. 果树原生质体培养研究进展. 分子植物育种,(6):143-148.
[18]	Ma W J, Yi F, Xiao Y, Yang G J, Chen F J, Wang J H. 2020. Isolation of leaf mesophyll protoplasts optimized by orthogonal design for transient gene expression in catalpa bungei. Scientia Horticulturae, 274:109684. doi: 10.1016/j.scienta.2020.109684 URL
[19]	Ochatt S J, Caso O H. 1986. Shoot Regeneration from leaf mesophyll protoplasts of wild pear(Pyrus communis var. pyraster L.). Journal of Plant Physiology, 122 (3):243-249. doi: 10.1016/S0176-1617(86)80123-7 URL
[20]	Ochatt S J, Power J B. 1988. Rhizogenesis in callus from conference pear(Pyrus communis L.)protoplasts. Plant Cell,Tissue and Organ Culture, 13 (2):159-164. doi: 10.1007/BF00034453 URL
[21]	Pan Zeng-guang, Deng Xiu-xin. 2000. Plant regeneration from protoplasts of apple. Acta Horticulturae Sinica,(2):95-101. (in Chinese)
	潘增光, 邓秀新. 2000. 苹果原生质体分离培养及植株再生. 园艺学报,(2):95-101.
[22]	Schapire A L, Lois L M. 2016. A simplified and rapid method for the isolation and transfection of arabidopsis leaf mesophyll protoplasts for large-scale applications. Methods in Molecular Biology(Clifton,N.J.),79-88.
[23]	Sheen J. 2001. Signal transduction in maize and Arabidopsis mesophyll protoplasts. Plant Physiology, 127 (4):1466-1475. pmid: 11743090
[24]	Shi Qing-hua, Liu Ping, Liu Meng-jun. 2012. Advances in ploidy breeding of fruit trees. Acta Horticulturae Sinica, 39 (9):1639-1654. (in Chinese)
	石庆华, 刘平, 刘孟军. 2012. 果树倍性育种研究进展. 园艺学报, 39 (9):1639-1654.
[25]	Shu Xiao-juan, Wen Teng-jian, Xing Jia-yi, Lu Long, Hu Jian-fang. 2015. Isolation of protoplast and establishment of transient expression system in grapevine. Acta Botanica Boreali-Occidentalia Sinica, 35 (6):1262-1268. (in Chinese)
	舒小娟, 温腾建, 邢佳毅, 卢龙, 胡建芳. 2015. 葡萄原生质体分离及瞬时转化体系的建立. 西北植物学报, 35 (6):1262-1268.
[26]	Song Ai-hua, Zhang Wen-bin, Sun Shu-lan, Li Ling-fei, Wang Xiao-jing. 2017. Preparation of protoplast and establishment of transient expression system in Gerbera hybrida. Chinese Bulletin of Botany, 52 (4):511-519. (in Chinese)
	宋爱华, 张文斌, 孙姝兰, 李凌飞, 王小菁. 2017. 非洲菊原生质体制备及瞬时转化系统的建立. 植物学报, 52 (4):511-519. doi: 10.11983/CBB16155
[27]	Sun He, Lang Zhi-hong, Zhu Li, Huang Da-fang. 2013. Optimized condition for protoplast isolation from maize,wheat and rice leaves. Chinese Journal of Biotechnology, 29:224-234. (in Chinese) pmid: 23697167
	孙鹤, 郎志宏, 朱莉, 黄大昉. 2013. 玉米、小麦、水稻原生质体制备条件优化. 生物工程学报, 29:224-234.
[28]	Wang H L, Wang T, Yang Q, Yin W L, Xia X L, Guo H W, Li Z H. 2021. Preparation and transfection of Populus tomentosa mesophyll protoplasts. BIO-PROTOCOL, 11 (22):e4220.
[29]	Wu J Z, Liu Q, Geng X S, Li K M, Luo L J, Liu J P. 2017. Highly efficient mesophyll protoplast isolation and PEG-mediated transient gene expression for rapid and large-scale gene characterization in cassava(Manihot esculenta Crantz). BMC Biotechnology, 17 (1):1-8. doi: 10.1186/s12896-016-0323-4
[30]	Yan Siyuan, Du Juan, Yang Fulong, Ren Miaomiao, Li Jiahong, Gu Peiwen. 2020. Establishment of genetic transformation system of Lycium barbarum endophytic fungus Fusarium nematophilum NQ8GⅡ4 using PEG-mediated method and transformants evaluation. Acta Horticulturae Sinica, 47 (12):2385-2396. (in Chinese)
	闫思远, 杜娟, 杨富龙, 任苗苗, 李嘉泓, 顾沛雯. 2020. PEG介导枸杞内生真菌NQ8GⅡ4遗传转化及转化子评价. 园艺学报, 47 (12):2385-2396.
[31]	Yoo S D, Cho Y H, Sheen J. 2007. Arabidopsis mesophyll protoplasts:a versatile cell system for transient gene expression analysis. Nature Protocols, 2 (7):1565-1572. doi: 10.1038/nprot.2007.199
[32]	Zhang Ning, Li Wei, Gu Zhao-yu, Chen Qiu-ju, Duan Xu-wei, Yang Qing, Li Tian-zhong. 2015. Preliminary study on the isolation of mature pollen protoplasts in‘Fuji’apple. Acta Horticulturae Sinica, 42 (6):1167-1174. (in Chinese) doi: 10.16420/j.issn.0513-353x.2015-0071
	张宁, 李威, 顾钊宇, 陈秋菊, 段续伟, 杨清, 李天忠. 2015. ‘富士’苹果花粉原生质体分离初探. 园艺学报, 42 (6):1167-1174. doi: 10.16420/j.issn.0513-353x.2015-0071

梨叶片原生质体制备方法的建立及其基因瞬时转化试验

Method for High Efficiency Isolation of Protoplasts from Pear Leaves and Its Utility for Gene Transient Expression

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梨叶片原生质体制备方法的建立及其基因瞬时转化试验

Method for High Efficiency Isolation of Protoplasts from Pear Leaves and Its Utility for Gene Transient Expression

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