棉花真叶原生质体分离及瞬时表达体系的优化

doi:10.3864/j.issn.0578-1752.2021.21.003

Abstract

Abstract:

【Objective】Cells of true leaves can well mimic the plant endogenous situation. It is an efficient way for expediting cotton functional study to establish an effective transient expression system using cotton protoplasts obtained from true leaves.【Method】The enzyme combination of cellulose and macerozyme were used to isolate protoplasts from true leaves of Gossypium hirsutum L. acc. TM-1. The effects of osmotic pressure, components of digestion buffer and digestion time on protoplast yield were studied and the validity of protoplasts were compared under different mannitol concentration and digestion time. To improve the transformation efficiency of cotton protoplast, the effects of mannitol and PEG concentration and buffers for protoplast culture were subsequently studied. In order to verify the optimized transient expression system, the vector 35S:LTP-GFP was constructed and transformed into protoplasts of Arabidopsis and cotton and tobacco epidermal cells followed by observation of fusion protein localization.【Result】High concentration of CaCl₂ in the digestion buffer significantly inhibited the isolation of protoplast from cotton true leaves, which was opposite of that using cotyledon. 10 mmol·L ^-1 CaCl₂ was employable for digestion buffer to isolate cotton protoplasts from true leaves. Mannitol concentration significantly affected protoplast yield that peaked under mannitol concentration of 0.5 mol·L^-1, and protoplast validity decreased moiety under 0.4 mol·L^-1 mannitol, suggesting that 0.5 mol·L^-1 mannitol was most suitable to maintain the osmotic pressure of cotton protoplasts. Cotton protoplasts displayed suitable size when isolated from newly flattened true leaves, while protoplast enlarged and yield decreased when produced from young leaves flattened 5 days. The protoplasts dissociate slowly until being digested 9 h when the yield reached the peak. The transformation efficiency was greatly improved under isotonic condition of 40% PEG buffer. While hypotonic condition that is commonly applied to facilitate transformation was against the entrance of exogenous DNA into cotton protoplasts. After transformation, the protoplast ruptured abundantly in WI buffer,whereas the shape maintained well in W5 buffer adding 0.5 mol·L^-1 mannitol. The transformation efficiency was improved to 90% using the optimized transient expression system. The subcellular location analysis results showed consistent GFP signal in protoplasts of cotton and Arabidopsis true leaf and epidermal cells of tobacco leaf.【Conclusion】Our study has optimized the cotton mesophyll protoplast transient expression system, which could produce 8.10×10 ⁶·mL^-1 fine protoplasts with validity above 95% and transformation efficiency reached to 90%. This system is applicable for analysis of subcellular location, protein interaction and research on metabolism and regulation network.

Key words: cotton, TM-1, isolation of protoplasts, transient expression

LI Qing,YU HaiPeng,ZHANG ZiHao,SUN ZhengWen,ZHANG Yan,ZHANG DongMei,WANG XingFen,MA ZhiYing,YAN YuanYuan. Optimization of Cotton Mesophyll Protoplast Transient Expression System[J].Scientia Agricultura Sinica, 2021, 54(21): 4514-4524.

0
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://www.chinaagrisci.com/EN/10.3864/j.issn.0578-1752.2021.21.003

https://www.chinaagrisci.com/EN/Y2021/V54/I21/4514

Figures/Tables 5

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

References 41

[1]	WAND K B, WAND 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, RUSSELL J K, RICHARD G P, JOHN Z Y, ZHU Y X, WANG J, YU S X. The draft genome of a diploid cotton Gossypium raimondii. Nature Genetics, 2012, 44(7052):1098-1103. doi: 10.1038/ng.2371
[2]	WANG K, WAND D H, ZHENG X M, QIN A, ZHOU J, GUO B Y, CHEN Y J, WEN X P, YE W, ZHOU Y, ZHU Y X. Multi-strategic RNA-seq analysis reveals a high-resolution transcriptional landscape in cotton. Nature Communications, 2019, 10(1):4714-4729. doi: 10.1038/s41467-019-12575-x
[3]	ZHANG T Z, HU Y, JIANG W K, FANG L, GUAN X Y, CHEN J D, ZHANG J B, SASKI C A, SCHEFFLER B E, STELLY D M, HULSE-KEMP A M, WAN Q, LIU B L, LIU C X, WANG S, PAN M Q, WANG Y K, WANG D W, YE W X, CHANG L J, ZHANG W P, SONG Q X, KIRKBRIDE R C, CHEN X Y, DENNIS E, LLEWELLYN D J, PETERSON D G, THAXTON P, JONES D C, WANG Q, XU X Y, ZHANG H, WU H T, ZHOU L, MEI G F, CHEN S Q, TIAN Y. Sequencing of allotetraploid cotton (Gossypium hirsutum L. acc. TM-1) provides a resource for fiber improvement. Nature Biotechnology, 2015, 33(5):531-537. doi: 10.1038/nbt.3207
[4]	WANG M J, TU L L, YUAN D J, ZHU D, SHEN C, LI J Y, LIU F Y, PEI L L, WANG P C, ZHAO G N, YE Z X, HUANG H, YAN F L, MA Y Z, ZHANG L, LIU M, YOU J Q, YANG Y C, LIU Z P, HUANG F, LI B Q, QIU P, ZHANG Q H, ZHU L F, JIN S X, YANG X Y, MIN L, LI G L, CHEN L L, ZHENG H K, LINDSEY K, LIN Z X, UDALL J A, ZHANG X L. Reference genome sequences of two cultivated allotetraploid cottons, Gossypium hirsutum and Gossypium barbadense. Nature Genetics, 2019, 51(2):224-229. doi: 10.1038/s41588-018-0282-x
[5]	HUANG G, WU Z G, RICHARD G. P, BAI M Z, LI Y, JAMES E. F, HU J, WANG K, JOHN Z. Y, ZHU Y X. Genome sequence of Gossypium herbaceum and genome updates of Gossypium arboreum and Gossypium hirsutum provide insights into cotton A-genome evolution. Nature Genetics, 2020, 52(5):516-524. doi: 10.1038/s41588-020-0607-4
[6]	JASON G W, ELI R, EDWARD S B. On the road to breeding 4.0: Unraveling the good, the bad and the boring of crop quantitative genomics. Annual Reveiew of Genetics, 2018, 52(26):1-24.
[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 Physiology, 2010, 154:1232-1243. doi: 10.1104/pp.110.157545
[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 Tissue and Organ Culture, 2010, 100:139-148. doi: 10.1007/s11240-009-9629-4
[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(2):224-234.
	SUN H, LANG Z H, ZHU L, HUANG D F. Optimized condition for protoplast isolation from maize, wheat and rice leaves. Chinese Journal of Biotechnology, 2013, 29(2):224-234. (in Chinese)
[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. Journal of Integrative Plant Biology, 2012, 54:500-510. doi: 10.1111/jipb.2012.54.issue-7
[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 dwarﬁsm and panicle enclosure in rice. Planta, 2012, 235:25-38. doi: 10.1007/s00425-011-1471-3
[12]	XIONG L, LI C, LI H Y, LYU X G, ZHAO T, LIU J, ZUO Z C, LIU B. A transient expression system in soybean mesophyll protoplasts reveals the formation of cytoplasmic GmCRY1 photobody-like structures. Science China Life Sciences, 2019, 62(8):1070-1077. doi: 10.1007/s11427-018-9496-5
[13]	GAO L, SHEN G J, ZHANG L D, QI J F, C ZHANG C P, MA C R, LI J, WANG L, SAIF U M, WU J Q. An efficient system composed of maize protoplast transfection and HPLC-MS for studying the biosynthesis and regulation of maize benzoxazinoids. Plant Methods, 2019, 15(1):144-157. doi: 10.1186/s13007-019-0529-2
[14]	PATIENCE C, MARIE E C R. A cassava protoplast system for screening genes associated with the response to South African Cassava Mosaic Virus. Virology Journal, 2020, 17(1):184-199. doi: 10.1186/s12985-020-01453-4
[15]	SU Y Y, CHEN Y E, CHEN J, ZHANG Z J, GUO J Y, CAI Y, ZHU C Y, LI Z Y, ZHANG H Y. Effectors of Puccinia striiformis f. sp. tritici suppressing the pathogenic-associated molecular pattern-triggered immune response were screened by transient expression of wheat protoplasts. International Journal of Molecular Sciences, 2021, 22(9):4985-5003. doi: 10.3390/ijms22094985
[16]	张宝红. 棉花原生质体培养研究进展. 四川农业大学学报, 1995, 13(1):55-61.
	ZHANG B H. Research progress on culture of cotton protoplasts. Journal of Sichuan Agricultural University, 1995, 13(1):55-61. (in Chinese)
[17]	孙玉强. 棉花原生质体培养和原生质体对称融合研究. 华中农业大学学报, 2011, 30(6):784-786.
	SUN Y Q. Research on cotton protoplasts culture and fusion. Journal of Huazhong Agricultural University, 2011, 30(6):784-786. In Chinese. (in Chinese)
[18]	李妮娜, 丁林云, 张志远. 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, 2014, 40(2):231-239.
	LI N N, DING L Y, ZHANG Z Y. Establishment of isolation of cotton mesophll protoplast and transient expression system of target gene. The Crop Journal, 2014, 40(2):231-239. (in Chinese)
[19]	李婧瑶, 刘龙飚, 丁兵, 杨海昕, 吴琼, 张旸. 植物原生质体分离及培养研究进展. 分子植物育种, 2021: 1-20. https://kns.cnki.net/kcms/detail/46.1068.S.20210308.1639.026.html .
	LI J Y, LIU L B, DING B, YANG H X, WU Q, ZHANG C. Research progress on plant protoplasts isolation and culture. Molecular PlantBreeding, 2021: 1-20. https://kns.cnki.net/kcms/detail/46.1068.S.20210308.1639.026.html . (in Chinese)
[20]	LI X Y. A transient expression assay using Arabidopsis mesophyll protoplasts. Bio-Protocol, 2011, 1(10):e70.
[21]	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 inter-specific asymmetric somatic hybrids obtained by donor-recipient fusion in cotton. Chinese Science Bulletin, 2009, 54:2219-2227.
[22]	EECKHAUT T, VAN H W, BRUZNICAN S, LEUS L, VAN H J. Somaclonal variation in chrysanthemum × morifolium protoplast regenerants. Frontiers in Plant Science, 2020, 11:607171-607187. doi: 10.3389/fpls.2020.607171
[23]	ELŻBIETA J, ALEKSANDRA N, ANNA M. Progress towards sugar beet improvement through somatic hybridization: I. Inactivation of nuclei and cytoplasm in donor and recipient protoplasts. Acta Societatis Botanicorum Poloniae, 1995, 64(4):341-347. doi: 10.5586/asbp.1995.044
[24]	谢鑫, 蒋君梅, 王勇, 任明见. 高粱原生质体的制备及转化方法研究. 种子, 2019, 38(8):43-46.
	XIE X, JIANG J M, WANG Y, REN M J. Study on the method of protoplast isolation and transformation of Sorghum bicolor. Seed, 2019, 30(8):43-46. (in Chinese)
[25]	SANDHYA D, JOGAM P, ALLINI V R, ABBAGANI S, ALOK A. The present and potential future methods for delivering CRISPR/Cas9 components in plants. Biotechnology & Genetic Engineering Reviews, 2020, 18(1):25-36.
[26]	NICOLIA A, FÄLT A, HOFVANDER P, ANDERSSON M. Protoplast-based method for genome editing in tetraploid potato. Methods in Molecular Biology, 2021, 2264(24):177-186.
[27]	WU S P, ZHU H C, LIU J X, YANG Q S, SHAO X H, BI F C, HU C H, HUO H Q, CHEN K L, YI G J. Establishment of a PEG-mediated protoplast transformation system based on DNA and CRISPR/Cas9 ribonucleoprotein complexes for banana. Plant Biology, 2020, 20(1):1-10. doi: 10.1111/plb.12676
[28]	JIN D M, CHOI S H, LEE M H, JIE E Y, AHN W S, JOO S J, AHN J W, JO Y D, AHN S J, KIM S W. Development of a rapid selection system for salt-resistant mutants of nicotiana benthamiana through protoplast culture after gamma irradiation. Plants, 2020, 9(12):1720-1733. doi: 10.3390/plants9121720
[29]	HUANG M K, ZHANG L, ZHOU L M, WANG M Z, YUNG W S, WANG Z L, DUAN S W, XIAO Z X, WANG Q W, WANG X, LI M W, LAM H M. An expedient survey and characterization of the soybean JAGGED 1 (GmJAG1) transcription factor binding preference in the soybean genome by modified ChIP mentation on soybean protoplasts. Genomics, 2021, 113(1):344-355. doi: 10.1016/j.ygeno.2020.12.026
[30]	SAHAB S, HAYDEN M J, MASON J, SPANGENBERG G. Mesophyll protoplasts and PEG-mediated transfections: Transient assays and generation of stable transgenic canola plants. Methods in Molecular Biology, 2019, 1864:131-152.
[31]	ZHOU Q Y, JIANG Z H, LI Y M, ZHANG T, ZHU H L, ZHAO F, ZHAO Z. Mesophyll protoplast isolation technique and flow cytometry analysis of ancient Platycladus orientalis. Turkish Journal of Agriculture and Forestry, 2019(3):275-287.
[32]	HU Y F, SONG D L, GAO L, BABATOPE S A, WANG Y B, HUANG H H, ZHANG J J, LIU H M, LIU Y H, YU G W, LIU Y J, LI Y P, HUANG Y B. Optimization of isolation and transfection conditions of maize endosperm protoplasts. Plant Methods, 2020, 16(1):1-15. doi: 10.1186/s13007-019-0534-5
[33]	赵严伟, 黄志刚, 李合松. 洗液对拟南芥叶原生质体分离的影响. 中国农学通报, 2011, 27(12):187-190.
	ZHAO Y W, HUANG Z G, LI H S. EffectS of washing solution on protoplast isolation from Arabidopsis thaliana leave. Chinese Agricultural Science Bulletin, 2011, 27(12):187-190. (in Chinese)
[34]	朱俊, 聂琼, 杨川龙, 陈茜. 不同质膜稳定剂对烟草原生质体细胞壁再生的影响. 山地农业生物学报, 2012, 31(3):222-227.
	ZHU J, NIE Q, YANG C L, CHEN X. Effects of different plasma membrane stabilizers on cell wall regeneration of tobacco protoplasts. Journal of Mountain Agriculture and Biology, 2012, 31(3):222-227. (in Chinese)
[35]	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. Production, culture and plant regeneration of protoplasts from embryogenic callus of Gossypium hirsutum. Journal of Integrative Plant Biology, 1998, 40:234-240. (in Chinese)
[36]	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 Tissue and Organ Culture, 2007, 89(2/3):225-235. doi: 10.1007/s11240-007-9245-0
[37]	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):36-37.
	LI R J, ZHANG Z X, SHI Y H. Reports of isolation of cotton mesophyll protoplast. Heridity, 1980, 2(4):36-37. (in Chinese)
[38]	曾弓剑, 程云伟, 韩少鹏, 吕阳, 陆业磊, 周超, 张德春, 沈祥陵. 高粱品种BTx623原生质体分离及瞬时表达体系的建立. 生物资源, 2021, 43(1):42-49.
	ZENG G J, CHENG Y W, HAN S P, LÜ Y, LU Y L, ZHOU C, ZHANG D C, SHEN X L. The establishment of protoplast isolation and transient expresion system in sorghum cultivar BTx623. Biotic Resources, 2021, 43(1):42-49. (in Chinese)
[39]	YOO S D, CHO Y, SHEEN J. Arabidopsis mesophyll protoplasts: A versatile cell system for transient gene expression analysis. Nature Protocols, 2007(2):1565-1572.
[40]	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(1):30-44. doi: 10.1186/1746-4811-7-30
[41]	GUO J J, MORRELL-FALVEY J L, LABBÉ J L, MUCHERO W, KALLURI U C, TUSKAN G A, CHEN J G. Highly efficient isolation of Populus mesophyll protoplasts and its application in transient expression assays. PLoS ONE, 2012, 7(9):44908-44917.

Related Articles 15

[1]	WANG CaiXiang,YUAN WenMin,LIU JuanJuan,XIE XiaoYu,MA Qi,JU JiSheng,CHEN Da,WANG Ning,FENG KeYun,SU JunJi. Comprehensive Evaluation and Breeding Evolution of Early Maturing Upland Cotton Varieties in the Northwest Inland of China [J]. Scientia Agricultura Sinica, 2023, 56(1): 1-16.
[2]	WANG JunJuan,LU XuKe,WANG YanQin,WANG Shuai,YIN ZuJun,FU XiaoQiong,WANG DeLong,CHEN XiuGui,GUO LiXue,CHEN Chao,ZHAO LanJie,HAN YingChun,SUN LiangQing,HAN MingGe,ZHANG YueXin,FAN YaPeng,YE WuWei. Characteristics and Cold Tolerance of Upland Cotton Genetic Standard Line TM-1 [J]. Scientia Agricultura Sinica, 2022, 55(8): 1503-1517.
[3]	YIN YanYu,XING YuTong,WU TianFan,WANG LiYan,ZHAO ZiXu,HU TianRan,CHEN Yuan,CHEN Yuan,CHEN DeHua,ZHANG Xiang. Cry1Ac Protein Content Responses to Alternating High Temperature Regime and Drought and Its Physiological Mechanism in Bt Cotton [J]. Scientia Agricultura Sinica, 2022, 55(23): 4614-4625.
[4]	XIE XiaoYu, WANG KaiHong, QIN XiaoXiao, WANG CaiXiang, SHI ChunHui, NING XinZhu, YANG YongLin, QIN JiangHong, LI ChaoZhou, MA Qi, SU JunJi. Restricted Two-Stage Multi-Locus Genome-Wide Association Analysis and Candidate Gene Prediction of Boll Opening Rate in Upland Cotton [J]. Scientia Agricultura Sinica, 2022, 55(2): 248-264.
[5]	WANG Juan, MA XiaoMei, ZHOU XiaoFeng, WANG Xin, TIAN Qin, LI ChengQi, DONG ChengGuang. Genome-Wide Association Study of Yield Component Traits in Upland Cotton (Gossypium hirsutum L.) [J]. Scientia Agricultura Sinica, 2022, 55(12): 2265-2277.
[6]	WANG Ning,FENG KeYun,NAN HongYu,ZHANG TongHui. Effects of Combined Application of Organic Fertilizer and Chemical Fertilizer on Root Characteristics and Yield of Cotton Under Different Water Conditions [J]. Scientia Agricultura Sinica, 2022, 55(11): 2187-2201.
[7]	QIN HongDe, FENG ChangHui, ZHANG YouChang, BIE Shu, ZHANG JiaoHai, XIA SongBo, WANG XiaoGang, WANG QiongShan, LAN JiaYang, CHEN QuanQiu, JIAO ChunHai. F₁ Performance Prediction of Upland Cotton Based on Partial NCII Design [J]. Scientia Agricultura Sinica, 2021, 54(8): 1590-1598.
[8]	TongYu HOU,TingLi HAO,HaiJiang WANG,Ze ZHANG,Xin LÜ. Advances in Cotton Growth and Development Modelling and Its Applications in China [J]. Scientia Agricultura Sinica, 2021, 54(6): 1112-1126.
[9]	LOU ShanWei,DONG HeZhong,TIAN XiaoLi,TIAN LiWen. The " Short, Dense and Early" Cultivation of Cotton in Xinjiang: History, Current Situation and Prospect [J]. Scientia Agricultura Sinica, 2021, 54(4): 720-732.
[10]	NIE JunJun,DAI JianLong,DU MingWei,ZHANG YanJun,TIAN XiaoLi,LI ZhaoHu,DONG HeZhong. New Development of Modern Cotton Farming Theory and Technology in China - Concentrated Maturation Cultivation of Cotton [J]. Scientia Agricultura Sinica, 2021, 54(20): 4286-4298.
[11]	ZHOU Meng,HAN XiaoXu,ZHENG HengBiao,CHENG Tao,TIAN YongChao,ZHU Yan,CAO WeiXing,YAO Xia. Remote Sensing Estimation of Cotton Biomass Based on Parametric and Nonparametric Methods by Using Hyperspectral Reflectance [J]. Scientia Agricultura Sinica, 2021, 54(20): 4299-4311.
[12]	WANG Na,ZHAO ZiBo,GAO Qiong,HE ShouPu,MA ChenHui,PENG Zhen,DU XiongMing. Cloning and Functional Analysis of Salt Stress Response Gene GhPEAMT1 in Upland Cotton [J]. Scientia Agricultura Sinica, 2021, 54(2): 248-260.
[13]	ZHOU JingLong,FENG ZiLi,WEI Feng,ZHAO LiHong,ZHANG YaLin,ZHOU Yi,FENG HongJie,ZHU HeQin. Biocontrol Effect and Mechanism of Cotton Endophytic Bacterium YUPP-10 and Its Secretory Protein CGTase Against Fusarium Wilt in Cotton [J]. Scientia Agricultura Sinica, 2021, 54(17): 3691-3701.
[14]	WEN Ming, LI MingHua, JIANG JiaLe, MA XueHua, LI RongWang, ZHAO WenQing, CUI Jing, LIU Yang, MA FuYu. Effects of Nitrogen, Phosphorus and Potassium on Drip-Irrigated Cotton Growth and Yield in Northern Xinjiang [J]. Scientia Agricultura Sinica, 2021, 54(16): 3473-3487.
[15]	ZHANG XiaoXue,SUN TianGe,ZHANG YingChun,CHEN LiHua,ZHANG XinYu,LI YanJun,SUN Jie. Identification of Xylosidase Genes from Verticillium dahliae and Functional Analysis Based on HIGS Technology [J]. Scientia Agricultura Sinica, 2021, 54(15): 3219-3231.

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed

Comments

Recommended 0

No Suggested Reading articles found!

Optimization of Cotton Mesophyll Protoplast Transient Expression System

RichHTML

PDF