Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (11): 2223-2236.doi: 10.3864/j.issn.0578-1752.2023.11.015

• RESEARCH NOTES • Previous Articles    

The Modification of Gene Editing Vector for Efficient GFPuv Fluorescence Screening and Its Application in Potato Genetic Transformation

DU JingYa1,2(), CHEN KaiYuan2, PU Jin2, ZHOU HuiYing3, ZHU GuangTao3, ZHANG ChunZhi2, DU Hui2()   

  1. 1 College of Life Sciences, Henan University, Kaifeng 475004, Henan
    2 Agricultural Genomics Institute, Chinese Academy of Agricultural Sciences, Shenzhen 518000, Guangdong
    3 Joint Academy of Potato Sciences, Yunnan Normal University, Kunming 650500
  • Received:2023-02-07 Accepted:2023-04-07 Online:2023-06-01 Published:2023-06-19


【Objective】The improvement and innovation of screening markers contributes to the development of transgenic technology, among which the visual screening markers are widely modified for better effect. Recent studies revealed that an enhanced Yellow Green Fluorescent like Protein (eYGFPuv (GFPuv)) obtained by mutation can emit strong and stable green fluorescence under 365 nm UV light irradiation and be easily observed. Constructing the gene editing vector with GFPuv fluorescence screening marker and carrying out experiment application and verifications in potato genetic transformation will provide technical support for the screening of positive transgenic plants in potato transformation, and lay the foundation for using genome editing technology to create potato male sterile lines in the future. 【Method】By using homologous recombination, the GFPuv expression framework and gene editing element Cas9_sgRNA were successively recombined into pCAMBIA2300 vector, and then with this new designed vector the Agrobacterium-mediated transient expression assay was conducted in tobacco plants. Six editing vectors with potato anther development conservative genes were constructed using this modified vector. The A. rhizogenes strains Ar qual and MSU440 harbouring these vectors were transformed into the potato stem segments respectively, and then the A. rhizogenes-induced hairy roots with green fluorescence were observed and counted under the portable UV lamp. The transformation efficiency and editing efficiency of these vectors were analyzed using hairy root transformation system in two different potato genotypes. In the end, the modified vectors were applied to produce transformed potato plants with modifications on target genes. 【Result】A novel gene editing vector pCAMBIA2300MGFPuv-sgRNACas harbouring a GFPuv fluorescence marker was successfully constructed, and the transient transformation in tobacco plants confirmed that the GFPuv expression framework was expressed successfully. The hairy roots with green fluorescence were screened after the transformation with two kinds of A. rhizogenes, and an additional supplement of kanamycin (Kan) significantly increased the proportion of positive fluorescent roots. Although the transformation rates of the two strains were not significantly different, the hairy roots of MSU440 formed faster. Furthermore, the transformation rates and editing rates of editing vectors for six potato anther development conservative genes in two different potato genotypes were the same, but the editing rates of six target sites differed significantly. Potato genetic transformation using the modified vector confirmed that GFPuv fluorescence could be used for the screening of transgenic callus and plants in potato. 【Conclusion】The hairy root transformation system mediated by A. rhizogenes is an essential approach to verifying the efficiency of gene editing, and GFPuv fluorescence can be used in the screening of transgenic plants in potato transformation.

Key words: potato, GFPuv, genome editing, hairy root, genetic transformation

Table 1

Primer names and sequences used for vector construction"

引物名称 Primer name 引物序列 Primer sequence (5′-3′)
CZ1F aagcactctttcctgtggATAGCACGTACATTG

Table 2

Primers for detecting the mutations of target genes"

Primer names
Primer sequences (5′-3′)
Amplification of StAMS target site
Amplification of StDYT1 target site
Amplification of StMS1a target site
Amplification of StMS1b target site
Amplification of StMYB80 target site
Amplification of StTDF1 target site
Hi-TOM sequencing amplification of StAMS target site1
TomAMSsite1F ggagtgagtacggtgtgcGCTGCTGTGGTGGAGCTGAG
TomAMSsite1R gagttggatgctggatggACATGTACCCGCAGTCTAGC
Hi-TOM sequencing amplification of StAMS target site2
TomAMSsite2F ggagtgagtacggtgtgcCTAGCAGAAGATGAGAAAGTC
TomAMSsite2R gagttggatgctggatggTGAATGGAGGAAGGAAGTTC
Hi-TOM sequencing amplification of StDYT1 target site
TomDYT1siteF ggagtgagtacggtgtgcGGAAGCAAATTGATGGTGAA
TomDYT1siteR gagttggatgctggatggGTCTCCGGTTCCTCCCCATG
Hi-TOM sequencing amplification of StMS1a target site
TomMS1asiteF ggagtgagtacggtgtgcGGGGCAACAATTTGATGTGC
TomMS1asiteR gagttggatgctggatggGTCCAATGCAAAGTCGATCCC
Hi-TOM sequencing amplification of StMS1b target site1
TomMS1bsite1F ggagtgagtacggtgtgcGCATAAGAGTAAAGGTGTGC
TomMS1bsite1R gagttggatgctggatggCCGACAAATCCCCAAAACCC
Hi-TOM sequencing amplification of StMS1b target site2
TomMS1bsite2F ggagtgagtacggtgtgcTACCAGCTGATAGTGAATGG
TomMS1bsite2R gagttggatgctggatggGTGCAAATACGATCCCAGAG
Hi-TOM sequencing amplification of StMYB80 target site
TomMYB80siteR gagttggatgctggatggGATGAAGCATTTCATCTTTG
Hi-TOM sequencing amplification of StTDF1 target site
TomTDF1siteF ggagtgagtacggtgtgcCTAACATCATTCATGTGCAGG
TomTDF1siteR gagttggatgctggatggGCATTTTGAATTGGGAGACC

Fig. 1

Construction of gene editing vector for GFPuv fluorescence screening and transient transformation validation in tobacco A: The diagram of pCAMBIA2300 original vector; B: pCAMBIA2300M is the diagram of the vector after mutating the restriction site BsaⅠ of pCAMBIA2300 vector; C: pCAMBIA2300MGFPuv is the diagram of pCAMBIA2300M vector adding GFPuv expression framework; D: pCAMBIA2300MGFPuv-sgRNACas is the diagram of pCAMBIA2300MGFPuv vector adding Cas9 and sgRNA; E and F are the tobacco plants transformed transiently, of which E is a photo in the bright field and F is a photo in the dark under a portable ultraviolet lamp, scale bars are 1 cm"

Fig. 2

Observation and comparison of hairy roots induced by Agrobacterium rhizogenes MSU440 and Ar qual in potato stem segments A and B are photos of hairy roots produced by potato stem segments, A is a photo in the bright field, and B is a photo in the dark under a portable ultraviolet lamp. Scale bars are 1 cm. Without and with 50 mg·L-1 Kan, MSU440 and Ar qual produced the number of fluorescent roots per stem segment (C) and the proportion of fluorescent roots to the total hairy roots produced (D). Values represent means ± SD (n=7). Lowercase letters above the bars indicate significant differences according to Tukey’s multiple comparisons test with P<0.05"

Fig. 3

Comparison of hairy roots induced by different explants by transformation of editing vectors of potato anther development-related genes The six editing vectors corresponding to the potato anther development-related genes StAMS, StDYT1, StMS1a, StMS1b, StMYB80 and StTDF1 were transformed into A056 and PG6359, A is the number of fluorescent roots produced by each stem segment and B is the proportion of fluorescent roots to the total hairy roots produced. Values represent means ± SD (n=5). ns indicates no significant differences according to Student’s T-test, and **(P<0.01) indicates extremely significant differences according to Student’s T-test"

Fig. 4

PCR identification of gene editing sites of some fluorescent roots The red box represents homozygous large fragment deletion lines, and the white box represents heterozygous large fragment deletion lines"

Table 3

Statistical analysis of PCR identification of gene editing sites of fluorescent roots"

No. of fluorescent root
带型 Band type
野生型 Wild type 杂合型 Heterozygous type 纯合突变型 Homozygous type
StAMS A056 48 43 3 1
PG6359 48 44 4 0
StDYT1 A056 48 37 8 3
PG6359 48 45 3 0
StMS1a A056 48 38 5 5
PG6359 48 40 5 3
StMS1b A056 48 37 9 2
PG6359 48 39 8 1
StMYB80 A056 48 35 7 6
PG6359 48 36 6 6
StTDF1 A056 48 44 3 1
PG6359 48 45 2 1

Fig. 5

Display of a Hi-TOM sequencing result of StMS1a gene"

Table 4

Mutation efficiencies of editing sites of fluorescent roots (%)"

A056 PG6359
靶点1 Target site 1 靶点2 Target site 2 靶点1 Target site 1 靶点2 Target site 2
StAMS 1.63 85.24 1.37 84.28
StDYT1 7.11 38.32 0.00 24.50
StMS1a 95.29 54.14 96.74 54.71
StMS1b 20.96 87.78 23.29 85.33
StMYB80 80.49 79.48 76.09 72.84
StTDF1 90.43 1.50 86.90 1.51

Fig. 6

Observation on callus and regenerated seedlings of potato genetic transformation by gene editing vector for GFPuv fluorescence screening A and B: Potato stem segments at differentiation stage; C: The PCR identification of transgenic lines, of which 1 and 2 are positive lines, the plasmid is the pCAMBIA2300MGFPuv-sgRNACas vector for transgenic using, and the control is a non-transgenic line; D and G: Transgenic positive lines 1; E and H: Transgenic positive lines 2; F and I: Non-transgenic controls. A and D-F are photos in the bright field, B and G-I are photos in the dark under the portable ultraviolet lamp, scale bars are 1 cm"

WALDRON C, MURPHY E B, ROBERTS J L, GUSTAFSON G D, ARMOUR S L, MALCOLM S K. Resistance to hygromycin B. Plant Molecular Biology, 1985, 5(2): 103-108.

doi: 10.1007/BF00020092
BRUKHIN V, CLAPHAM D, ELFSTRAND M, VON ARNOLD S. Basta tolerance as a selectable and screening marker for transgenic plants of Norway spruce. Plant Cell Reports, 2000, 19(9): 899-903.

doi: 10.1007/s002990000217 pmid: 30754927
JEFFERSON R A. The GUS reporter gene system. Nature, 1989, 342(6251): 837-838.

doi: 10.1038/342837a0
JEFFERSON R A, KAVANAGH T A, BEVAN M W. GUS fusions: Beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. The EMBO Journal, 1987, 6(13): 3901-3907.

doi: 10.1002/embj.1987.6.issue-13
OW D W, DE WET J R, HELINSKI D R, HOWELL S H, WOOD K V, DELUCA M. Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants. Science, 1986, 234(4778): 856-859.

doi: 10.1126/science.234.4778.856 pmid: 17758108
SHANER N C, CAMPBELL R E, STEINBACH P A, GIEPMANS B N G, PALMER A E, TSIEN R Y. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nature Biotechnology, 2004, 22(12): 1567-1572.

doi: 10.1038/nbt1037
HARPER B K, MABON S A, LEFFEL S M, HALFHILL M D, RICHARDS H A, MOYER K A, STEWART C N. Green fluorescent protein as a marker for expression of a second gene in transgenic plants. Nature Biotechnology, 1999, 17(11): 1125-1129.

doi: 10.1038/15114 pmid: 10545923
SHIMIZU A, SHIRATORI I, HORII K, WAGA I. Molecular evolution of versatile derivatives from a GFP-like protein in the marine copepod Chiridius poppei. PLoS ONE, 2017, 12(7): e0181186.

doi: 10.1371/journal.pone.0181186
CHIN D P, SHIRATORI I, SHIMIZU A, KATO K, MII M, WAGA I. Generation of brilliant green fluorescent petunia plants by using a new and potent fluorescent protein transgene. Scientific Reports, 2018, 8(1): 16556.

doi: 10.1038/s41598-018-34837-2 pmid: 30410086
李颖, 李广存, 李灿辉, 屈冬玉, 黄三文. 二倍体杂种优势马铃薯育种的展望. 中国马铃薯, 2013, 27(2): 96-99.
LI Y, LI G C, LI C H, QU D Y, HUANG S W. Prospects of diploid hybrid breeding in potato. Chinese Potato Journal, 2013, 27(2): 96-99.. (in Chinese)
LI D W, LU X Y, ZHU Y H, PAN J, ZHOU S Q, ZHANG X Y, ZHU G T, SHANG Y, HUANG S W, ZHANG C Z. The multi‐omics basis of potato heterosis. Journal of Integrative Plant Biology, 2022, 64(3): 671-687.

doi: 10.1111/jipb.v64.3
LINDHOUT P, MEIJER D, SCHOTTE T, HUTTEN R C B, VISSER R G F, VAN ECK H J. Towards F1 hybrid seed potato breeding. Potato Research, 2011, 54(4): 301-312.

doi: 10.1007/s11540-011-9196-z
ZHANG C Z, YANG Z M, TANG D, ZHU Y H, WANG P, LI D W, ZHU G T, XIONG X Y, SHANG Y, LI C H, HUANG S W. Genome design of hybrid potato. Cell, 2021, 184(15): 3873-3883.

doi: 10.1016/j.cell.2021.06.006 pmid: 34171306
GÓMEZ J F, TALLE B, WILSON Z A. Anther and pollen development: A conserved developmental pathway. Journal of Integrative Plant Biology, 2015, 57(11): 876-891.

doi: 10.1111/jipb.12425
WAN X Y, WU S W, LI Z W, DONG Z Y, AN X L, MA B, TIAN Y H, LI J P. Maize genic male-sterility genes and their applications in hybrid breeding: Progress and perspectives. Molecular Plant, 2019, 12(3): 321-342.

doi: S1674-2052(19)30020-6 pmid: 30690174
JIANG Y L, AN X L, LI Z W, YAN T W, ZHU T T, XIE K, LIU S S HOU Q C, ZHAO L N, WU S W, LIU X Z, ZHANG S W, HE W, LI F, LI J P, WAN X Y. CRISPR/Cas9-based discovery of maize transcription factors regulating male sterility and their functional conservation in plants. Plant Biotechnology Journal, 2021, 19(9): 1769-1784.

doi: 10.1111/pbi.13590 pmid: 33772993
曹贞菊, 李飞, 陈明俊, 罗小波, 李标, 尹旺. 农杆菌介导几种不同马铃薯外植体转化研究. 种子, 2021, 40(9): 52-56.
CAO Z J, LI F, CHEN M J, LUO X B, LI B, YIN W. Study on Agrobacterium tumefaciens-mediated transformation of several potato explants. Seed, 2021, 40(9): 52-56. (in Chinese)
蒋继滨, 高冬丽, 朱曦鉴, 李灿辉. 二倍体马铃薯基因编辑载体快速验证体系的建立. 种子, 2019, 38(10): 29-33.
JIANG J B, GAO D L, ZHU X J, LI C H. Establishment of a rapid verification system for diploid potato gene editing vector. Seed, 2019, 38(10): 29-33. (in Chinese)
叶明旺, 张春芝, 黄三文. 二倍体栽培马铃薯高效遗传转化体系的建立. 中国农业科学, 2018, 51(17): 3249-3257.

doi: 10.3864/j.issn.0578-1752.2018.17.002
YE M W, ZHANG C Z, HUANG S W. Construction of high efficient genetic transformation system for diploid potatoes. Scientia Agricultura Sinica, 2018, 51(17): 3249-3257. (in Chinese)

doi: 10.3864/j.issn.0578-1752.2018.17.002
张西英, 张爱萍, 刘江娜. 马铃薯遗传转化体系的优化建立及其主要影响因素. 基因组学与应用生物学, 2019, 38(7): 3174-3179.
ZHANG X Y, ZHANG A P, LIU J N. Optimization establishment of the genetic transformation system of potato and its main influencing factors. Genomics and Applied Biology, 2019, 38(7): 3174-3179. (in Chinese)
LIU Q, WANG C, JIAO X Z, ZHANG H W, SONG L L, LI Y X, GAO C X, WANG K J. Hi-TOM: A platform for high-throughput tracking of mutations induced by CRISPR/Cas systems. Science China Life Sciences, 2019, 62(1): 1-7.

doi: 10.1007/s11427-018-9402-9 pmid: 30446870
BUTLER N M, JANSKY S H, JIANG J M. First-generation genome editing in potato using hairy root transformation. Plant Biotechnology Journal, 2020, 18(11): 2201-2209.

doi: 10.1111/pbi.v18.11
KIRYUSHKIN A S, ILINA E L, GUSEVA E D, PAWLOWSKI K, DEMCHENKO K N. Hairy CRISPR: Genome editing in plants using hairy root transformation. Plants (Basel), 2021, 11(1): 51.
CAO X S, XIE H T, SONG M L, LU J H, MA P, HUANG B Y, WANG M G, TIAN Y F, CHEN F PENG J, LANG Z B, LI G F, ZHU J K. Cut-dip-budding delivery system enables genetic modifications in plants without tissue culture. The Innovation, 2023, 4(1): 100345.

doi: 10.1016/j.xinn.2022.100345
李勤霞, 刘亚楠, 张译文, 程敏, 薛晓东. 绿色和红色荧光蛋白基因在二穗短柄草中的应用. 分子植物育种, 2022:
LI Q X, LIU Y N, ZHANG Y W, CHENG M, XUE X D. Application of green and red fluorescent protein gene in Brachypodium distachyon. Molecular Plant Breeding, 2022: (in Chinese)
HRAŠKA M, RAKOUSKÝ S, ČURN V. Green fluorescent protein as a vital marker for non-destructive detection of transformation events in transgenic plants. Plant Cell, Tissue and Organ Culture, 2006, 86(3): 303-318.

doi: 10.1007/s11240-006-9131-1
TOINGA-VILLAFUERTE S, JANGA M R, ISABEL VALES M, RATHORE K S. Green fluorescent protein gene as a tool to examine the efficacy of Agrobacterium-delivered CRISPR/Cas9 reagents to generate targeted mutations in the potato genome. Plant Cell, Tissue and Organ Culture (PCTOC), 2022, 150(3): 587-598.

doi: 10.1007/s11240-022-02310-8
YUAN G L, LU H W, TANG D, HASSAN M M, LI Y, CHEN J G, TUSKAN G A, YANG X H. Expanding the application of a UV-visible reporter for transient gene expression and stable transformation in plants. Horticulture Research, 2021, 8: 234.

doi: 10.1038/s41438-021-00663-3 pmid: 34719678
[1] WEN YuanYuan, LI Yan, LI JianGuo, WANG MeiMei, YU ChangHui, SHEN YiZhao, GAO YanXia, LI QiuFeng, CAO YuFeng. Effects of Holstein Bulls Fed Mixed Silage of Potato Chips Processing by Product with Rice Straw on Fattening Performance and Blood Biochemical Indexes [J]. Scientia Agricultura Sinica, 2023, 56(9): 1800-1812.
[2] ZHANG ZhiPeng, TAN YunXiu, LI BaoJun, LI YongCai, BI Yang, LI ShouQiang, WANG XiaoJing, ZHANG Yu, HU Dan. Effects of Exogenous Abscisic Acid Treatment on Periderm Suberification of Postharvest Mini-Tuber Potato from Aeroponic System and Its Possible Mechanisms [J]. Scientia Agricultura Sinica, 2023, 56(6): 1154-1167.
[3] YE Nan, ZHU Yan, ZHAO YuanShou, ZHU JianNing, MEN JiaWei, CHEN Fu, KONG DeYuan, ZHANG WeiBing, ZONG YuanYuan, LI YongCai. Effects of Seed Soaking with Chitooligosaccharide on the Growth of Sprout and Endogenous Phytohormone Content in Potato Minitubers [J]. Scientia Agricultura Sinica, 2023, 56(4): 788-800.
[4] ZHAO HaiXia,XIAO Xin,DONG QiXin,WU HuaLa,LI ChengLei,WU Qi. Optimization of Callus Genetic Transformation System and Its Application in FtCHS1 Overexpression in Tartary Buckwheat [J]. Scientia Agricultura Sinica, 2022, 55(9): 1723-1734.
[5] PENG Xue,GAO YueXia,ZHANG LinXuan,GAO ZhiQiang,REN YaMei. Effects of High-Energy Electron Beam Irradiation on Potato Storage Quality and Bud Eye Cell Ultrastructure [J]. Scientia Agricultura Sinica, 2022, 55(7): 1423-1432.
[6] CUI Peng,ZHAO YiRen,YAO ZhiPeng,PANG LinJiang,LU GuoQuan. Starch Physicochemical Properties and Expression Levels of Anabolism Key Genes in Sweetpotato Under Low Temperature [J]. Scientia Agricultura Sinica, 2022, 55(19): 3831-3840.
[7] XiaoChuan LI,ChaoHai WANG,Ping ZHOU,Wei MA,Rui WU,ZhiHao SONG,Yan MEI. Deciphering of the Genetic Diversity After Field Late Blight Resistance Evaluation of Potato Breeds [J]. Scientia Agricultura Sinica, 2022, 55(18): 3484-3500.
[8] ZHANG XiaoPing,SA ShiJuan,WU HanYu,QIAO LiYuan,ZHENG Rui,YAO XinLing. Leaf Stomatal Close and Opening Orchestrate Rhythmically with Cell Wall Pectin Biosynthesis and Degradation [J]. Scientia Agricultura Sinica, 2022, 55(17): 3278-3288.
[9] FAN WenJing,LIU Ming,ZHAO Peng,ZHANG QiangQiang,WU DeXiang,GUO PengYu,ZHU XiaoYa,JIN Rong,ZHANG AiJun,TANG ZhongHou. Screening of Sweetpotato Varieties Tolerant to Low Nitrogen at Seedling Stage and Evaluation of Different Nitrogen Efficiencies [J]. Scientia Agricultura Sinica, 2022, 55(10): 1891-1902.
[10] YuXin LIANG,JianXiang WU,XiaoYu LI,ChunYu ZHANG,JiChao HOU,XuePing ZHOU,YongZhi WANG. Mapping of Epitopes and Establishment of Rapid DAS-ELISA for Potato Virus Y Coat Protein [J]. Scientia Agricultura Sinica, 2021, 54(6): 1154-1162.
[11] JianZhao TANG,Jing WANG,DengPan XIAO,XueBiao PAN. Research Progress and Development Prospect of Potato Growth Model [J]. Scientia Agricultura Sinica, 2021, 54(5): 921-932.
[12] LI KaiFeng,YIN YuHe,WANG Qiong,LIN TuanRong,GUO HuaChun. Correlation Analysis of Volatile Flavor Components and Metabolites Among Potato Varieties [J]. Scientia Agricultura Sinica, 2021, 54(4): 792-803.
[13] WANG Xin,LI Qiang,CAO QingHe,MA DaiFu. Current Status and Future Prospective of Sweetpotato Production and Seed Industry in China [J]. Scientia Agricultura Sinica, 2021, 54(3): 483-492.
[14] ZHANG MengDi,YAN JunJie,GAO YuLin. The Adaptive Analysis of Phthorimaea operculella to Different Potato Tuber Varieties [J]. Scientia Agricultura Sinica, 2021, 54(3): 536-546.
[15] LI Xiang,ZHANG XiaoJiao,XIAO Chun,DONG WenXia. Electroantennogram Responses of Phthorimaea operculella of Different Sexes and Mating States to Potato Volatiles [J]. Scientia Agricultura Sinica, 2021, 54(3): 547-555.
Full text



No Suggested Reading articles found!