高效GFPuv荧光筛选基因编辑载体的改造及其在马铃薯遗传转化中的应用

doi:10.3864/j.issn.0578-1752.2023.11.015

摘要/Abstract

摘要：

【目的】转基因技术的发展离不开筛选标记的完善与创新，其中的可视化筛选标记在转基因中的应用越来越广泛。近年来，经过突变获得的增强型黄绿荧光蛋白（an enhanced Yellow Green Fluorescent like Protein (eYGFP) under ultraviolet (UV)，eYGFPuv（GFPuv））在365 nm紫外光线下能够发出强烈且稳定的绿色荧光，便于观察。构建GFPuv荧光筛选的基因编辑载体，并在马铃薯遗传转化中进行应用验证，为GFPuv荧光筛选在马铃薯转化中广泛应用提供技术支持，同时为后期利用基因组编辑技术创制马铃薯雄性不育系奠定基础。【方法】利用同源重组技术将GFPuv表达框架和基因编辑元件Cas9_sgRNA依次与pCAMBIA2300载体连接，并进行农杆菌介导的烟草瞬时转化试验；通过发根农杆菌Ar qual和MSU440转化马铃薯茎段，在便携紫外灯下观察，并统计荧光根；利用改造载体构建了6个马铃薯花药发育保守基因的编辑载体，通过发根农杆菌体系转化2种马铃薯材料，验证转化效率和编辑效率；利用改造载体对马铃薯进行遗传转化。【结果】成功构建了GFPuv荧光筛选的基因编辑载体pCAMBIA2300MGFPuv-sgRNACas，瞬时转化烟草证实GFPuv表达框架能正常表达；2种发根农杆菌的转化均筛选到绿色荧光的毛状根，加入卡那霉素（kanamycin，Kan）显著提高阳性荧光根的比例，2种菌的转化效率差异不大，但MSU440的发根形成速度更快；6个马铃薯花药发育保守基因的编辑载体在2种马铃薯材料中的转化效率和编辑效率是一致的，但不同靶位点的编辑效率差异较大；改造载体遗传转化马铃薯证实GFPuv荧光可用于马铃薯愈伤组织和再生苗的筛选。【结论】发根农杆菌遗传转化体系是基因编辑效率验证的重要途径，GFPuv荧光可用于马铃薯转化的筛选。

关键词: 马铃薯, GFPuv, 基因组编辑, 毛状根, 遗传转化

Abstract:

【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

杜静雅, 陈凯园, 普金, 周会英, 祝光涛, 张春芝, 杜慧. 高效GFPuv荧光筛选基因编辑载体的改造及其在马铃薯遗传转化中的应用[J]. 中国农业科学, 2023, 56(11): 2223-2236.

DU JingYa, CHEN KaiYuan, PU Jin, ZHOU HuiYing, ZHU GuangTao, ZHANG ChunZhi, DU Hui. The Modification of Gene Editing Vector for Efficient GFPuv Fluorescence Screening and Its Application in Potato Genetic Transformation[J]. Scientia Agricultura Sinica, 2023, 56(11): 2223-2236.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2023.11.015

https://www.chinaagrisci.com/CN/Y2023/V56/I11/2223

图/表 10

表1

表2

靶点检测引物"

用途 Purposes	引物名称 Primer names	引物序列 Primer sequences (5′-3′)
StAMS靶位点扩增 Amplification of StAMS target site	AMSsiteCeF	CTCATGGAGAGGCTTAGGCC
StAMS靶位点扩增 Amplification of StAMS target site	AMSsiteCeR	GCATCCATCTCACTCATGTACC
StDYT1靶位点扩增 Amplification of StDYT1 target site	DYT1siteCeF	ATGGTAGTAATTAGGTGTGC
StDYT1靶位点扩增 Amplification of StDYT1 target site	DYT1siteCeR	AGCTTAGTTGGGCCAATGTG
StMS1a靶位点扩增 Amplification of StMS1a target site	MS1asiteCeF	TCGTCAATAGTGATGGAGCC
StMS1a靶位点扩增 Amplification of StMS1a target site	MS1asiteCeR	CAATTGCGGCACTCTAACTCC
StMS1b靶位点扩增 Amplification of StMS1b target site	MS1bsiteCeF	AGAGAAGAAGAAGGGAGTGTCG
StMS1b靶位点扩增 Amplification of StMS1b target site	MS1bsiteCeR	GCTTCCTGGATACATCTTCC
StMYB80靶位点扩增 Amplification of StMYB80 target site	MYB80siteCeF	GTATGTTGCTCAGACTCTCTG
StMYB80靶位点扩增 Amplification of StMYB80 target site	MYB80siteCeR	TACTAGATGTTGCTCCTACAC
StTDF1靶位点扩增 Amplification of StTDF1 target site	TDF1siteCeF	GCATTCATTACATCCGTCTTGTG
StTDF1靶位点扩增 Amplification of StTDF1 target site	TDF1siteCeR	CCCTTGTGTAGATTTGAGTGGAG
StAMS靶位点1的Hi-TOM测序扩增 Hi-TOM sequencing amplification of StAMS target site1	TomAMSsite1F	ggagtgagtacggtgtgcGCTGCTGTGGTGGAGCTGAG
	TomAMSsite1R	gagttggatgctggatggACATGTACCCGCAGTCTAGC
StAMS靶位点2的Hi-TOM测序扩增 Hi-TOM sequencing amplification of StAMS target site2	TomAMSsite2F	ggagtgagtacggtgtgcCTAGCAGAAGATGAGAAAGTC
	TomAMSsite2R	gagttggatgctggatggTGAATGGAGGAAGGAAGTTC
StDYT1靶位点的Hi-TOM测序扩增 Hi-TOM sequencing amplification of StDYT1 target site	TomDYT1siteF	ggagtgagtacggtgtgcGGAAGCAAATTGATGGTGAA
	TomDYT1siteR	gagttggatgctggatggGTCTCCGGTTCCTCCCCATG
StMS1a靶位点的Hi-TOM测序扩增 Hi-TOM sequencing amplification of StMS1a target site	TomMS1asiteF	ggagtgagtacggtgtgcGGGGCAACAATTTGATGTGC
	TomMS1asiteR	gagttggatgctggatggGTCCAATGCAAAGTCGATCCC
StMS1b靶位点1的Hi-TOM测序扩增 Hi-TOM sequencing amplification of StMS1b target site1	TomMS1bsite1F	ggagtgagtacggtgtgcGCATAAGAGTAAAGGTGTGC
	TomMS1bsite1R	gagttggatgctggatggCCGACAAATCCCCAAAACCC
StMS1b靶位点2的Hi-TOM测序扩增 Hi-TOM sequencing amplification of StMS1b target site2	TomMS1bsite2F	ggagtgagtacggtgtgcTACCAGCTGATAGTGAATGG
	TomMS1bsite2R	gagttggatgctggatggGTGCAAATACGATCCCAGAG
StMYB80靶位点的Hi-TOM测序扩增 Hi-TOM sequencing amplification of StMYB80 target site	TomMYB80siteF	ggagtgagtacggtgtgcTATGATTTACGCGTAATTGAACAG
	TomMYB80siteR	gagttggatgctggatggGATGAAGCATTTCATCTTTG
StTDF1靶位点的Hi-TOM测序扩增 Hi-TOM sequencing amplification of StTDF1 target site	TomTDF1siteF	ggagtgagtacggtgtgcCTAACATCATTCATGTGCAGG
	TomTDF1siteR	gagttggatgctggatggGCATTTTGAATTGGGAGACC

表2

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表3

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参考文献 28

[1]	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
[2]	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
[3]	JEFFERSON R A. The GUS reporter gene system. Nature, 1989, 342(6251): 837-838. doi: 10.1038/342837a0
[4]	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
[5]	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
[6]	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
[7]	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
[8]	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
[9]	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
[10]	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)
[11]	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
[12]	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
[13]	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
[14]	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
[15]	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
[16]	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
[17]	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)
[18]	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)
[19]	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
[20]	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)
[21]	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
[22]	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
[23]	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.
[24]	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
[25]	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: https://kns.cnki.net/kcms/detail/46.1068.S.20221010.1626.026.html.
	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: https://kns.cnki.net/kcms/detail/46.1068.S.20221010.1626.026.html. (in Chinese)
[26]	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
[27]	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
[28]	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

引物名称 Primer name	引物序列 Primer sequence (5′-3′)
CZ1F	aagcactctttcctgtggATAGCACGTACATTG
CZ1R	ccacaggaaagagtgcttTTCGACCTTTTTCCCCTG
CZ2F	cgagagtgtcgtgctccaccatgttggTGAGACTTTTCAACAAAGGGTAATATC
CZ2R	ggctagagcagcttgccaacatggtggTGTGTACAGATATATGTTGAATTATTGAGC
CZ3F	gagctcggtacccggggatccGATACCGTCGAATCTTGCTGAAA
CZ3R	tgcctgcaggtcgactctagaGCTGCAAGGCGATTAAGTTGG
StAMSCZF	tcgaagtagtgattgTGCTTGTGATTTACTGGCTCGTTTTAGAGCTAGAAATAGC
StAMSCZR	ttctagctctaaaacACAGACAGTCTCCCCTGCAGCAATCTCTTAGTCGACTCTAC
StDYT1CZF	tcgaagtagtgattgGGCGTCAAAAACTTAGCGAAGTTTTAGAGCTAGAAATAGC
StDYT1CZR	ttctagctctaaaacAAGTGAGCAGCTTCTTGAAACAATCTCTTAGTCGACTCTAC
StMS1aCZF	tcgaagtagtgattgATTGGAGCTTGTGTAGAAGGGTTTTAGAGCTAGAAATAGC
StMS1aCZR	ttctagctctaaaacGGCAAGTCAGAAGAAGTAGGCAATCTCTTAGTCGACTCTAC
StMS1bCZF	tcgaagtagtgattgTGTGATCACTGTCGATGTGCGTTTTAGAGCTAGAAATAGC
StMS1bCZR	ttctagctctaaaacCCCTCAATTCCATTAAGTGACAATCTCTTAGTCGACTCTAC
StMYB80CZF	tcgaagtagtgattgGTGATAGCTGCTCAACTTCCGTTTTAGAGCTAGAAATAGC
StMYB80CZR	ttctagctctaaaacAGTGCTGCTTCAGCCAAATGCAATCTCTTAGTCGACTCTAC
StTDF1CZF	tcgaagtagtgattgGAAAAAGCTGTAGGCTGAGAGTTTTAGAGCTAGAAATAGC
StTDF1CZR	ttctagctctaaaacTACTCAGCAAAGACCTGAGCCAATCTCTTAGTCGACTCTAC

基因 Gene	A056		PG6359
基因 Gene	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