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

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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

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
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

Table 2

Primers for detecting the mutations of target genes"

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
AMSsiteCeR GCATCCATCTCACTCATGTACC
StDYT1靶位点扩增
Amplification of StDYT1 target site		 DYT1siteCeF ATGGTAGTAATTAGGTGTGC
DYT1siteCeR AGCTTAGTTGGGCCAATGTG
StMS1a靶位点扩增
Amplification of StMS1a target site		 MS1asiteCeF TCGTCAATAGTGATGGAGCC
MS1asiteCeR CAATTGCGGCACTCTAACTCC
StMS1b靶位点扩增
Amplification of StMS1b target site		 MS1bsiteCeF AGAGAAGAAGAAGGGAGTGTCG
MS1bsiteCeR GCTTCCTGGATACATCTTCC
StMYB80靶位点扩增
Amplification of StMYB80 target site		 MYB80siteCeF GTATGTTGCTCAGACTCTCTG
MYB80siteCeR TACTAGATGTTGCTCCTACAC
StTDF1靶位点扩增
Amplification of StTDF1 target site		 TDF1siteCeF GCATTCATTACATCCGTCTTGTG
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

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"

基因
Gene		 材料
Materials		 荧光根数
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 (%)"

基因
Gene		 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"

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