Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (2): 63-69.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0848
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LU Zhen-wan1,2(), LI Xue-qi1,2,3, HUANG Jin-guang3, ZHOU Huan-bin1,2()
Received:
2022-07-10
Online:
2023-02-26
Published:
2023-03-07
LU Zhen-wan, LI Xue-qi, HUANG Jin-guang, ZHOU Huan-bin. Creation of Glyphosate-tolerant Rice by Cytosine Base Editing[J]. Biotechnology Bulletin, 2023, 39(2): 63-69.
名称 Name | 抗性/菌株 Resistance/Species | 来源 Source | |
---|---|---|---|
Plasmid | pENTR4:sgRNA5 | KanR | Lab stock |
pUbi:rBE22 | KanR | Lab stock | |
Strain | JM109 | Escherichia coli | Lab stock |
EHA105 | Agrobacterium tumefaciens | Lab stock |
Table 1 Bacterial strains and plasmids
名称 Name | 抗性/菌株 Resistance/Species | 来源 Source | |
---|---|---|---|
Plasmid | pENTR4:sgRNA5 | KanR | Lab stock |
pUbi:rBE22 | KanR | Lab stock | |
Strain | JM109 | Escherichia coli | Lab stock |
EHA105 | Agrobacterium tumefaciens | Lab stock |
Fig. 1 Amino acid sequence alignment of rice OsEPSPS and Eleusine indica EiEPSPS EiEPSPS-R(CAD01095.1)and EiEPSPS-S(CAD01096.1)were isolated from the resistant(R)and sensitive(S)Eleusine indica biotypes, respectively[22]. EiEPSPS-P106 site is highlighted in red box
Fig. 2 Identification of base editing materials in T0 A: Design of the rice OsEPSPS gene target site. B: Detected results of the OsEPSPS target base editing in T0 transgenic plants. PAM sequences, target sequences, the target bases and target amino acids, and the mutated predicted bases and corresponding amino acids are highlighted in green, bold, red, and blue, respectively. The mutated bases in the sequencing chromatograms are underlined
Fig. 3 Identification of base editing materials OsEPSPS(P177L)without exogenous components and homozygous mutations A: Separation detection of exogenous T-DNA elements(sgRNA, Hyg and Cas9)from self-progeny of base editing material #105. Kit is wild-type Kitaake, and lane 1-15 is the T1 plants of #105. B: Sequencing results of the target sites in transgene-free T1 progeny plants. PAM sequences, target sequence, the target bases and target amino acids, and the mutated predicted bases/corresponding amino acids are highlighted in green, bold, red, and blue, respectively. The mutated bases in the sequencing chromatograms are underlined
Fig. 4 Glyphosate-tolerance assay of base editing materials OsEPSPS(P177L)plants A: Germination assay of OsEPSPS(P177L)seeds in cylinders containing 1/2MS with glyphosate, and photos were taken after 14 d treatment. Bar=5 cm. B: Glyphosate-tolerance assay of OsEPSPS(P177L)seedling by spraying glyphosate with spray tower, photographs were taken at 14 d respectively,the recommended dose of glyphosate in the field was 1 350 g a.i./ha as 1×
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