Acta Agriculturae Zhejiangensis ›› 2023, Vol. 35 ›› Issue (8): 1823-1833.DOI: 10.3969/j.issn.1004-1524.20230491
• Plant Protection • Previous Articles Next Articles
Characterization of transgenic insect resistance and glyphosate tolerance rice expressing cry1Ab-vip3Af2 and cp4-epsps
ZHANG Siyu1(
), LIN Chaoyang1,2, YE Yuxuan1,2, SHEN Zhicheng1,*()
- 1. Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, China
2. The Rural Development Academy, Zhejiang University, Hangzhou 310058, China
-
Received:2023-04-18Online:2023-08-25Published:2023-08-29
CLC Number:
Cite this article
ZHANG Siyu, LIN Chaoyang, YE Yuxuan, SHEN Zhicheng. Characterization of transgenic insect resistance and glyphosate tolerance rice expressing cry1Ab-vip3Af2 and cp4-epsps[J]. Acta Agriculturae Zhejiangensis, 2023, 35(8): 1823-1833.
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Fig.1 Diagram of the T-DNA structure
Fig.1 Diagram of the T-DNA structure
Table 1 Primers of PCR
| 引物 Primer | 引物序列 Primer sequences(5'→3') |
|---|---|
| cry1Ab-vip3Af2-F | ACCTTCTTCTTGTTCAGGTCGATCTC |
| cry1Ab-vip3Af2-R | CATCAACACCATGCTGCACATCTACC |
| cp4-epsps-F | TTCATGAGGACGTTCAGGATGGTCAC |
| cp4-epsps-R | AGATGGGCGTACAGGTCAAATCCGA |
Table 1 Primers of PCR
| 引物 Primer | 引物序列 Primer sequences(5'→3') |
|---|---|
| cry1Ab-vip3Af2-F | ACCTTCTTCTTGTTCAGGTCGATCTC |
| cry1Ab-vip3Af2-R | CATCAACACCATGCTGCACATCTACC |
| cp4-epsps-F | TTCATGAGGACGTTCAGGATGGTCAC |
| cp4-epsps-R | AGATGGGCGTACAGGTCAAATCCGA |
Table 2 Primers of Hi-Tail PCR
| 引物 Primer | 引物序列 Primer sequences(5'→3') |
|---|---|
| LAD1 | ACGATGGACTCCAGAGCGGCCGCVNVNNNGGAA |
| LAD2 | ACGATGGACTCCAGAGCGGCCGCBNBNNNGGTT |
| LAD3 | ACGATGGACTCCAGAGCGGCCGCVVNVNNNCCAA |
| LAD4 | ACGATGGACTCCAGAGCGGCCGCBDNBNNNCGGT |
| RB-1 | GGCACTGGCCGTCGTTTTACAACGTCGT |
| RB-2 | ACGATGGACTCCAGTCCGGCCGCGTTACCCAACTTAA- TCGCCTTGCAGC |
| RB-3 | GCACCGATCGCCCTTCCCAACAGTTGC |
| LB-1 | TTTCTCCATAATAATGTGTGAGTAGTTCCC |
| LB-2 | ACGATGGACTCCAGTCCGGCCCTCATGTGTTGAGCAT- ATAAGAAACCCTTAG |
| LB-3 | CTAAAACCAAAATCCAGTACTAAAATCC |
| AC1 | ACGATGGACTCCAGAG |
Table 2 Primers of Hi-Tail PCR
| 引物 Primer | 引物序列 Primer sequences(5'→3') |
|---|---|
| LAD1 | ACGATGGACTCCAGAGCGGCCGCVNVNNNGGAA |
| LAD2 | ACGATGGACTCCAGAGCGGCCGCBNBNNNGGTT |
| LAD3 | ACGATGGACTCCAGAGCGGCCGCVVNVNNNCCAA |
| LAD4 | ACGATGGACTCCAGAGCGGCCGCBDNBNNNCGGT |
| RB-1 | GGCACTGGCCGTCGTTTTACAACGTCGT |
| RB-2 | ACGATGGACTCCAGTCCGGCCGCGTTACCCAACTTAA- TCGCCTTGCAGC |
| RB-3 | GCACCGATCGCCCTTCCCAACAGTTGC |
| LB-1 | TTTCTCCATAATAATGTGTGAGTAGTTCCC |
| LB-2 | ACGATGGACTCCAGTCCGGCCCTCATGTGTTGAGCAT- ATAAGAAACCCTTAG |
| LB-3 | CTAAAACCAAAATCCAGTACTAAAATCC |
| AC1 | ACGATGGACTCCAGAG |
Table 3 The laboratory bioassay of target pests
| 靶标昆虫 Target pests | 被试组织 Subject tissue | 幼虫数量 Quantity of larvae | 水稻生长期 Rice growth stage | 观察时间 Observation time/h |
|---|---|---|---|---|
| 大螟 Sesamia inferens | VB3叶片 VB3 leaves | 20 | 分蘖期、抽穗期、灌浆期 Tillering stage, heading stage, filling stage | 24 |
| 秀水134叶片 Xiushui 134 leaves | ||||
| 二化螟 Chilo suppressalis | VB3叶片 VB3 leaves | 20 | 分蘖期、抽穗期、灌浆期 Tillering stage, heading stage, filling stage | 24 |
| 秀水134叶片 Xiushui 134 leaves | ||||
| 稻纵卷叶螟 Cnaphalocrocis medinalis | VB3叶片 VB3 leaves | 20 | 分蘖期、抽穗期、灌浆期 Tillering stage, heading stage, filling stage | 24 |
| 秀水134叶片 Xiushui 134 leaves |
Table 3 The laboratory bioassay of target pests
| 靶标昆虫 Target pests | 被试组织 Subject tissue | 幼虫数量 Quantity of larvae | 水稻生长期 Rice growth stage | 观察时间 Observation time/h |
|---|---|---|---|---|
| 大螟 Sesamia inferens | VB3叶片 VB3 leaves | 20 | 分蘖期、抽穗期、灌浆期 Tillering stage, heading stage, filling stage | 24 |
| 秀水134叶片 Xiushui 134 leaves | ||||
| 二化螟 Chilo suppressalis | VB3叶片 VB3 leaves | 20 | 分蘖期、抽穗期、灌浆期 Tillering stage, heading stage, filling stage | 24 |
| 秀水134叶片 Xiushui 134 leaves | ||||
| 稻纵卷叶螟 Cnaphalocrocis medinalis | VB3叶片 VB3 leaves | 20 | 分蘖期、抽穗期、灌浆期 Tillering stage, heading stage, filling stage | 24 |
| 秀水134叶片 Xiushui 134 leaves |
Table 4 Glyphosate tolerance determination
| 除草剂剂量 Herbicide concentration/ (mL·hm-2) | 被试组织 Subject tissue | 观察时间 Observation time/d |
|---|---|---|
| 0 | VB3幼苗期3~5叶 3-5 leaves at seedling stage of VB3 | 7 |
| 秀水134幼苗期3~5叶 3-5 leaves at seedling stage of Xiushui 134 | ||
| 20 | VB3幼苗期3~5叶 3-5 leaves at seedling stage of VB3 | 7 |
| 秀水134幼苗期3~5叶 3-5 leaves at seedling stage of Xiushui 134 | ||
| 40 | VB3幼苗期3~5叶 3-5 leaves at seedling stage of VB3 | 7 |
| 秀水134幼苗期3~5叶 3-5 leaves at seedling stage of Xiushui 134 | ||
| 80 | VB3幼苗期3~5叶 3-5 leaves at seedling stage of VB3 | 7 |
| 秀水134幼苗期3~5叶 3-5 leaves at seedling stage of Xiushui 134 |
Table 4 Glyphosate tolerance determination
| 除草剂剂量 Herbicide concentration/ (mL·hm-2) | 被试组织 Subject tissue | 观察时间 Observation time/d |
|---|---|---|
| 0 | VB3幼苗期3~5叶 3-5 leaves at seedling stage of VB3 | 7 |
| 秀水134幼苗期3~5叶 3-5 leaves at seedling stage of Xiushui 134 | ||
| 20 | VB3幼苗期3~5叶 3-5 leaves at seedling stage of VB3 | 7 |
| 秀水134幼苗期3~5叶 3-5 leaves at seedling stage of Xiushui 134 | ||
| 40 | VB3幼苗期3~5叶 3-5 leaves at seedling stage of VB3 | 7 |
| 秀水134幼苗期3~5叶 3-5 leaves at seedling stage of Xiushui 134 | ||
| 80 | VB3幼苗期3~5叶 3-5 leaves at seedling stage of VB3 | 7 |
| 秀水134幼苗期3~5叶 3-5 leaves at seedling stage of Xiushui 134 |
Fig.2 The expression level of exogenous proteins in T0 transformants of rice A, The expression level of Cry1Ab-Vip3Af2, the dotted line showed the Cry1Ab-Vip3Af2 expression level was 5 μg·g-1, which was used as the screening standard; B, The expression level of CP4-EPSPS, the dotted line showed the CP4-EPSPS expression level was 150 μg·g-1, which was used as the screening standard.
Fig.2 The expression level of exogenous proteins in T0 transformants of rice A, The expression level of Cry1Ab-Vip3Af2, the dotted line showed the Cry1Ab-Vip3Af2 expression level was 5 μg·g-1, which was used as the screening standard; B, The expression level of CP4-EPSPS, the dotted line showed the CP4-EPSPS expression level was 150 μg·g-1, which was used as the screening standard.
Fig.3 PCR analysis of T1, T2 generations of VB3 A, PCR analysis of cry1Ab-vip3Af2; B, PCR analysis of cp4-epsps; M, DNA molecular marker;-, Non-transgenic rice control;+, Positive plasmids; 1-3, Samples of T1 and T2 generations of VB3.
Fig.3 PCR analysis of T1, T2 generations of VB3 A, PCR analysis of cry1Ab-vip3Af2; B, PCR analysis of cp4-epsps; M, DNA molecular marker;-, Non-transgenic rice control;+, Positive plasmids; 1-3, Samples of T1 and T2 generations of VB3.
Fig.4 Identification and analyzation of VB3 border sequences A, The electrophoresis result of Hi-Tail PCR about VB3 border sequences. R1, Round 1 products; R2, Round 2 products; R3, Round 3 products; M, DNA molecular marker. B, The identification of VB3 border sequences. M, DNA molecular marker.
Fig.4 Identification and analyzation of VB3 border sequences A, The electrophoresis result of Hi-Tail PCR about VB3 border sequences. R1, Round 1 products; R2, Round 2 products; R3, Round 3 products; M, DNA molecular marker. B, The identification of VB3 border sequences. M, DNA molecular marker.
Fig.5 Southern bloting of transgenic rice VB3 A, Hybridization with cry1Ab full-length gene as probe; B, Hybridization with cp4-epsps full-length gene as probe;+, Positive plasmids; NT, Non-transgenic rice control digested with the same enzyme; M, DNA molecular maker.
Fig.5 Southern bloting of transgenic rice VB3 A, Hybridization with cry1Ab full-length gene as probe; B, Hybridization with cp4-epsps full-length gene as probe;+, Positive plasmids; NT, Non-transgenic rice control digested with the same enzyme; M, DNA molecular maker.
Fig.6 The expression level of exogenous proteins in different tissues of VB3 A, The expression level of Cry1Ab-Vip3Af2 in different tissues of VB3; B, The expression level of CP4-EPSPS in different tissues of VB3.
Fig.6 The expression level of exogenous proteins in different tissues of VB3 A, The expression level of Cry1Ab-Vip3Af2 in different tissues of VB3; B, The expression level of CP4-EPSPS in different tissues of VB3.
Fig.7 Western blotting about Cry1Ab-Vip3Af2 and CP4-EPSPS in T1 and T2 generations of VB3 A, Western blotting of Cry1Ab-Vip3Af2; B, Western blotting of CP4-EPSPS; M, Protein pageruler;+, Protein expressed by E.coli;-, Non-transgenic rice control; L, Leaves; St, Stem; Sd, Seed.
Fig.7 Western blotting about Cry1Ab-Vip3Af2 and CP4-EPSPS in T1 and T2 generations of VB3 A, Western blotting of Cry1Ab-Vip3Af2; B, Western blotting of CP4-EPSPS; M, Protein pageruler;+, Protein expressed by E.coli;-, Non-transgenic rice control; L, Leaves; St, Stem; Sd, Seed.
Table 5 Mortality of Sesamia inferens, Chilo suppressalis, Cnaphalocrocis medinalis fed on VB3 and non-transgenic rice leaves
| 时期 Stage | 水稻株系 Rice line | 大螟Sesamia inferens | 二化螟Chilo suppressalis | 稻纵卷叶螟Cnaphalocrocis medinalis | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| 死亡率Mortality/% | 死亡率Mortality/% | 死亡率Mortality/% | ||||||||
| 24 h | 48 h | 72 h | 24 h | 48 h | 72 h | 24 h | 48 h | 72 h | ||
| 分蘖期 Tillering stage | VB3 VB3 | 29.37± 3.00** | 91.30± 2.50** | 100** | 31.40± 2.44** | 85.07± 2.04** | 100** | 32.57± 2.68** | 92.10± 1.97** | 100** |
| 秀水134 Xiushui 134 | 3.06± 0.57 | 5.80± 1.45 | 7.33± 0.81 | 2.80± 0.46 | 3.77± 0.45 | 7.60± 0.56 | 3.30± 0.44 | 5.57± 0.76 | 8.50± 0.53 | |
| 抽穗期 Heading stage | VB3 VB3 | 23.63± 2.65** | 88.73± 1.23** | 100** | 22.53± 1.78** | 86.73± 2.87** | 100** | 23.43± 1.25** | 87.03± 1.69** | 100** |
| 秀水134 Xiushui 134 | 3.47± 0.40 | 4.67± 0.40 | 8.30± 0.9 | 2.57± 0.23 | 3.97± 0.42 | 6.87± 0.25 | 3.03± 0.23 | 5.67± 0.57 | 7.83± 0.81 | |
| 灌浆期 Filling stage | VB3 VB3 | 30.37± 2.57** | 82.97± 2.32** | 100** | 32.60± 2.04** | 93.77± 1.17** | 100** | 26.53± 0.96** | 89.00± 2.26** | 100** |
| 秀水134 Xiushui 134 | 2.77± 0.42 | 4.57± 0.61 | 7.37± 0.55 | 4.00± 0.46 | 5.17± 0.45 | 7.50± 0.17 | 2.80± 0.36 | 4.67± 0.32 | 8.17± 0.25 | |
Table 5 Mortality of Sesamia inferens, Chilo suppressalis, Cnaphalocrocis medinalis fed on VB3 and non-transgenic rice leaves
| 时期 Stage | 水稻株系 Rice line | 大螟Sesamia inferens | 二化螟Chilo suppressalis | 稻纵卷叶螟Cnaphalocrocis medinalis | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| 死亡率Mortality/% | 死亡率Mortality/% | 死亡率Mortality/% | ||||||||
| 24 h | 48 h | 72 h | 24 h | 48 h | 72 h | 24 h | 48 h | 72 h | ||
| 分蘖期 Tillering stage | VB3 VB3 | 29.37± 3.00** | 91.30± 2.50** | 100** | 31.40± 2.44** | 85.07± 2.04** | 100** | 32.57± 2.68** | 92.10± 1.97** | 100** |
| 秀水134 Xiushui 134 | 3.06± 0.57 | 5.80± 1.45 | 7.33± 0.81 | 2.80± 0.46 | 3.77± 0.45 | 7.60± 0.56 | 3.30± 0.44 | 5.57± 0.76 | 8.50± 0.53 | |
| 抽穗期 Heading stage | VB3 VB3 | 23.63± 2.65** | 88.73± 1.23** | 100** | 22.53± 1.78** | 86.73± 2.87** | 100** | 23.43± 1.25** | 87.03± 1.69** | 100** |
| 秀水134 Xiushui 134 | 3.47± 0.40 | 4.67± 0.40 | 8.30± 0.9 | 2.57± 0.23 | 3.97± 0.42 | 6.87± 0.25 | 3.03± 0.23 | 5.67± 0.57 | 7.83± 0.81 | |
| 灌浆期 Filling stage | VB3 VB3 | 30.37± 2.57** | 82.97± 2.32** | 100** | 32.60± 2.04** | 93.77± 1.17** | 100** | 26.53± 0.96** | 89.00± 2.26** | 100** |
| 秀水134 Xiushui 134 | 2.77± 0.42 | 4.57± 0.61 | 7.37± 0.55 | 4.00± 0.46 | 5.17± 0.45 | 7.50± 0.17 | 2.80± 0.36 | 4.67± 0.32 | 8.17± 0.25 | |
Fig.8 The glyphosate tolerance of VB3 in green house
Fig.8 The glyphosate tolerance of VB3 in green house
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