Chinese Bulletin of Botany ›› 2025, Vol. 60 ›› Issue (4): 586-596.DOI: 10.11983/CBB24148 cstr: 32102.14.CBB24148
• TECHNIQUES AND METHODS • Previous Articles Next Articles
Can Ye, Linbo Yao, Ying Jin, Rong Gao, Qi Tan, Xuying Li, Yanjun Zhang, Xifeng Chen, Bojun Ma, Wei Zhang*(), Kewei Zhang*(
)
Received:
2024-09-25
Accepted:
2024-12-14
Online:
2025-07-10
Published:
1900-01-01
Contact:
Wei Zhang, Kewei Zhang
Can Ye, Linbo Yao, Ying Jin, Rong Gao, Qi Tan, Xuying Li, Yanjun Zhang, Xifeng Chen, Bojun Ma, Wei Zhang, Kewei Zhang. Establishment and Application of a High-throughput Screening Method for Salicylic Acid Metabolic Mutants in Rice[J]. Chinese Bulletin of Botany, 2025, 60(4): 586-596.
Figure 1 Standard curve of luminescence of Acinetobacter sp. ADPWH_lux strain Equation: Y=235621X-77045, Coefficient of determination: R2=0.9723. Data are means ± SD (n=3).
Figure 2 Modified manipulating process for high-throughput determination of salicylic acid (SA) content using SA biosensor Acinetobacter sp. ADPWH_lux strain in rice (adapted from Marek et al., 2010) (1) Rice was hydroponic for about 3 weeks in 96-well PCR plates (The bottom of tube was cut) (As controls, 3 wild type,1 oss5hDM/NIP and S5H1-OE plants were added to each plate); (2) Took two leaves of 5 cm (about 0.015 g) from each rice seedling; (3) Kneaded the leaf into a 2 mL 96-well plate with 600 μL LB, and the positions of the 96-well PCR plate and the 96-well 2 mL plate were one-to-one corresponding; (4) The 2 mL 96-well plate was bathed in water at 95°C for 30 minutes (the lid of the 96-well plate should be re-closed every 5 minutes, because high temperature would make the lid open); (5) After the leaf extract liquid from the 2 mL 96-well plate was cooled to room temperature, 50 μL of the extract liquid was pipetted into the black enzyme-labeled plate that had been added with 50 μL biosensor in advance, and the position of the enzyme-labeled plate was one-to-one corresponding to the 96-well 2 mL plate (two plates were repeated for each rice seedling in each experiment); (6) Incubation at 37°C in a incubator for 1.5 hours in the dark; (7) The relative luminescence value was measured with an enzyme-linked immunodetector (each enzyme plate needs to be measured twice).
Figure 3 The salicylic acid (SA) luminescence (A) and SA content (B) of wild type (NIP), oss5hDM/NIP and S5H1-OE Rice plants were cultured in greenhouse, and the culture conditions were the same. Data are means ± SD (n=3). The statistical significances were calculated by t-test, * P<0.05; **P<0.01
Figure 4 The salicylic acid (SA) luminescence of all rice on the 96-well PCR plate (A) and SA luminescence in the selected candidate mutants (B) Data are means ± SD (n=3). Statistical significance was calculated by t-test, * P<0.05; ** P<0.01; *** P<0.001
No. | Sample name | Luminescence | SA concentration in extracting solution (nmol·mL-1) | Estimated SA content in rice leaves (nmol·g-1) |
---|---|---|---|---|
1 | NIP | 240906.67±33164.52 | 1.35±0.14 | 53.98±5.63 |
41-4A | 373098.67±23587.55** | 1.91±0.10* | 76.42±4.00* | |
2 | NIP | 251577.33±48090.31 | 1.39±0.20 | 55.79±8.16 |
133-3H | 7778.33±1563.11*** | 0.36±0.01*** | 13.21±0.27*** | |
3 | NIP | 253330.67±50076.11 | 1.40±0.21 | 56.09±8.50 |
134-6D | 21748.67±2827.30** | 0.42±0.01** | 16.77±0.48** | |
4 | NIP | 232799.67±29917.16 | 1.32±0.13 | 52.60±5.08 |
144-5B | 44528±4907.68*** | 0.52±0.02*** | 20.64±0.83*** | |
5 | NIP | 232799.67±29917.16 | 1.32±0.13 | 52.60±5.08 |
144-5F | 72798.67±7982.44*** | 0.64±0.03*** | 25.44±1.36*** |
Table 1 The salicylic acid (SA) concentration corresponding to the SA luminescence of mutant in the standard curve and estimated SA content in rice leaves
No. | Sample name | Luminescence | SA concentration in extracting solution (nmol·mL-1) | Estimated SA content in rice leaves (nmol·g-1) |
---|---|---|---|---|
1 | NIP | 240906.67±33164.52 | 1.35±0.14 | 53.98±5.63 |
41-4A | 373098.67±23587.55** | 1.91±0.10* | 76.42±4.00* | |
2 | NIP | 251577.33±48090.31 | 1.39±0.20 | 55.79±8.16 |
133-3H | 7778.33±1563.11*** | 0.36±0.01*** | 13.21±0.27*** | |
3 | NIP | 253330.67±50076.11 | 1.40±0.21 | 56.09±8.50 |
134-6D | 21748.67±2827.30** | 0.42±0.01** | 16.77±0.48** | |
4 | NIP | 232799.67±29917.16 | 1.32±0.13 | 52.60±5.08 |
144-5B | 44528±4907.68*** | 0.52±0.02*** | 20.64±0.83*** | |
5 | NIP | 232799.67±29917.16 | 1.32±0.13 | 52.60±5.08 |
144-5F | 72798.67±7982.44*** | 0.64±0.03*** | 25.44±1.36*** |
Figure 5 Histogram of salicylic acid (SA) content in wild type (NIP) and 5 SA rice mutant plants FW: Fresh weight. Data are means ± SD (n=3). The statistical significance was calculated by t-test, **P<0.01; ***P<0.001
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