水稻水杨酸代谢突变体高通量筛选方法的建立与应用

doi:10.11983/CBB24148

摘要/Abstract

摘要： 水杨酸(SA)是植物免疫的关键防御信号分子。植物SA的定量分析对于SA代谢途径及其生物学功能研究至关重要。利用高效液相色谱仪(HPLC)和液相-质谱联用仪(LC-MS)测定SA含量是目前常用方法, 但难以实现高通量测定。水稻(Oryza sativa)中SA合成代谢途径目前尚未完全解析, 高效筛选水稻SA相关突变体对于阐明其代谢途径具有重要意义。该文对已有基于SA生物传感菌株Acinetobacter sp. ADPWH_lux估算SA的分析方法进行了改良, 建立了水稻SA高通量估算方法, 简化了样品采集和提取过程, 省去样品称重、组织研磨及离心等耗时步骤, 整个操作流程便捷且高效。同时, 利用已报道的水稻SA代谢相关遗传材料验证了该方法的可行性, 并使用该方法筛选了钴-60诱变的水稻突变体库, 获得一批水稻SA含量发生显著变化的突变体, 采用HPLC法对突变体内源SA进行了验证。该方法可用于SA代谢突变体的遗传筛选及其代谢相关酶鉴定, 对水稻等作物的SA代谢及生物学功能研究具有重要的应用价值。

关键词: 水杨酸测定, 生物传感器, 高通量方法, 水杨酸代谢突变体, 水稻

Abstract: INTRODUCTION Salicylic acid (SA) plays an important role in the plant immune system. The quantitative analysis of SA in plants is fundamental to studying SA metabolism and its biological functions. Although high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC/MS) are widely used for SA determination, their low throughput limits their suitability for large-scale analysis. However, the SA biosynthetic pathway in rice is not well understood, highlighting the need for efficient methods to screen SA-related mutants and elucidate SA metabolic pathways. RATIONALECurrent methods for measuring endogenous SA levels, such as HPLC and LC/MS, involve labor-intensive sample preparation, making them unsuitable for high-throughput analysis. While a lux gene-based SA biosensor has been successfully used in tobacco and Arabidopsis, a reliable and efficient method for SA detection in rice remains unavailable. To address this problem, we optimized sample processing and operational workflows to enable high- throughput SA quantification in rice plants.RESULTS We developed a streamlined, high-throughput method for SA quantification in rice, eliminating time-consuming steps such as sample weighing, tissue grinding, and centrifugation. This approach significantly simplifies the process while maintaining efficiency and accuracy. We validated the method’s feasibility using published rice SA metabolic mutants. We then applied it to screen a Cobalt-60 induced rice mutant library, identifying mutants with altered SA metabolism. Endogenous SA levels in these mutants were confirmed using HPLC. The results demonstrate the method’s effectiveness in screening SA-related metabolic mutants, providing a valuable tool for studying SA metabolism and its roles in rice and other crops. The method was validated using known SA genetic materials. SA content-altered mutants were successfully isolated for further research.CONCLUSION This study establishes a rapid and cost-effective method for measuring SA content in rice tissues using the SA biosensor Acinetobacter sp. ADPWH_lux. Given the pivotal role of SA in plant defense, our method adopts streamlined sampling process, requiring only leaf clipping and boiling in LB medium, and dramatically reduces the time and effort associated with tissue collection and processing. This high-throughput approach is well-suited for large-scale screening of greenhouse-grown or hydroponic plants, providing a powerful platform for advancing research on SA metabolism and its biological functions in crops.
Modified manipulating process for high-throughput determination of salicylic acid (SA) content using SA biosensor Acinetobacter sp. ADPWH_lux strain in rice

Key words: salicylic acid assay, biosensor, high throughput method, salicylic acid metabolism mutant, rice

叶灿, 姚林波, 金莹, 高蓉, 谭琪, 李旭映, 张艳军, 陈析丰, 马伯军, 章薇, 张可伟. 水稻水杨酸代谢突变体高通量筛选方法的建立与应用. 植物学报, 2025, 60(4): 586-596.

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. Chinese Bulletin of Botany, 2025, 60(4): 586-596.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB24148

https://www.chinbullbotany.com/CN/Y2025/V60/I4/586

图/表 6

图1 Acinetobacter sp. ADPWH_lux菌株的发光强度标准曲线标准曲线方程是Y=235621X-77045, 决定系数R2 =0.9723。数据为平均值±标准差(n=3)。

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).

图2 利用水杨酸(SA)生物传感器Acinetobacter sp. ADPWH_ lux菌株高通量测定水稻SA含量的改良操作流程(改自Marek et al., 2010) (1) 水稻在96孔PCR板(管底经切割处理)水培约3周(每板加入3株野生型, 1株oss5hDM/NIP和S5H1-OE作为对照); (2) 剪取每株水稻苗5 cm的叶片2枚(约0.015 g); (3) 揉成团塞入对应的已加600 μL LB的96孔2 mL板中, 96孔PCR板与96孔2 mL板位置一一对应; (4) 将96孔2 mL板在95°C下水浴30分钟, 每隔5分钟将96孔板盖子重新盖好(高温会使盖子顶开); (5) 待96孔2 mL板中的叶片提取液冷却至室温后, 吸取50 μL叶片提取液至已提前加入50 μL生物传感器的黑色酶标板中, 酶标板与96孔2 mL板位置一一对应(每次实验每板水稻苗做2板重复); (6) 37°C下恒温黑暗静置培养1.5小时; (7) 用酶联免疫检测仪测定相对发光值(每一酶标板需测2次)。

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).

图3 NIP、oss5hDM/NIP和S5H1-OE的水杨酸(SA)发光强度(A)及内源性SA含量(B) 水稻在温室条件下培养, 培养条件完全相同。数据为平均值±标准差(n=3)。使用t检验进行统计学分析, *P<0.05; **P<0.01

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

图4 96孔PCR板上所有水稻的水杨酸(SA)发光强度(A)及筛选候选突变体的SA发光强度(B) 数据为平均值±标准差(n=3)。使用t检验进行统计学分析, * P<0.05; **P<0.01; ***P<0.001

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

表1 突变体水杨酸(SA)发光强度在标准曲线中对应的SA浓度及估算的水稻叶片SA含量

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
1	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
2	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
3	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
4	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
5	144-5F	72798.67±7982.44***	0.64±0.03***	25.44±1.36***

图5 野生型(NIP)和5个水杨酸(SA)水稻突变体的SA含量柱状图 FW: 鲜重。数据为平均值±标准差(n=3)。使用t检验进行统计学分析, **P<0.01; ***P<0.001

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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