大豆根瘤菌菌株5873 PCR快速检测方法的建立及应用效果评价

doi:10.3864/j.issn.0578-1752.2023.14.010

摘要/Abstract

摘要：

【目的】大豆根瘤菌（Bradyrhizobium japonicum）是微生物肥料重要的功能菌种之一，可通过生物固氮为大豆生长提供氮素，实现节本增效，菌株5873作为其典型代表，已广泛应用于农业生产。筛选并鉴定其特异引物，建立大豆根瘤菌5873菌株水平的快速检测方法，对微生物肥料生产菌种鉴定、产品质量检测和功能评价至关重要。【方法】以商业化菌株大豆根瘤菌5873为供试材料，基于其全基因组序列和NCBI数据库中大豆根瘤菌种内参比菌株相关序列，以及与其高度同源（基因组ANI值大于99.95%）的大豆根瘤菌USDA 6^T差异片段，通过多重序列比对，进行特异引物设计和筛选，获得特异性引物对，并通过对PCR反应条件/体系优化、特异性及灵敏度检测，建立大豆根瘤菌5873的快速检测方法。然后，采用盆栽试验，将大豆根瘤菌5873与其他根瘤菌菌株混合接种于大豆根际，应用上述方法对大豆根瘤菌5873竞争结瘤能力进行评价和方法验证。【结果】筛选获得了一组特异引物4-4和Q1（4-4-F 5′-GATAAGGCCACGGGTGAACA-3′/4-4-R 5′-CACTCGATAAGCTCCGCTGT-3′和Q1-F 5′-CCGGTCGTGACTGGAATGAT-3′/Q1-R 5′-TCGAGGCCTACAAGAACGTC-3′），优化并建立了PCR快速检测方法，即反应体系：Premix Taq^TM 12.5 μL，引物各1.0 μL，基因组DNA 15 ng左右，加ddH₂O补足至25 μL；反应条件：95 ℃ 预热5 min，94 ℃ 变性45 s，61 ℃ 退火45 s，72 ℃ 延伸60 s，30个循环，再72 ℃ 延伸10 min。通过凝胶电泳检测目的条带的有无（355和218 bp），即可实现大豆根瘤菌5873的快速检测，该方法检出灵敏度为1 850 CFU/µL。此外，借助该方法可以成功地评价大豆根瘤菌5873竞争结瘤能力，与传统BOX-PCR评价结果一致。【结论】大豆根瘤菌5873快速鉴定方法的建立实现了以菌体发酵液或根瘤破碎提取液为模板，直接进行PCR扩增的鉴定操作，省去了根瘤的分离、根瘤菌分离纯化、培养、DNA提取及测序鉴定等繁琐的环节，大大减少了工作量，只需短短几个小时便可准确检测大豆根瘤菌5873，为微生物肥料中根瘤菌菌剂的产品质量检测和竞争结瘤能力评价提供了参考。

关键词: 大豆根瘤菌, 特异引物, PCR扩增

Abstract:

【Objective】Bradyrhizobium japonicum is one of the important functional bacteria in microbial fertilizer products. As a commercialized strain, B. japonicum 5873 was wildly used in agricultural production and played important roles in symbiotic nitrogen fixation. Therefore, it is necessary to establish a rapid identification method at the strain level by screening and identifying specific primers for rhizobia, and is of great importance in the aspects of microbial fertilizer product quality inspection, strain identification and function evaluation.【Method】According to the whole genome sequencing of B. japonicum 5873 and other strain related sequences from NCBI, as well as the differential fragments of B. japonicum USDA 6^T which is highly homologous to B. japonicum 5873 (the ANI value of the genome is greater than 99.95%), specific PCR primers were designed and obtained. After optimization of PCR reaction conditions and the detection of sensitivity and specificity, a rapid detection method for B. japonicum 5873 was established. Then, using pot experiment, B. japonicum 5873 was mixed with other rhizobium strains and inoculated in the soybean rhizosphere, and the above method was used to evaluate the competitive nodulation ability of B. japonicum 5873.【Result】A set of species-specific primers 4-4 and Q1 (4-4-F 5′-GATAAGGCCACGGGTGAACA-3′/4-4-R 5′-CACTCGATAAGCTCCGCTGT-3′ and Q1-F 5′-CCGGTCGTGACTGGAATGAT-3′/Q1-R 5′-TCGAGGCCTACAAGAACGTC -3′), were successfully designed to selectively amplify 355 and 218 bp amplicon from B. japonicum 5873. Well specificity was demonstrated by reference strains, from which only the targeted bands of B. japonicum 5873 were observed. Amplifications were performed in a 25 μL reaction mixture, which contained Premix Taq^TM 12.5 μL, template DNA (15 ng of genomic DNA), primers 4-4 and Q1 (1.0 μL, respectively). The PCR cycling consisted of one cycle of 5 min at 95 ℃ and 30 cycles of 45 s at 94 ℃, 45 s at 61 ℃, and 60 s at 72 ℃. A final extension step was run for 10 min at 72 ℃. The sensitivity was 1 850 CFU/µL. In addition, the method mentioned above was successfully used to evaluate the competitive nodulation ability of B. japonicum 5873, which was consistent with the results of traditional BOX-PCR identification.【Conclusion】The established rapid detection method can directly use the bacterial solution or squeezed nodules as templates, which will eliminate laborious tasks, such as processes of nodule isolation, purification, culture, DNA extraction, sequencing and sequence comparison. It only takes a few hours to accurately detect B. japonicum 5873, which provides a reference for product quality detection and competitive nodulation ability evaluation of microbial fertilizers.

Key words: Bradyrhizobium japonicum, strain-specific primer pair, PCR amplification

马鸣超, 姜昕, 王鹏辉, 关大伟, 李俊. 大豆根瘤菌菌株5873 PCR快速检测方法的建立及应用效果评价[J]. 中国农业科学, 2023, 56(14): 2751-2760.

MA MingChao, JIANG Xin, WANG PengHui, GUAN DaWei, LI Jun. Rapid Identification for Bradyrhizobium japonicum 5873 by PCR and Its Evaluation in Application[J]. Scientia Agricultura Sinica, 2023, 56(14): 2751-2760.

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种Species	菌株编号No. of strain
大豆根瘤菌 Bradyrhizobium japonicum	USDA 6^T, 5821, 5841, 5876, 5547, 5588, 5665, 5129, 4892, 5452, 5599, 5016, 5437, 4129, 4470, 4530, 4438, 5248, 4784, 5528, 5412, 5260, 4503, 5039, 4248, 5240, 5190, 5742, 4855, 4794, 4291, 4698, 5128, 4770, 4296, 4207, 4543
有效慢生根瘤菌Bradyrhizobium diazoefficiens	USDA 110^T, 5119
埃氏慢生根瘤菌Bradyrhizobium elkanii	5136, 5161, 5109
弗氏中华根瘤菌Sinorhizobium fredii	USDA 205^T, 4475
奥氏根瘤菌Rhizobium alamii	4370

引物Primer	序列Sequence (5′-3′)	长度Length (bp)	Tm	GC (%)
4-4 Forward primer	GATAAGGCCACGGGTGAACA	20	60.04	55.00
4-4 Reverse primer	CACTCGATAAGCTCCGCTGT	20	59.90	55.00
Q1 Forward primer	CCGGTCGTGACTGGAATGAT	20	59.82	55.00
Q1 Reverse primer	TCGAGGCCTACAAGAACGTC	20	59.48	55.00

复合接种 Co-inoculation	占瘤率Nodule occupancy (%)
复合接种 Co-inoculation	快速检测技术 Rapid identification	BOX-PCR
B. diazoefficiens USDA 110^T+ B. japonicum 5873	78.3±1.2	76.9±2.2
B. japonicum 5136+B. japonicum 5873	73.7±1.6	76.0±1.7
B. japonicum 5665+B. japonicum 5873	84.2±0.9	82.1±2.5
B. japonicum 5841+B. japonicum 5873	87.0±1.6	85.5±2.6
B. japonicum 5821+B. japonicum 5873	95.2±1.9	92.9±0.9
B. japonicum 5119+B. japonicum 5873	87.5±1.3	85.8±1.9