一株源自新疆盐碱棉田土壤吡蚜酮降解菌株的分离鉴定及其降解途径

Isolation, identification, and degradation pathway prediction of a pymetrozine-degrading strain from a saline cotton field in Xinjiang

摘要:

【背景】 吡蚜酮是一种在碱性条件下稳定且具有潜在致癌性的杀虫剂，现有吡蚜酮降解菌株大都是从高污染环境或农药生产厂环境中分离而来。【目的】 获取能适用于碱性土壤环境修复吡蚜酮污染的菌株。【方法】 采集新疆长期连作棉田土壤，通过先富集后纯化的方法分离吡蚜酮降解菌，然后开展其降解特性、途径及关键基因等研究。【结果】 获得1株吡蚜酮高效降解菌3BR11-2，将该菌以2% (OD₆₀₀=1.0)的接种量加入装有100 mL吡蚜酮浓度为50 mg/L、pH 8.0基础无机盐培养基的250 mL三角瓶中，37 ℃、120 r/min培养36 h时可将吡蚜酮完全降解。超高效液相色谱四极杆飞行时间串联质谱仪(ultra high performance liquid chromatography quadrupole time-of-flight mass spectrometry, UHPLC-Q-TOF/MS)检测菌株3BR11-2在降解吡蚜酮过程中产生了3-吡啶甲醇、烟酸(nicotinic acid, NA)和6-羟基烟酸(6-hydroxynicotinic acid, 6HNA) 3种代谢产物。根据16S rRNA基因序列对比分析，该菌株与简单类诺卡氏菌(Nocardioides simplex)相似度达99.29%。以菌株3BR11-2基因组为模板，依据菌株假单胞菌(Pseudomonas sp.) BYT-1吡蚜酮水解酶基因pyzH设计引物PCR扩增得到846 bp片段，经测序分析发现该片段与基因pyzH相似度为99.65% (843/846)。通过氨基酸序列比对，菌株3BR11-2基因pyzH表达的吡蚜酮水解酶第195丙氨酸残基A和第212半胱氨酸C转变为丝氨酸残基S，第263丙氨酸残基A转变为苏氨酸残基T。【结论】 本研究丰富了吡蚜酮高效降解菌资源，有利于盐碱特殊环境下吡蚜酮的生物修复应用。

Abstract:

[Background] Pymetrozine is a pesticide that exhibits stability under alkaline conditions and possesses potential carcinogenic properties. Most existing strains capable of degrading pymetrozine have been isolated from highly contaminated environments or pesticide production facilities. [Objective] To obtain strains suitable for bioremediation of pymetrozine-contaminated alkaline soil. [Methods] We collected soil samples from long-term continuous cropping cotton fields in Xinjiang. Pymetrozine-degrading bacteria were isolated by enrichment followed by purification. Subsequently, their degradation characteristics, metabolic pathways, and key genes were investigated. [Results] An efficient pymetrozine-degrading bacterial strain 3BR11-2 was successfully isolated. This strain could completely degrade pymetrozine when it was inoculated in a 250 mL Erlenmeyer flask containing 100 mL basic inorganic salt medium (50 mg/L pymetrozine, pH 8.0) at an inoculum size of 2% (OD₆₀₀=1.0) and cultured at 37 ℃ and 120 r/min for 36 h. UHPLC-Q-TOF/MS revealed the production of three metabolites during the degradation process by strain 3BR11-2. These metabolites were identified as 3-pyridinemethanol, nicotinic acid (NA), and 6-hydroxynicotinic acid (6HNA). The phylogenetic analysis based on 16S rRNA gene sequence comparison showed that the strain shared 99.29% similarity with Nocardioides simplex. With the genome of strain 3BR11-2 as a template, primers were designed based on the pymetrozine hydrolase gene pyzH from strain Pseudomonas sp. BYT-1. The gene pyzH was then amplified by PCR, which yielded a fragment of 846 bp. This fragment shared the similarity of 99.65% (843/846 nucleotides) with pyzH. Furthermore, amino acid sequence alignment indicated that the pymetrozine hydrolase expressed by pyzH of strain 3BR11-2 had three residue changes: A195S, C212S, and A263T. [Conclusion] This study enriches the repertoire of highly efficient pymetrozine-degrading bacteria and holds promise for the application of bioremediation strategies targeting pymetrozine in unique saline-alkali environments.