生物技术进展 ›› 2019, Vol. 9 ›› Issue (4): 384-395.DOI: 10.19586/j.2095-2341.2019.0025

• 研究论文 • 上一篇    下一篇

除草剂2,4-D降解菌株的分离、筛选与鉴定

栗旭阳,黄丽玲,郭倩楠,高如雨,张维,陈明,陆伟,周正富*   

  1. 中国农业科学院生物技术研究所, 北京 100081
  • 收稿日期:2019-03-08 出版日期:2019-07-25 发布日期:2019-04-16
  • 通讯作者: 周正富,副研究员,博士,主要从事极端环境微生物分子生物学研究。E-mail:zhouzhengfu@caas.cn
  • 作者简介:栗旭阳,硕士研究生,主要从事环境微生物基因工程研究。E-mail:lixuyangwsx@163.com。
  • 基金资助:
    国家转基因生物新品种培育重大专项(2016ZX08009003-002);中央级公益性科研院所基本科研业务专项(1610392019006)资助。

Isolating, Screening and Identification Involved in 2,4-D Degradation

LI Xuyang, HUANG Liling, GUO Qiannan, GAO Ruyu, ZHANG Wei, CHEN Ming, LU Wei, ZHOU Zhengfu   

  1. Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2019-03-08 Online:2019-07-25 Published:2019-04-16

摘要: 2,4-二氯苯氧乙酸(2,4-dichlorophenoxyacetic acid,2,4-D)是一类广泛应用于单子叶作物田间杂草防除的除草剂,但其大量施用导致的环境残留已对生态环境造成严重威胁。通过富集培养的方法,从2,4-D污染土壤样品中筛选分离到85株耐受菌,16S rDNA分析显示其分属假单胞菌属(Pseudomonas)、芽孢杆菌属(Bacillus)、赖氨酸杆菌属(Lysinibacillus)、类芽孢杆菌属(Paenibacillus)、短杆菌属(Brevibacillus)、沙雷氏菌属(Serratia)、甲基杆菌属 (Methylobacterium)、贪铜菌属(Cupriavidus)、红球菌属(Rhodococcus)、拉恩氏菌属(Rahnella)、葡萄球菌属(Staphylococcus)、阿氏肠杆菌属(Enterobacter)和分支杆菌属(Mycobacterium)。其中一株假单胞菌属细菌2,4-D最高耐受浓度达8 g/L。选取部分菌株以2,4-D为唯一碳源培养,通过检测2,4-D降解产物(4-aminoantipyrine显色测量和高效液相色谱)测定菌株的降解能力,并利用质谱(LC/MS、LC-QTOF)检测其代谢产物。共确定11株菌株具有较高的2,4-D降解能力,其中菌株L1、L2、L4和L6在培养72 h后2,4-D的利用率分别为17.3%、28.07%、38.97%和49.02%;LC/MS和LC-QTOF分析检测表明上述菌株将2,4-D脱去侧链基团转化为酚类物质,推测其降解主要为2,4-二氯苯酚(2,4-DCP)途径。

关键词: 除草剂, 2, 4-D, 分离鉴定, 除草剂降解, 降解途径

Abstract: 2,4-dichlorophenoxyacetic acid (2,4-D) is a systemic and broad-spectrum foliar herbicide which can control  broadleaf weeds. But because of its frequent use, 2,4-D has resulted in considerable pollution to water and soil and thus threatened the ecological environment and human health. In this paper, 85 strains were isolated and screened from 2,4-D contaminated soil samples (inorganic salt medium with 2,4-D as the sole carbon source) by enrichment culture. 16S rDNA analysis showed that it belongs to Pseudomonas, Bacillus, Lysinibacillus, Paenibacillus, Brevibacillus, Serratia, Methylobacterium, Cupriavidus, Rhodococcus, Larender (Rahnella), Staphylococcus, Enterobacter and Mycobacterium. The strain herbicide tolerance test showed that Pseudomonas can tolerate a higher concentration of 2,4-D with a maximum tolerated concentration of 8 g/L. Some strains were selected to be cultured with 2,4-D as the sole carbon source. The degradation ability of the strain was determined by detecting 2,4-D degradation products (4-aminoantipyrine colorimetric measurement and high performance liquid chromatography), the LC/MS and LC-QTOF were used to detected its metabolites. A total of 11 strains were identified with highly 2,4-D degradation ability. The utilization rate of L1, L2, L4 and L6 cultured for 72 h was 17.3%, 28.07%, 38.97% and 49.02% respectively. The preliminary analysis and structural prediction of the metabolites of the above selected strains were carried out by LC/MS and LC-QTOF. The results showed that a total of 4 strains could convert 2,4-D de-chained groups into phenolic substances, presumably the product was 2,4-dichlorophenol (2,4-DCP).

Key words: herbicide, 2,4-D, isolation and identification of the strains, herbicide degradation, degradation pathway