不动杆菌3-苯氧基苯甲酸降解基因的克隆与表达

Cloning and Expression of 3-Phenoxybenzoic Acid Biodegrading Gene from Acinetobacter sp.

3-苯氧基苯甲酸(3-PBA)作为拟除虫菊酯类农药的非特异性降解中间产物,具有抗雌激素特性,可扰乱生物体内分泌系统,比菊酯类农药迁移更广,半衰期更长,生物毒性更大,是拟除虫菊酯类农药在生物体中暴露的标志。通过构建4-D菌(Acinetobacter sp.)基因组文库,混合池驯化筛选得到4-D菌中降解3-PBA的关键酶基因,其开放阅读框为921 bp,编码306个氨基酸,Genbank登录号为KR024742。经同源比对和酶活验证,证实该酶为邻苯二酚双加氧酶。根据该ORF序列设计引物,引物两端分别加上NdeⅠ和HindⅢ酶切位点,以4-D菌基因组DNA为模板,成功克隆到D34基因序列。构建表达载体p ET-21b-D34并转化进宿主大肠杆菌BL21(DE3),经IPTG(0.1 mmol/L)诱导后,表达重组菌对3-PBA降解率为18.7%。研究结果为3-PBA污染的微生物环境修复提供了理论参考。

3-Phenoxybenzoic acid（ 3-PBA） is known as non-specific intermediate products of pyrethriods pesticide,which has antiestrogenic activity,and can disturb the endocrine system in vivo. 3-PBA has wider migration,longer half-life period,and higher biotoxicity than the pyrethroid pesticide,so it has been used as a marker for pyrethroids exposure. With the contruction and screening of 4-D（ Acinetobacter sp.） genomic library,the key gene having a ORF of 921 bp and encoding an amino acid of 306 aa for degrading 3-PBA was screened,its Gen Bank accession number was KR024742. Homolog comparison results and substrate experiments inferred that it was catechol dioxygenase. The primer was designed on the authority of opening reading frames（ ORF）and added with restriction sites of NdeⅠ and HindⅢ. With genomic DNA of 4-D as template,the D34 gene was cloned. The recombinant expression vector p ET-21b-D34 plasmid was constructed and transformed into competent cell BL21（ DE3）. After inducing by IPTG（ 0. 1 mmol / L）,the degration rate of 3-PBA was 18. 7%. Results provided theory reference for microbial environmental restoration of 3-PBA population.