红球菌R04联苯/多氯联苯代谢相关调控蛋白RHOGL007659的生理功能

Physiological function of regulatory protein RHOGL007659 involved in metabolism biphenyl/polychlorinated biphenyl in Rhodococcus sp. R04

【目的】研究红球菌(Rhodococcus sp.)R04调控蛋白RHOGL007659的生理功能及其缺陷菌株的代谢特性,初步探究红球菌R04降解联苯的调控机制。【方法】通过基因同源重组敲除红球菌R04联苯代谢相关基因RHOGL007659。比较红球菌R04(野生型)和缺陷型菌株R04Δ7659(基因RHOGL007659缺陷型的R04)在不同碳源培养下的生长情况,HPLC分析R04和R04Δ7659转化联苯的能力。提取R04和R04Δ7659的总RNA,实时荧光定量PCR检测联苯降解关键基因的转录表达。纯化Bph B(联苯降解脱氢酶)和Bph D(联苯降解水解酶),制备多克隆抗体。Western blot分析Bph B和Bph D蛋白在R04和R04Δ7659中的表达水平。【结果】获得了RHOGL007659基因的缺陷型菌株R04Δ7659,与R04相比,R04Δ7659在联苯培养条件下的生物量趋近于零。HPLC分析表明,RHOGL007659基因的缺失使红球菌R04丧失转化联苯的能力。实时荧光定量PCR结果表明,在联苯培养条件下,缺失RHOGL007659后的R04,其联苯降解关键基因均有不同程度的下调表达。Western blot分析显示RHOGL007659缺失后,联苯降解关键酶Bph B和Bph D表达量均降低,这与实时荧光定量PCR结果相一致。【结论】RHOGL007659是红球菌R04联苯降解关键基因簇的调控蛋白,该蛋白对红球菌R04代谢联苯过程具有正调控作用。

[Objective] The physiological function of regulatory protein RHOGL007659 in Rhodococcus sp. R04 and the metabolic properties of the mutant strain were investigated to explore its regulation mechanism of biphenyl degradation. [Methods] The RHOGL007659 gene was knocked out by homologous recombination, and the growth of the wild-type strain and deficient mutant strain in different carbon sources were compared. The transformation of biphenyl by the wild-type strain and the deficient mutant strain was detected by HPLC. Total RNAs were extracted from the wild-type strain and deficient mutant strain, and quantitative RT-PCR was carried out to analyze the transcriptional variation of bph A, bph B, bph C and bph D between the wild-type strain and the deficient mutant strain. Bph B and Bph D were expressed in Escherichia coli BL 21（DE3） and polyclonal antibodies were prepared after purification. Bph B and Bph D expression levels in the wild strain and deficient mutant strains were detected by Western blot. [Results] The deficient mutant strain R04Δ7659 of RHOGL007659 gene was obtained. The biomass of the deficient mutant strain significantly reduced in biphenyl culture, and almost did not grow. HPLC analysis showed that the deletion of RHOGL007659 gene resulted in inability to transform biphenyl by Rhodococcus sp. R04. Q-RT PCR experiment disclosed that the key genes of biphenyl degradation were down regulated in different degrees under biphenyl culture condition in the RHOGL007659 deficient mutant strain, the same results were obtained by Western blot. [Conclusion] RHOGL007659 is a regulatory protein of the upper biphenyl degradation gene in Rhodococcus sp. R04, and functions as a positive regulatory factor of the metabolism of biphenyl by Rhodococcus sp. R04.