首页 > 核心刊物 > 中国生物工程杂志 2018.1 Vol 38, No.1 > 地塞米松降解新基因的探讨

地塞米松降解新基因的探讨

张进,斯丹,杨致邦,熊玉霞,马廉举,李津阳,蒋仁举

1 重庆医科大学基础医学院病原生物学教研室 重庆 400016 2 重庆医科大学药学实验教学中心 重庆 400016 3 重庆医科大学临床医学专业一系 重庆 400016

 
Key Words:  伯克霍尔德菌; 地塞米松; 降解; 基因; 实时荧光定量PCR
 

Abstract

根据新发现的地塞米松降解菌Burkholderia sp.CQ001(B.CQ001)全基因组测序和生物信息学分析,筛选疑似与地塞米松代谢相关的基因。提取B.CQ001总RNA逆转录为cDNA,以cDNA为模板,利用PCR技术快速克隆经实时荧光定量PCR技术(RT-qPCR)验证筛选出的新基因,测序鉴定后连接原核表达载体pET-28a-c(+),构建重组质粒pET-28a-Ivd。将pET-28a-Ivd转化感受态B.CQ001,高效液相色谱(HPLC)验证表达菌降解能力的提升。 从B.CQ001基因组中筛选出3个未知基因,分别为ORF05499、ORF05827、ORF06535,其表达产物分别为过氧化物还原酶、甾酮异构酶、异戊酰辅酶A脱氢酶;RT-qPCR分析显示,3个目的基因在地塞米松诱导后均有不同程度表达上调,基因ORF06535表达上调明显;在B.CQ001中过表达基因ORF06535,HPLC检测显示,表达菌对地塞米松磷酸钠和地塞米松的降解率可达到89.0%和80.0%,相比原菌B.CQ001有明显提升。 在B.CQ001中发现新的地塞米松降解相关基因ORF06535,并完成基因克隆和功能验证,为制备地塞米松及甾体激素污染的生物修复剂提供了新的基因信息。


 

Abstract

To investigate the new genes associated with dexamethasone metabolism in order to provide an idea for the construction of an efficient and stable dexamethasone degrading engineering bacterium. Target genes which were suspectly associated with dexamethasone metabolism were screened according to the results of the whole genome sequencing and bioinformatics analysis of the newly discovered dexamethasone degrading bacteria Burkholderia sp.CQ001 (B.CQ001). Total RNA of B.CQ001 was extracted from B.CQ001wasprocessed by RT-PCR to produce cDNA. Using cDNA as a template, the new gene was rapidly cloned subsequently by PCR technology which was verified by real-time fluorescence quantitative PCR (RT-qPCR), then connected with prokaryotic expression vector pET-28a-c (+), after DNA sequencing. The recombinant plasmid pET-28a-Ivd was transformed into B.CQ001. Using high performance liquid chromatography (HPLC) to evaluate the biodegradability of the expressed bacteria for dexmethas one sodium phosphate. Three unknown genes were screened out from B.CQ001: ORF05499, ORF05827, ORF06535, and their expressed products were: peroxiredoxin, ketosteroid isomerase, isovaleryl coenzyme A dehydrogenase; Real time quantitative PCR analysis showed that the expression levels of three genes were upregulated in different degrees after induced by dexamethasone ,especially gene ORF06535; HPLC showed that the degradation of dexamethasone sodium phosphate and dexamethasone rate can reach to 89% and 80% after overexpression of ORF06535 in B.CQ001. Compared with the original strain B.CQ001, the degradation ability had improved significantly. New gene ORF06535 associated with dexamethasone degradation was discovered in B.CQ001 which had been cloned and functional verified. It provides new genetic information for preparation of dexamethasone and steroid hormone bioremediation.


期刊简介
 
主办单位:
中国生物工程学会(CSBT)、中国生物技术发展中心(CNCBD)、中国科学院文献情报中心。
编委会:
最新一届编辑委员会由国家生物技术研究开发计划的评审专家及有关院士组成,代表中国生物技术的最高学术水平。
编委会主任:陈志南
通信地址:北京市中关村北四环西路33号《中国生物工程杂志》编辑部(100190)
国内刊号:CN11-4816/Q
国际刊号:ISSN1671-8135
邮发代号:82-673