地塞米松降解新基因的探讨

doi:10.13523/j.cb.20180102

中国生物工程杂志

2018, Vol. 38

Issue (1): 15-24 DOI: 10.13523/j.cb.20180102

研究报告

地塞米松降解新基因的探讨

张进¹,斯丹¹,杨致邦¹,熊玉霞¹,马廉举²,李津阳³,蒋仁举¹(

)

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

Study on New Genes Associated with Dexamethasone Degradation

Jin ZHANG¹,Dan SI¹,Zhi-bang YANG¹,Yu-xia XIONG¹,Lian-ju MA²,Jin-yang LI³,Ren-ju JIANG¹(

)

1 Department of Pathogenic Biology, Basic Medical College, Chongqing Medical University, Chongqing 400016,China
2 Center of Pharmacy Experimental Teaching, College of Pharmacy, Chongqing Medical University, Chongqing 400016, China
3 Clinical Medical College, Chongqing Medical University, Chongqing 400016, China

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摘要： 目的

探讨地塞米松降解代谢新基因,为构建高效、稳定的地塞米松降解基因工程菌提供思路。

方法

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。

结果

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。

结论

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。

关键词： 伯克霍尔德菌; 地塞米松; 降解; 基因; 实时荧光定量PCR

Abstract: Objective:

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.

Methods:

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.

Results:

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.

Conclusion:

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.

Key words: Burkholderia Dexamethasone Degradation Gene RT-qPCR

收稿日期: 2017-09-02 出版日期: 2018-01-31

ZTFLH:

Q78

基金资助: 重庆市渝中区科委科技计划资助项目(20160110)

作者简介: 通讯作者蒋仁举, E-mail: 743322835@qq.com

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引用本文:

张进,斯丹,杨致邦,熊玉霞,马廉举,李津阳,蒋仁举. 地塞米松降解新基因的探讨[J]. 中国生物工程杂志, 2018, 38(1): 15-24.

Jin ZHANG,Dan SI,Zhi-bang YANG,Yu-xia XIONG,Lian-ju MA,Jin-yang LI,Ren-ju JIANG. Study on New Genes Associated with Dexamethasone Degradation. China Biotechnology, 2018, 38(1): 15-24.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180102 或 https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I1/15

表1 B. CQ001 3个未知基因实时荧光定量PCR引物

表2 待验证的B.CQ001 3个未知基因

图1 待验证的B. CQ001 3个未知基因基因簇排列情况

图2 待验证的B. CQ001 3个未知基因基因位置图

图3 目的基因ORF06535氨基酸序列与降解相关蛋白41kDa 5条肽段氨基酸序列比对结果

表3 待验证的B. CQ001 3个未知基因生物信息学分析结果

表3(续表) 待验证的B. CQ001 3个未知基因生物信息学分析结果

图4 RT-qPCR技术验证地塞米松诱导组和蔗糖无机盐对照组3个基因差异表达

图5 基因ORF06535 PCR产物的琼脂糖凝胶电泳结果

图6 pUCm-Ivd琼脂糖凝胶电泳结果

图7 pET-28a-Ivd琼脂糖凝胶电泳结果

图8 驯化冻存菌B.CQ001和表达菌B.CQ002降解过程HPLC色谱图

图9 B.CQ001和B.CQ002地塞米松磷酸钠和地塞米松的降解曲线

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