采用重叠PCR构建高灵敏度酵母细胞传感器评估遗传毒性化合物  

A highly sensitive yeast cell sensor constructed by overlap PCR was used to evaluate genotoxic compounds

作　　者：何颖[1,2] 夏星雅 魏嘉利 郑枫[1,2] HE Ying;XIA Xingya;WEI Jiali;ZHENG Feng(Department of Pharmaceutical Analysis;Key Laboratory of Drug Quality Control and Pharmacovigilance,Ministry of Education,China Pharmaceutical University,Nanjing 210009,China)

机构地区：[1]中国药科大学药物分析学教研室 [2]中国药科大学教育部药品安全与预警重点实验室,南京210009

出　　处：《中国药科大学学报》2021年第2期236-244,共9页Journal of China Pharmaceutical University

基　　金：国家自然科学基金资助项目(No.82073815)。

摘　　要：采用重叠PCR(overlap PCR)方法构建了pdr5与snq2基因敲除组件,研究了pdr5、snq2基因突变对酵母细胞传感器评估遗传毒性的影响。考察了野生型、pdr5单基因突变、snq2单基因突变与pdr5、snq2双基因突变酵母细胞传感器暴露于系列浓度甲磺酸甲酯(MMS)、甲磺酸乙酯(EMS)、顺铂、4-硝基喹啉-N-氧化物(4NOQ)、5-氟尿嘧啶(5-FU)、羟基脲、水杨酸和葡萄糖溶液24 h后的细胞生长抑制情况与16 h后的荧光诱导情况。研究结果表明,overlap PCR方法能够高效率构建基因突变酵母细胞传感器;snq2单基因突变与pdr5、snq2双基因突变细胞传感器检测遗传毒性的准确度为100%,高于野生型与pdr5单基因突变细胞传感器(87.5%);pdr5、snq2双基因突变酵母细胞传感器表现出最高的遗传毒性检测灵敏度,为构建高准确度与灵敏度的酵母细胞传感器提供了思路与方法,为酵母细胞膜转运蛋白基因pdr5与snq2的进一步功能研究奠定了基础。pdr5 and snq2 gene knockout was constructed by overlap PCR,and the effects of pdr5 and snq2 muta⁃tions on the accuracy and sensitivity of RNR2 promoter-regulated yeast cell sensors in detecting genotoxic compounds were studied.The yeast cell sensors of wild-type,single-gene mutation of pdr5,single-gene mutation of snq2,and double-gene mutation of pdr5 and snq2 were studied.The cell growth inhibition and the fluores⁃cence induction factor of the yeast cell sensors exposed to a series of concentrations of methyl methanesulfonate(MMS),ethyl methanesulfonate(EMS),cisplatin,4-nitroquinoline-N-oxide(4NOQ),5-fluorouracil(5-FU),hydroxy⁃urea,salicylic acid and glucose solution were investigated.The results showed that overlap PCR method could efficiently construct the mutant yeast cell sensor.The accuracy of cell sensors of single-gene mutation of snq2 and double-gene mutation of pdr5 and snq2 were both 100%,higher than that of cell sensors of wild-type and single-gene mutation of pdr5(87.5%).The yeast cell sensor of double-gene mutation of pdr5 and snq2 showed the highest sensitivity in detecting genotoxicity.This study provides guidance for the construction of high accuracy and sensitivity yeast cell sensor,and foundation for further functional research of yeast cell membrane transporter gene pdr5 and snq2.

关 键 词：重叠PCR 酵母细胞传感器 RNR2启动子 遗传毒性 

分 类 号：R917[医药卫生—药物分析学]