小番茄内生菌耐药基因检测及种植模型中GFP标记菌转移研究

摘要

[目的]调查广州市售小番茄内生菌中常用抗菌药物耐药基因流行情况及基于绿色荧光蛋白(GFP)标记的大肠埃希菌人工栽种模型探讨耐药基因转移进入小番茄果实内的可能性.[方法]采用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)法分离鉴定市售小番茄内生菌,PCR定性检测内生菌中氯霉素类(cmlA)、四环素类(tetA和tetM)、磺胺类(sul1、sul2和sul3)和喹诺酮类(oqxA 、oqxB 、qnrB 、qnrS和qepA)等药物耐药基因的流行情况;采用GFP标记–大肠埃希菌人工种植模型检测小番茄果实、叶片和根系中目标GFP菌,探讨内生菌及GFP标记进入小番茄内部的可能性.[结果]市售小番茄分离的52株内生菌菌株中,肠杆菌属Enterobacter和克雷伯菌属Klebsiella占有较高比例,几乎所有菌株均携带oqxB基因(总阳性率达92.31％),在人工种植模型第27天采集的果实中分离出含有GFP标记的内生菌.[结论]小番茄内生菌中oqxB基因最为流行,耐药基因可通过根系浇灌的方式转移到小番茄果实内.%[Objective]To investigate the prevalence of antibiotic resistant genes of endophytic bacteria in cherry tomatoes sold in Guangzhou,and discuss the possibilities of resistant genes transferring into cherry tomatoes based on GFP-tagged Escherichia coli artificial plantation model.[Method]The endophytic bacteria in commercially available cherry tomatoes was identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS).Polymerase chain reaction was used to qualitatively detect the prevalence of resistant genes including chloromycetin(cmlA),tetracycline(tetA and tetM),sulfonamide(sul1,sul2 and sul3)and quinolones(oqxA,oqxB,qnrB,qnrS and qepA).The GFP-tagged E.coli artificial plantation model was used to detect target GFP-bacteria in cherry tomato fruits,leaves and roots,and explore the possibilities of entophytic bacteria and GFP tag transferring into cherry tomatoes.[Result]Enterobacter and Klebsiella accounted for a relatively high proportion in 52 endophytic bacterial strains,and almost all strains carried oqxB gene with a total positive rate of 92.31％.The endophytic bacterial strain containing GFP tag was isolated from the fruit collected on the 27th day in artificial plantation model.[Conclusion]oqxB gene is most prevalent in cherry tomato endophytic bacteria.The antibiotic resistant genes can transfer into cherry tomato fruits through root irrigation.