第41卷第1期2023年2月广东医科大学学报JOURNALOFGUANGDONGMEDICALUNIVERSITYVol.41No.1Feb.20237酵母PHO8与PHO13双基因缺失酵母菌株构建崔晓静1，2，杨晓迪1，2，袁頔3，王俊芳4，崔红晶1，4*（广东医科大学1.医学分子诊断重点实验室衰老研究所；2.医学技术学院；3.第二临床医学；4.基础医学院，广东东莞523808）摘要：目的构建碱性磷酸酶PHO8与PHO13双基因缺失酵母菌株。方法依据基因同源重组原理，采用一步基因置换法构建编码Pho8蛋白N端60个氨基酸残基（包括跨膜结构域）缺失的PHO8基因缺失酵母（pho8∆60）菌株；线性化载体pRS303-pho13-ko转化pho8∆60菌株，进一步缺失PHO13基因，构建pho8∆60和PHO13双基因缺失（pho8∆60pho13∆）菌株；通过缺陷型培养基筛选方法，PCR鉴定阳性克隆基因组DNA。结果pho8∆60菌株电泳条带为1151bp和180bp，pho8∆60pho13∆菌株电泳条带分别为1872bp和174bp。结论成功构建pho8∆60pho13∆酵母菌株。关键词：酿酒酵母；碱性磷酸酶；自噬；衰老中图分类号：Q255文献标志码：A文章编号：2096-3610（2023）01-0007-05Constructionofyeaststrainswithdouble-genedeletionofPHO8andPHO13CUIXiao-jing1,2,YANGXiao-di1,2,YUANDi3,WANGJun-fang4,CUIHong-jing1,4*(1.TheKeyLaboratoryofMedicalMolecularDiagnostics,InstituteofAgingResearch;2.CollegeofMedicalTechnology;3.TheSecondSchoolofClinicalMedicine;4.SchoolofBasicMedicine,GuangdongMedicalUniversity,Dongguan523808,China)Abstract:ObjectiveToconstructayeaststrainwithdouble-genedeletionofalkalinephosphatasePHO8andPHO13.MethodsAccordingtotheprincipleofgenehomologousrecombination,theencodingsequencesoftheN-terminal60aminoacidresidues(includingthetransmembranedomain)ofPho8proteinwasdeletedbyone-stepgenedisruption,resultingthatPHO8deletion(pho8∆60)strainwasestablished.Thedouble-genedeletion(pho8∆60pho13∆)strainwasestablishedfromthelinearvectorpRS303-pho13-kotransformingintothepho8∆60strain,resultingthatthePHO13genewasfurtherdeletedinthepho8∆60strain.PCRwasusedtoidentifythegenomicDNAofthepositiveclonesfromthedefectivemedium.ResultsThe1151bpand180bpbandsweredetectedbyagarosegelelectrophoresisfromthePCRproductsofthegenomicDNAinthepho8strain,and1872bpand174bpbandsweredetectedinthepho...
