中华皮肤科杂志
中華皮膚科雜誌
중화피부과잡지
Chinese Journal of Dermatology
2014年
8期
559-562
,共4页
陈昕宜%杨国玲%刘建雯%刘金鹏%张芳芳%赵晓宣
陳昕宜%楊國玲%劉建雯%劉金鵬%張芳芳%趙曉宣
진흔의%양국령%류건문%류금붕%장방방%조효선
犬小孢子菌%RNA干扰%基因,PQ-LRP%基因沉默
犬小孢子菌%RNA榦擾%基因,PQ-LRP%基因沉默
견소포자균%RNA간우%기인,PQ-LRP%기인침묵
Microsporum canis%RNA interference%Genes,PQ-LRP%Gene silencing
目的 构建犬小孢子菌PQ-LRP基因RNA干扰载体,探讨RNA干扰载体对犬小孢子菌PQ-LRP基因表达的影响.方法 用根癌农杆菌介导的T-DNA插入突变技术,加入多克隆位点,引入潮霉素抗性基因,先后构建PUC-PLULT、PCB309-PLULT载体.应用实时PCR方法检测PCB309-PLULT载体对犬小孢子菌PQ-LRP基因表达的干扰作用.结果 构建了中间载体PUC-PLUT、PUC-PLULT,并通过PCR、基因测序进行鉴定.成功构建了长为8 825 bp的干扰载体PCB309-PLULT,并通过酶切测定为该目的载体;筛选出犬小孢子菌转化子的潮霉素最佳浓度为300 mg/L;用实时定量PCR方法对犬小孢子菌PQ-LRP基因干扰前后表达量进行鉴定:以干扰前PQ-LRP相对表达量1.0为标准,干扰后PQ-LRP相对表达量为0.39,干扰后下调61％.结论 干扰载体PCB309-PLULT可以明显抑制犬小孢子菌PQ-LRP基因的表达.
目的 構建犬小孢子菌PQ-LRP基因RNA榦擾載體,探討RNA榦擾載體對犬小孢子菌PQ-LRP基因錶達的影響.方法 用根癌農桿菌介導的T-DNA插入突變技術,加入多剋隆位點,引入潮黴素抗性基因,先後構建PUC-PLULT、PCB309-PLULT載體.應用實時PCR方法檢測PCB309-PLULT載體對犬小孢子菌PQ-LRP基因錶達的榦擾作用.結果 構建瞭中間載體PUC-PLUT、PUC-PLULT,併通過PCR、基因測序進行鑒定.成功構建瞭長為8 825 bp的榦擾載體PCB309-PLULT,併通過酶切測定為該目的載體;篩選齣犬小孢子菌轉化子的潮黴素最佳濃度為300 mg/L;用實時定量PCR方法對犬小孢子菌PQ-LRP基因榦擾前後錶達量進行鑒定:以榦擾前PQ-LRP相對錶達量1.0為標準,榦擾後PQ-LRP相對錶達量為0.39,榦擾後下調61％.結論 榦擾載體PCB309-PLULT可以明顯抑製犬小孢子菌PQ-LRP基因的錶達.
목적 구건견소포자균PQ-LRP기인RNA간우재체,탐토RNA간우재체대견소포자균PQ-LRP기인표체적영향.방법 용근암농간균개도적T-DNA삽입돌변기술,가입다극륭위점,인입조매소항성기인,선후구건PUC-PLULT、PCB309-PLULT재체.응용실시PCR방법검측PCB309-PLULT재체대견소포자균PQ-LRP기인표체적간우작용.결과 구건료중간재체PUC-PLUT、PUC-PLULT,병통과PCR、기인측서진행감정.성공구건료장위8 825 bp적간우재체PCB309-PLULT,병통과매절측정위해목적재체;사선출견소포자균전화자적조매소최가농도위300 mg/L;용실시정량PCR방법대견소포자균PQ-LRP기인간우전후표체량진행감정:이간우전PQ-LRP상대표체량1.0위표준,간우후PQ-LRP상대표체량위0.39,간우후하조61％.결론 간우재체PCB309-PLULT가이명현억제견소포자균PQ-LRP기인적표체.
Objective To build a RNA interference vector for PQ-loop repeat protein (LRP) gene,and to evaluate the effect of the vector on the expression of PQ-LRP gene in Microsporum canis.Methods The PUC-PLULT and PCB309-PLULT vectors were constructed sequentially by using Agrobacterium tumefaciens-mediated T-DNA insertional mutagenesis,adding multiple cloning sites,and introducing the hygromycin-resistance gene.Microsporum canis was transformed with the PCB309-PLULT vector followed by a series of passages and hygromycin selection.Real-time quantitative PCR was performed to measure the expression of PQ-LRP gene in Microsporum canis before and after transformation.Results The intermediate vectors PUC-PLUT and PUC-PLULT were constructed and identified by PCR and gene sequencing.The 8 825-bp interference vector PCB309-PLULT was successfully built and confirmed by enzyme digestion.The optimum concentration of hygromycin for screening for Microsporum canis transformants was determined as 300 mg/L.The mRNA expression level of PQ-LRP was decreased by 61％ in the transformants as compared with untransformed Microsporum canis (0.39 vs.1.00).Conclusion The constructed PCB309-PLULT interference vector can effectively inhibit the expression of PQ-LRP gene in Microsporum canis.
