白念珠菌RAS1基因高表达菌株的构建及鉴定

›› 2016, Vol. 7 ›› Issue (2): 72-75.

白念珠菌RAS1基因高表达菌株的构建及鉴定

章珍珍¹,夏金萍¹,²,马鸣³,魏昕³

1. 南京医科大学口腔医学院
2.
3. 江苏省口腔医院

收稿日期:2016-01-08 修回日期:2016-05-14 出版日期:2016-06-25 发布日期:2016-06-22
通讯作者: 魏昕 E-mail:weixinart@163.com
基金资助:
国家自然科学基金;江苏高校优势学科建设工程资助项目;江苏省高校“青蓝工程”

Construction and identification of RAS1-over strain of Candida albicans

Received:2016-01-08 Revised:2016-05-14 Online:2016-06-25 Published:2016-06-22

摘要/Abstract

摘要： 目的：构建白念珠菌RAS1基因高表达菌株pCaEXP-RAS1-CAI4。方法：将白念珠菌RAS1基因的开放阅读框（ORF）置于高表达载体pCaEXP的启动子MET3之后，构建高表达质粒载体pCaEXP-RAS1。将整合的重组质粒转化进入大肠杆菌DH5α，经扩增后采用质粒抽提试剂盒大量制备pCaEXP-RAS1质粒。单酶切线性化质粒，将线性化的pCaEXP-RAS1经醋酸锂法转化入白念珠菌CAI4细胞内，随后在SD-ura-met-cys选择性培养基获得转化子，构建pCaEXP-RAS1-CAI4菌株。挑取单克隆菌落，经菌落PCR验证阳性转化子，并采用实时定量PCR检验RAS1基因在pCaEXP-RAS1-CAI4菌株的表达水平。结果：酶切及测序鉴定表明高表达质粒载体pCaEXP-RAS1构建成功，实时定量PCR检验转化子RAS1基因在pCaEXP-RAS1-CAI4菌株的表达水平，表明pCaEXP-RAS1-CAI4菌株构建成功。结论：通过高表达质粒载体pCaEXP-RAS1可以成功构建pCaEXP-RAS1-CAI4菌株。

Abstract: Objective：To construct the over RAS1 gene of Candida albicans. Methods：We put the open reading frame of RAS1 gene under the control of MET3 promoter in plasmid pCaEXP to construct recombinant plasmid pCaEXP-RAS1 which can overexpress RAS1 gene. The plasmid pCaEXP-RAS1 was transformed into Escherichia coli DH5α for amplification and extracted by Plasmid Maxi Kit. Then the recombinant plasmid was linearized by cutting of restriction enzyme, and the linear recombinant plasmid was introduced into Candida albicans CAI4 by lithium acetate to select positive colonies on the plates of SD-ura-met-cys selective culture medium. Realtime RT-PCR was used to detect the level of mRNA of RAS1gene. Results：RAS1-over plasmid was exactly established by restriction enzyme digestion. RAS1-over strain was constructed as confirmed by quantitative real-time PCR. RAS1-over strain was selected by quantitative real-time PCR. Conclusions：The RAS1-over strain can be successfully constructed by recombinant pCaEXP-RAS1 plasmid.

章珍珍夏金萍马鸣魏昕. 白念珠菌RAS1基因高表达菌株的构建及鉴定[J]. , 2016, 7(2): 72-75.

参考文献

[1]Leberer E,Harcus D,Dignard D,et al.Ras links cellular morphogenesis to virulence by regulation of the MAP kinase and cAMP signalling pathways in the pathogenic fungus Candida albicans[J].Mol Microbiol,2001,42(3):673-687.
[2]Piispanen AE,Grahl N,Hollomon JM,et al.Regulated proteolysis of Candida albicans Ras1 is involved in morphogenesis and quorum sensing regulation[J].Mol Microbiol,2013,89(1):166-178.
[3]Shapiro RS,Robbins N,Cowen LE.Regulatory circuitry governing fungal development, drug resistance, and disease[J].Microbiol Mol Biol Rev,2011,75(2):213-267.
[4] Inglis DO,Sherlock G.Ras signaling gets fine-tuned: regulation of multiple pathogenic traits of Candida albicans[J].Eukaryot Cell,2013,12(10):1316-1325.
[5]Rowbottom L,Munro CA,Gow NA.Candida albicans mutants in the BNI4 gene have reduced cell-wall chitin and alterations in morphogenesis[J].Microbiology,2004,150(Pt 10):3243-3252.
[6]Kullberg BJ,Filler SG.Candida and candidiasis. ASM Press,Washington,DC,2002
[7]Wisplinghoff H,Bischoff T,Tallent SM,et al.Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study[J].Clin Infect Dis,2004,39(3):309-317.
[8]Whiteway M,Oberholzer U.Candida morphogenesis and host-pathogen interactions[J].Curr Opin Microbiol,2004,7(4):350-357.
[9]Miller MS,Miller LD.RAS Mutations and Oncogenesis: Not all RAS Mutations are Created Equally[J].Front Genet,2012,2:100-.
[10]Jones S,Zhang X,Parsons DW,et al.Core signaling pathways in human pancreatic cancers revealed by global genomic analyses[J].Science,2008,321(5897):1801-1806.
[11]Mountain HA,Bystrom AS,Larsen JT,et al.Four major transcriptional responses in the methionine/threonine biosynthetic pathway of Saccharomyces cerevisiae[J].Yeast,1991,7(8):781-803.
[12]Ono B,Kijma K,Ishii N,et al.Regulation of sulphate assimilation in Saccharomyces cerevisiae[J].Yeast,1996,12(11):1153-1162.
[13]Care RS, Trevethick J, Binley KM,et al.The MET3 promoter: a new tool for Candida albicans molecular genetics[J].Mol Microbiol,1999,34(4):792-798.