Connolly Mary S, Sakihama Yasuko, Phuntumart Vipaporn, Jiang Yinjun, Warren Franklin, Mourant Lindsay, Morris Paul F
Biology Sciences, Bowling Green State University, OH, USA.
Curr Genet. 2005 Dec;48(6):356-65. doi: 10.1007/s00294-005-0015-4. Epub 2005 Nov 5.
A system for the expression of an ATP binding cassette (ABC) transporter from the soybean pathogen Phytophthora sojae is described. Pdr1, an ABC transporter with homology to the pleiotropic drug resistance (PDR) family of transporters, was cloned by primer walking from a P. sojae genomic library. Reverse transcriptase PCR assays showed that the transcript disappeared after encystment of zoospores and was not detected in hyphal germlings in dilute salts, in hyphae growing in liquid V8 media, or in tissue extracts from infected hypocotyls. BLAST analysis of Pdr1 against the P. sojae EST database also revealed that this gene was present only in zoospore libraries. Comparison of the number of hits to Pdr1 with that of a set of housekeeping genes revealed that Pdr1 was expressed at rates two- to threefold higher than other transcripts. To test the hypothesis that Pdr1p functions as a broad substrate membrane transporter, Pdr1 was transformed into yeast mutants deficient in several drug resistance transporters. Yeast mutants transformed with Pdr1 possessed partial drug resistance against only 5 of 17 chemically distinct compounds. Thus, when expressed in yeast, this transporter has a significantly narrower substrate specificity in comparison to the yeast transporters, Pdr5p, Yorlp, and Snq2p.
本文描述了一种用于表达来自大豆疫霉病原体的ATP结合盒（ABC）转运蛋白的系统。通过引物步移法从大豆疫霉基因组文库中克隆了Pdr1，它是一种与多药耐药性（PDR）转运蛋白家族具有同源性的ABC转运蛋白。逆转录酶PCR分析表明，游动孢子包囊化后该转录本消失，在稀盐中的菌丝幼体、液体V8培养基中生长的菌丝或受感染下胚轴的组织提取物中均未检测到。对大豆疫霉EST数据库进行Pdr1的BLAST分析还表明，该基因仅存在于游动孢子文库中。将Pdr1的命中数与一组管家基因的命中数进行比较，发现Pdr1的表达速率比其他转录本高两到三倍。为了验证Pdr1p作为一种广泛底物膜转运蛋白发挥功能的假设，将Pdr1转化到几种耐药转运蛋白缺陷的酵母突变体中。用Pdr1转化的酵母突变体仅对17种化学性质不同的化合物中的5种具有部分耐药性。因此，当在酵母中表达时，与酵母转运蛋白Pdr5p、Yorlp和Snq2p相比，这种转运蛋白的底物特异性明显更窄。
