Identifying secreted proteins of Marssonina brunnea by degenerate PCR
Identifying secreted proteins of Marssonina brunnea by degenerate PCR
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通过简并 PCR 鉴定 Marssonina brunnea 的分泌蛋白
DOI：
10.1002/pmic.200900844
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发表时间：
2010-07-01
期刊：
影响因子：
3.4
通讯作者：
Huang, Minren
中科院分区：
文献类型：
--
作者：
Cheng, Qiang;Cao, Youzhi;Huang, Minren
Marssonina brunnea是杨属重要的真菌病原菌，为了进一步了解M.brunnea的发病机制，我们启动了对真菌分泌组的蛋白质组水平的研究。通过MS/MS从头测序，我们得到了32个蛋白质点的多肽序列。通过与公共数据库中保守蛋白的序列同源性，利用MS-Driven BLAST鉴定了4个蛋白质。为了鉴定额外的蛋白质斑点，我们结合了基于共识的简并-简并杂交寡核苷酸引物法(CODEHOP)的简并PCR方法和快速扩增cDNAEnd的方法来完成编码凝胶中所识别的蛋白质的全长cDNA片段。用这种方法，我们克隆了编码11个布氏支原体特异蛋白的全长cDNA片段。该方法为鉴定未测序生物的物种特异性蛋白质提供了一种有效的途径。此外，我们还分析了这些基因在感染过程中的表达模式。我们发现，大多数已鉴定的分泌蛋白在菌丝进入杨树质外体间隙后，都能在人工培养基中被诱导。我们认为，对于寄主专化的布氏支原体，菌丝的伸长与质外体蛋白的分泌密切相关。
Marssonina brunnea is an important fungal pathogen of the Populus genus To further our understanding of the pathogenesis of M. brunnea, we initiated a proteome-level study of the fungal secretome. Using de novo peptide sequencing by MS/MS, we obtained peptide sequences for 32 protein spots. Four proteins were identified by sequence homology to conserved proteins in public databases using MS-driven BLAST. To identify additional protein spots, we combined a degenerate PCR method, based on the Consensus-DEgenerate Hybrid Oligonucleotide Primer (CODEHOP) method, and a rapid amplification of cDNA ends method to done the full-length cDNA fragments encoding the proteins identified in the gel. Using this method, we cloned the full-length cDNA fragments encoding 11 M. brunnea-specific proteins. This method provides an efficient approach to identification of species-specific proteins of non-sequenced organisms. Furthermore, we analyzed the expression patterns of these genes during infection. We found that most of the identified secreted proteins could be induced in artificial medium after hyphae entered poplar apoplast spaces. We propose that for the host-specialized M. brunnea, the elongation of hyphae has evolved closely with the secretion of apoplastic proteins.