黄孢原毛平革菌寡肽转运蛋白基因家族研究

项目名称： 黄孢原毛平革菌寡肽转运蛋白基因家族研究

项目编号： No.30871344

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 生物科学

项目作者： 张义正

作者单位： 四川大学

项目金额： 33万元

中文摘要： 黄孢原毛平革菌（Phanerochaete chrysosporium）降解木质纤维素是一个十分复杂的次生代谢过程，已知许多基因参与该过程。我们曾发现该菌的寡肽转运蛋白（PcOPT）基因表达也具有代谢阶段特异性。利用PCR、DNA重组和转录组测序等方法对该菌的OPT基因和编码区进行了克隆和测序，获得该菌OPT基因家族16个基因及编码序列。这些基因所编码的蛋白质长度约为700个氨基酸，具有14-16个跨膜结构域。根据蛋白质序列同源性分析，Pcopt基因家族可分为3个亚家族。利用RT-PCR方法对16个基因在20种培养条件下的表达规律进行了研究，结果发现，有9个基因的表达不具有代谢阶段特异性；其余7个基因则存在明显的初级阶段或次生代谢阶段表达特异性。Pcopt基因功能研究结果表明，PcOPT蛋白可使酿酒酵母寡肽转运系统突变体在含有4肽培养基上生长，显示了转运寡肽的功能。Pcopt-gfp融合基因在酿酒酵母中表达的蛋白质被定位在膜上。对大肠杆菌谷胱甘肽（三肽）转运系统的4个基因进行了克隆和表达，已获得大量的表达产物用于结构测定。上述研究结果有助于进一步阐明寡肽转运蛋白的作用。

中文关键词： 黄孢原毛平革菌；寡肽转运蛋白家族；基因克隆；基因表达；基因功能

英文摘要： Phanerochaete chrysosporium degrades lignin through a very complicated secondary metabolic process, in which a lot of genes have been known to be involved. Our previous results displayed that oligopeptide transporter (PcOPT) genes were specifically expressed in the secondary metabolism. In this study all sixteen genes and their coding sequences of the oligopeptide transporter (OPT) family were obtained from P. chrysosporium by using PCR, DNA cloning, transcriptomic and bioinfomatic analyses. The proteins predicted from these genes have about 700 amino acid residues and contain 14-16 transmembrane domains, implying that they are membrane proteins. Sixteen Pcopt genes can be divided into 3 sub-families on the basis of the sequence homology analyses. The results from RT-PCR analyses showed that nine Pcopt genes could be expressed without specificity while the other 7 Pcopt genes displayed primary or secondary metabolism-specific expression when P.chrysosporium was grown under different conditions. Saccharomyces cerevisiae cells transformed with some Pcopt genes could be grown on the media supplemented with tetrapeptides（KLGL）, implying that the PcOPT protein plays the role in oligopeptide transportation. The expressed OPT-GFP fused proteins in S.cerevisiae were localized in the membrane, which is accorded with above prediction of membrane proteins. Four genes of glutathione transporter of Escherichia coli were also cloned and expressed for the structure determination. All above results could be very useful in the further study of understanding the function of PcOPT in the secondary metabolism of P.chrysosporium.

英文关键词： Phanerochaete chrysosporium; oligopeptide transporter family;gene cloning;gene expression;gene function