Yi Ruirong, Mukaiyama Hiroyuki, Tachikawa Takashi, Shimomura Norihiro, Aimi Tadanori
Faculty of Agriculture, Tottori University, 4-101 Koyama-cho Minami, Tottori 680-8553, Japan.
Eukaryot Cell. 2010 Jul;9(7):1109-19. doi: 10.1128/EC.00374-09. Epub 2010 May 7.
In the bipolar basidiomycete Pholiota microspora, a pair of homeodomain protein genes located at the A-mating-type locus regulates mating compatibility. In the present study, we used a DNA-mediated transformation system in P. microspora to investigate the homeodomain proteins that control the clamp formation. When a single homeodomain protein gene (A3-hox1 or A3-hox2) from the A3 monokaryon strain was transformed into the A4 monokaryon strain, the transformants produced many pseudoclamps but very few clamps. When two homeodomain protein genes (A3-hox1 and A3-hox2) were transformed either separately or together into the A4 monokaryon, the ratio of clamps to the clamplike cells in the transformants was significantly increased to ca. 50%. We therefore concluded that the gene dosage of homeodomain protein genes is important for clamp formation. When the sip promoter was connected to the coding region of A3-hox1 and A3-hox2 and the fused fragments were introduced into NGW19-6 (A4), the transformants achieved more than 85% clamp formation and exhibited two nuclei per cell, similar to the dikaryon (NGW12-163 x NGW19-6). The results of real-time reverse transcription-PCR confirmed that sip promoter activity is greater than that of the native promoter of homeodomain protein genes in P. microspora. Thus, we concluded that nearly 100% clamp formation requires high expression levels of homeodomain protein genes and that altered expression of the A-mating-type genes alone is sufficient to drive true clamp formation.
在双极性担子菌小孢鳞伞中，位于A交配型位点的一对同源结构域蛋白基因调控交配亲和性。在本研究中，我们利用小孢鳞伞的DNA介导转化系统来研究控制锁状联合形成的同源结构域蛋白。当将来自A3单核菌株的单个同源结构域蛋白基因（A3-hox1或A3-hox2）转化到A4单核菌株中时，转化体产生了许多假锁状联合，但锁状联合很少。当将两个同源结构域蛋白基因（A3-hox1和A3-hox2）单独或一起转化到A4单核菌株中时，转化体中锁状联合与类锁状细胞的比例显著增加至约50%。因此，我们得出结论，同源结构域蛋白基因的基因剂量对锁状联合的形成很重要。当sip启动子与A3-hox1和A3-hox2的编码区连接，并将融合片段导入NGW19-6（A4）时，转化体实现了超过85%的锁状联合形成，并且每个细胞表现出两个细胞核，类似于双核体（NGW12-163×NGW19-6）。实时逆转录PCR结果证实，sip启动子活性大于小孢鳞伞中同源结构域蛋白基因的天然启动子活性。因此，我们得出结论，近乎100%的锁状联合形成需要同源结构域蛋白基因的高表达水平，并且仅改变A交配型基因的表达就足以驱动真正的锁状联合形成。
