Molecular evolution of Aphanomyces cochlioides flavone receptor protein which functions in host recognition of the oomycetes zoospores.

大肠隐菌（Aphanomyces cohlioides）是一种利用甜菜和菠菜特有的次生代谢产物5-羟基-6,7-亚甲基二氧黄酮（cochliophilin a）作为宿主识别信号化合物的植物病原体。我们寻找能引起b环非取代单黄酮强引诱活性的受体蛋白。利用亲和柱层析法对黄酮类受体候选蛋白进行了纯化。利用该蛋白n端氨基酸序列设计的简并引物，成功鉴定其EST区为32 kDa的β-桶型跨膜蛋白，命名为Aphanomyces cochliophilin a receptor protein 1 （ACCR1）。从mRNA文库中获得其cDNA的全长。ACCR1的氨基酸序列与电压依赖性阴离子通道蛋白（VDAC）具有较高的同源性，但在VDAC的c端缺失70AA。我们对ACCR1的免疫荧光分析显示，ACCR1仅位于细胞质膜上，而不在线粒体上。综上所述，我们提出了耳蜗隐菌ACCR1的分子进化假说。

Aphanomyces cohlioides is a phytopathogen that attacks beet and spinach, using their characteristic secondary metabolite5-hydroxy-6,7-methylenedioxyflavone (cochliophilin A) as host recognition signal compound. We searched receptor protein to cause strong attractant activity of the B-ring non-substituted simple flavone. By using affinity column chromatography of solubilized membrane-binding proteins obtained from A. chochlioides zoospores, we successfully purified one candidate of the flavonoid receptor protein. Using a degenerate primer designed from the amino-acid sequence at the N-terminal of this protein, EST region of the target protein was successfully characterized to be aβ-barrel-type transmembrane protein of a 32 kDa, and named Aphanomyces cochliophilin A receptor protein 1 (ACCR1). From the mRNA library, full length of its cDNA was obtained. Amino acid sequence of ACCR1 had a high homology with voltage-dependent anion channel protein (VDAC), but ACCR1 ismissing 70AA from the C-terminus of VDAC. Our immunofluorescence assay for ACCR1 revealed that ACCR1 locates only cytoplasmic membrane but not on mitochondrion. All together, we proposed a hypothesis on molecular evolution of ACCR1 in Aphanomyces cochlioides.