研究人类基因4-羟苯丙酮酸二加氧酶(4-hydroxyphenylpyruvate dioxygenase,HPD)的表达对酿酒酵母(Saccharomyces cerevisiae)产孢过程及酵母孢子壁组装的影响。将HPD导入酿酒酵母中,比较人源化HPD酵母与野生型酵母的表型差异,分析HPD对酵母产孢及孢子壁的影响。与野生型酵母相比,人源化HPD酵母对产孢率无影响,但孢子自身荧光强度下降,乙醚敏感性增加,荧光增白剂(calcofluor white,CFW)染色荧光强度增强,该结果表明孢子壁出现缺陷,其二酪氨酸层组装过程中部分缺失。进一步研究发现,人源化HPD酵母产孢时,Hpd蛋白的催化产物尿黑酸(homogentisate,HGA)在产孢过程中会被氧化从而引起培养基颜色变化,且二酪氨酸复合物会大量泄漏至培养基中,最终导致二酪氨酸层不能正确组装。该研究结果为酿酒酵母二酪氨酸层的形成机制提供了新的认知。
This study analyzed the effects of expressing human gene 4-hydroxyphenylpyruvate dioxygenase (HPD) on sporulation and spore wall assembly in Saccharomyces cerevisiae. HPD was introduced into S. cerevisiae, and the phenotype differences between humanized HPD yeast and wild-type yeast were compared. It was found that compared with wild-type yeast, humanized HPD yeast had no effect on the sporulation rate, while the fluorescence intensity of spores decreased, ether sensitivity increased, and the fluorescence intensity of calcofluor white increased. This indicated that HPD caused the defect of spore wall and part of the dityrosine layer missing during the assembly process. Moreover, expressed HPD gene changed the sporulation medium color and increased the fluorescence intensity, as homogentisate, the catalytic product of HPD, was oxidized during sporulation. Furthermore, a large number of dityrosine complex leaked into the sporulating medium and dityrosine layer could not assemble correctly in consequence. Overall, this study provides a new insight into the mechanisms of forming dityrosine layer in S. cerevisiae.
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