Expression analysis of alternative oxidase gene (aox1) with enhanced green fluorescent protein as marker in citric acid-producing Aspergillus niger
Expression analysis of alternative oxidase gene (aox1) with enhanced green fluorescent protein as marker in citric acid-producing Aspergillus niger
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DOI：
10.1263/jbb.102.210
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发表时间：
2006-09-01
影响因子：
2.8
通讯作者：
Kino, Kuniki
中科院分区：
文献类型：
--
作者：
Kirimura, Kohtaro;Ogawa, Satoshi;Kino, Kuniki
在产柠檬酸的丝状真菌黑曲霉 WU-2223L 中，除了细胞色素途径外，线粒体中还有一种对氰化物和抗霉素 A 不敏感且对水杨基异羟肟酸敏感的呼吸途径发挥作用，并由替代氧化酶 (AOX) 催化。我们构建了表达融合基因 aox1-egfp 的黑曲霉转化菌株 AOXEGFP-1，该融合基因编码 AOX 和增强型绿色荧光蛋白 (EGFP)，以便在不破坏菌丝体的情况下直观地分析 aox1 的表达水平。在菌株AOXEGFP-1中，清楚地证实了融合蛋白AOX-EGFP在线粒体中的定位，因为AOX-EGFP的绿色荧光位点与MitoTracker Red CMXRos染色的线粒体的红色荧光位点一致。当用抗霉素 A 培养菌株 AOXEGFP-1 时，抗霉素 A 抑制细胞色素 bc(1) 至细胞色素 c 水平的细胞色素途径并增加 aox1 的表达水平，EGFP 荧光强度随着 AOX 活性（以杜罗喹诺氧化酶活性测量）的增加而增加。此外，无论培养基中的葡萄糖浓度如何，菌株AOXEGFP-1中都检测到EGFP荧光：例如，当用10、30、60和120g/l葡萄糖进行培养时，通常在线粒体中检测到EGFP荧光。这些结果表明，无论黑曲霉中的葡萄糖浓度如何，aox1 都会持续表达。
In a citric acid-producing filamentous fungus Aspergillus niger WU-2223L, a cyanide- and antimycin A-insensitive and salicylhydroxamic acid-sensitive respiratory pathway functions in the mitochondria besides the cytochrome pathway and is catalyzed by alternative oxidase (AOX). We constructed an A. niger transformant strain AOXEGFP-1 expressing a fusion gene, aox1-egfp, encoding AOX and enhanced green fluorescent protein (EGFP) to visually analyze the expression levels of aox1 without disruption of mycelia. In strain AOXEGFP-1, the localization of the fusion protein AOX-EGFP in the mitochondria was clearly confirmed because the sites of the green fluorescence by AOX-EGFP were in agreement with those of the red fluorescence of the mitochondria stained with MitoTracker Red CMXRos. When strain AOXEGFP-1 was cultivated with antimycin A, which inhibits the cytochrome pathway at the level of cytochrome bc(1) to cytochrome c and increases the expression level of aox1, EGFP fluorescence intensity increased with an increase in AOX activity measured as duroquinol oxidase activity. Moreover, EGFP fluorescence was detected in strain AOXEGFP-1 regardless of the glucose concentration in the cultivation media: for example, when cultivations were performed with 10, 30, 60 and 120 g/l glucose, EGFP fluorescence was usually detected in the mitochondria. These results indicate that aox1 was constitutively expressed regardless of the glucose concentration in A. niger.