Functional expression and sub-cellular localization of the early aflatoxin pathway enzyme Nor-1 in Aspergillus parasiticus.
Functional expression and sub-cellular localization of the early aflatoxin pathway enzyme Nor-1 in Aspergillus parasiticus.
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DOI：
10.1016/j.mycres.2009.01.013
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发表时间：
2009-05
影响因子：
--
通讯作者：
Linz, John E.
中科院分区：
文献类型：
--
作者：
Hong, Sung-Yong;Linz, John E.
关键词：
寄生曲霉中黄曲霉毒素的生物合成需要至少17种酶活性（来自乙酸）。虽然大多数黄曲霉毒素生物合成酶的活性已经确定，但这些酶的转运和亚细胞定位的机制尚不清楚。我们开发了表达Nor-1融合到绿色荧光蛋白报告基因（EGFP）的质粒构建体，以监测寄生曲霉中真实的时间内这种早期途径酶的运输和定位。将表达与Nor-1融合的EGFP的质粒导入A.寄生虫B62（携带非功能性Nor-1）。在椰子琼脂培养基上筛选增加的黄曲霉毒素积累（恢复Nor-1活性）和使用荧光显微镜进行EGFP表达的转化体。通过TLC和ELISA证实黄曲霉毒素积累增加。Nor-1在N-或C-末端与EGFP融合，在功能上补充B62中的非功能性Nor-1，并将黄曲霉毒素合成增加至野生型（N-末端）或更低水平（C-末端）。我们在黄曲霉毒素积累增加的转化体中检测到全长Nor-1融合蛋白（Western印迹），并确定表达质粒在这些细胞中整合在Nor-1位点（Southern印迹）。共聚焦激光扫描显微镜（CLSM）显示，Nor-1融合蛋白定位于细胞质和空泡的真菌菌丝生长在黄曲霉毒素诱导的固体培养基上48小时;对照EGFP（没有Nor-1）没有本地化到空泡，直到72小时。黄曲霉毒素合成的最高速率与Nor-1融合蛋白运输到液泡的最高速率一致，这强烈表明Nor-1是在细胞质中合成的，并运输到液泡中进行黄曲霉毒素合成的早期步骤。
Aflatoxin biosynthesis in Aspergillus parasiticus requires at least 17 enzyme activities (from acetate). Although the activities of most aflatoxin biosynthetic enzymes have been established, the mechanisms that govern transport and sub-cellular localization of these enzymes are not clear. We developed plasmid constructs that express Nor-1 fused to a green fluorescent protein reporter (EGFP) to monitor transport and localization of this early pathway enzyme in real time in Aspergillus parasiticus. Plasmids expressing EGFP fused to Nor-1 were introduced into A. parasiticus B62 (carries non-functional Nor-1). Transformants were screened for increased aflatoxin accumulation (restored Nor-1 activity) on coconut agar medium and for EGFP expression using fluorescence microscopy. Increased aflatoxin accumulation was confirmed by TLC and ELISA. Nor-1 fused to EGFP at either the N- or C- terminus functionally complemented non-functional Nor-1 in B62 and increased aflatoxin synthesis to wild-type (N-terminus) or lower levels (C-terminus). We detected full-length Nor-1 fusion proteins in transformants with increased aflatoxin accumulation (Western blot) and determined that the expression plasmid integrated at the nor-1 locus in these cells (Southern blot). Confocal laser scanning microscopy (CLSM) demonstrated that Nor-1 fusion proteins localized in the cytoplasm and vacuoles of fungal hyphae grown on aflatoxin-inducing solid media for 48 h; control EGFP (no Nor-1) did not localize to vacuoles until 72 h. The highest rate of aflatoxin synthesis coincided with the highest rate of transport of Nor-1 fusion proteins to the vacuole strongly suggesting that Nor-1 is synthesized in the cytoplasm and transported to the vacuole to carry out an early step in aflatoxin synthesis.
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DOI：
10.1016/s0165-7992(85)80021-x
发表时间：
1985-01-01
期刊：
MUTATION RESEARCH
影响因子：
--
作者：
MORI, H;KITAMURA, J;HAMASAKI, T
通讯作者：
HAMASAKI, T
影响因子：
3.5
作者：
OAKLEY, BR;RINEHART, JE;MAY, GS
通讯作者：
MAY, GS
影响因子：
4.4
作者：
SKORY, CD;CHANG, PK;LINZ, JE
通讯作者：
LINZ, JE
影响因子：
2.8
作者：
Lee, LW;Chiou, CH;Linz, JE
通讯作者：
Linz, JE
影响因子：
2
作者：
REYNOLDS, G;PESTKA, JJ
通讯作者：
PESTKA, JJ