红曲菌(Monascus)是我国传统的发酵菌株,在发酵过程中会产生一种具有肾毒性的真菌毒素——桔霉素(Citrinin,CIT).桔霉素的存在极大地限制了红曲制品的应用与发展.明确红曲菌合成CIT的机制,对于开发抑制CIT合成的方法具有十分重要的意义.小G蛋白Rab1可以介导调控丝状真菌中蛋白的转运,ctnE基因编码红曲菌AS3.4384中CIT合成基因簇中的脱氢酶.然而,目前尚不清楚Rab1蛋白是否参与了 CtnE蛋白的转运.通过农杆菌介导转化的方法构建了 AS3.4384::Rab1-GFP(绿色荧光蛋白,Green Fluorescent Protein)和AS3.4384::Rab1-GFP::ctnE-mCherry(红色荧光蛋白,monomeric cherry red fluorescent protein)两种红曲菌株,并通过荧光共定位确定了 Rab1蛋白和CtnE蛋白在菌体中的分布情况.结果表明,CtnE-mCherry蛋白大多聚集于红曲菌丝体的顶端以及分生孢子中,并在菌丝体内呈均匀弥散状分布.Rab1蛋白和CtnE蛋白在红曲菌体内的分布位点基本一致,表明CIT的合成与囊泡转运密切相关.通过Rab1蛋白与CtnE蛋白的荧光共定位情况进一步解析了CIT合成位点.
Subcellular localization of Rab1 and CtnE proteins in Monascus aurantiacus
Monascus,a traditional fermentation strain in China,produces a nephrotoxic fungal toxin called citrinin(CIT)during fermentation.The presence of citrinin greatly limits the application and development of Monascus products.Understand-ing the mechanism of CIT synthesis by Monascus is of great significance for developing methods to inhibit its synthesis.The small G protein Rab1 can mediate the regulation of protein transport in filamentous fungi,and the ctnE gene encodes a dehydro-genase in the CIT biosynthetic gene cluster of Monascus AS3.4384.However,it is currently unclear whether the Rab1 protein is involved in the transport of the CtnE protein.Two Monascus strains,AS3.4384::Rab1-GFP(Green Fluorescent Protein)and AS3.4384::Rab1-GFP::ctnE-mCherry(monomeric cherry red fluorescent protein),were constructed using Agrobacterium-mediated transformation method.Fluorescence co-localization was used to determine the distribution of Rab1 and CtnE proteins in the fungal cells.The results showed that the CtnE-mCherry protein mostly accumulated at the tips of Monascus hyphae and in the developing spores,with a uniform distribution within the hyphae.The distribution sites of Rab1 and CtnE proteins in Monas-cus cells were essentially consistent,indicatied a close relationship between CIT synthesis and vesicle transport.Further analysis of the fluorescence co-localization of Rabl and CtnE proteins elucidated the sites of CIT synthesis.
万方数据
维普
