Abstract:

Zeng Rong, Tong Jianbo, Liu Yuzhen, Chen Qing, Duan Zhimin, Liu Caixia, Lyu Guixia, Lin Tong, Li Min Laser Department, Hospital of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs, Nanjing 210042, China （Zeng R, Liu YZ, Lin T）; Department of Mycology, Hospital of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs, Nanjing 210042, China （Tong JB, Duan ZM, Liu CX, Lyu GX, Li M）; Jiangsu Province Blood Center （Chen Q） Corresponding authors: Lin Tong, Email: ddlin@hotmail.com; Li Min, Email: drlimin@sina.cn 【Abstract】 Objective To construct a native promoter-regulated Aspergillus fumigatus strain containing red fluorescent protein-labeled calmodulin（CaM-RFP）, and to observe the dynamic distribution of calmodulin during the growth of Aspergillus fumigatus. Methods Bilateral flanking sequences of Aspergillus fumigatus calmodulin gene were designed, and plasmids containing the two flanking sequences and mRFP-Aspergillus fumigatus pyrG gene （mRFP-AfpyrG） were amplified separately. The final linear PCR product for transformation was generated from the above three PCR products by fusion PCR. Then, the above linear fragment was transferred into the Aspergillus fumigatus strain by protoplast transformation, so as to construct the CaM-RFP Aspergillus fumigatus strain. The monoclonal colony was picked from the screening medium and subjected to culture. Then, the stablest fluorescent monoxenic strain of Aspergillus fumigatus was selected, and the transformant was verified by PCR. The recombinant strain and wild-type stain were cultured on solid nutrient media separately, and the morphology of these strains was observed by fluorescence microscopy at different time points. Additionally, the above 2 strains were cultured in liquid media separately, and XTT assay was performed to evaluate the growth activity of strains. Microscopy was also conducted to dynamically observe the CaM-RFP Aspergillus fumigatus strain, and analyze the spatial and temporal distribution of calmodulin during the growth and development of Aspergillus fumigatus. Results The fluorescent phenotype and PCR identification results both indicated the successful construction of the CaM-RFP Aspergillus fumigatus strain. The growth activity at 24 hours did not differ between the recombinant strain and wild-type stain （A490: 0.689 ± 0.081 vs. 0.678 ± 0.054, t = 1.32, P > 0.05）, so did the morphology. During the polarized growth of Aspergillus fumigatus, calmodulin was always at the top of the hyphae, germination site of the hyphal branch and the top of new branches. Conclusion Calmodulin may be involved in the regulation of spore germination and polar hyphal growth of Aspergillus fumigatus.