烟曲霉钙调蛋白的红色荧光蛋白标记及其在菌株极性生长的动态分布  

Dynamic distribution of calmodulin labeled with red fluorescent protein in the polar growth of Aspergillus fumigatus

作　　者：曾荣[1] 童建波 刘宇甄 陈青[3] 段志敏[2] 刘彩霞[2] 吕桂霞[2] 林彤[1] 李岷[2] 

机构地区：[1]中国医学科学院北京协和医学院皮肤病研究所激光科江苏省皮肤性病学分子生物学重点实验室,南京210042 [2]中国医学科学院北京协和医学院真菌科,南京210042 [3]江苏省血液中心

出　　处：《中华皮肤科杂志》2018年第1期43-47,共5页Chinese Journal of Dermatology

基　　金：国家自然科学基金（81502739、81371750）;国家重点基础研究发展计划（973）项目（2013CB531605）;江苏省自然科学基金（BK20150068）;北京协和医学院青年基金（3332016108）;中国医学科学院医学与健康科技创新工程项目（2016-I2M-1-003、2017-I2M-1-017）

摘　　要：目的 构建自身启动子调控、含有红色荧光蛋白（RFP）标记的钙调蛋白烟曲霉菌株，观察菌株生长过程中钙调蛋白的动态分布。方法 设计烟曲霉钙调蛋白基因的两端侧翼序列，对两序列及含有RFP-烟曲霉pyrG 营养标记（mRFP-AfpyrG）的质粒分别进行PCR扩增，再通过融合PCR对上述3个片段进行整合，形成转化用的线性片段。随后采用原生质体转化法将上述片段转入烟曲霉受体菌株中，构建钙调蛋白-RFP烟曲霉菌株。在营养筛选培养基平板上挑取单克隆菌落培养，选取荧光表型最稳定的单菌菌落对其转化子进行PCR验证。将重组菌株和野生菌株分别接种于固体营养培养基中，培养不同时间后荧光显微镜下观察菌株形态学差异。将上述两株菌分别接种于液体培养基中，甲基四氮盐（XTT）方法观察其生长活力差异，并对红色荧光标记菌株进行动态活体观察，分析钙调蛋白在烟曲霉生长发育过程中的时空分布。结果 重组菌株的荧光表型及转化子的PCR鉴定结果表明，钙调蛋白-RFP烟曲霉菌株构建成功。重组菌株与野生菌株在24 h时生长活力（A490值）分别为0.689 ± 0.081和0.678 ± 0.054（t = 1.32，P〉0.05），差异无统计学意义，且形态学方面也无显著差异。钙调蛋白在烟曲霉生长过程中始终处于极性生长过程中的菌丝顶端、分枝出芽点及新形成的分枝顶端。结论 钙调蛋白可能参与调控烟曲霉孢子萌发及菌丝极性生长等重要生物学过程。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 bran

关 键 词：烟曲霉菌 钙调蛋白 菌丝 荧光蛋白质示踪 红色荧光蛋白 

分 类 号：R379[医药卫生—病原生物学]