禾谷炭疽菌CgRab5A的亚细胞定位研究

Study on Subcellular Localization of CgRab5A In Colletotrichum graminicola

为明确禾谷炭疽菌(Colletotrichum graminicola)中CgRab5A(GLRG00019)蛋白在细胞内的定位特点,利用多片段一步克隆法将CgRab5A自身启动子、GFP片段和CgRab5A开放阅读框(ORF)片段克隆到pKNT载体上,转化野生型禾谷炭疽菌的原生质体后,通过新霉素抗性筛选获得阳性候选转化子,利用共聚焦显微镜观察GFP-CgRab5A在菌丝和孢子内的定位情况。结果表明,一步克隆法获得的pKNT-GFP-CgRab5A重组载体经测序后序列完全正确,亚细胞定位观察发现GFP-CgRab5A在菌丝和孢子细胞内均呈点状分布,定位于早期内涵体上,暗示其可能参与早期内涵体的相关调控过程。

In order to clarify the cellular characteristics of CgRab5 A(GLRG00019)from Colletotrichum graminicola,firstly native promoter and ORF of CgRab5 A,GFP were cloned to pKNT vector by multis one step cloning method. And then the recombinant plasmid was transferred to protoplast of wild type C. graminicola and positive candidates were screened by neomycin resistance. Finally,the localization of GFP-CgRab5 A in mycelia and spores was observed by confocal microscope. The results showed that the recombinant vector of pKNT-GFP-CgRab5 A was completely right by sequencing,and the subcellular localization observation showed that GFP-CgRab5 A was distributed in the mycelia and spore cells in a spot-like manner and located on the early endosomes,suggesting that it may play important roles in regulating early endosome. This study provided clues for the further analysis of the functions of CgRab5 A,and also lays a theoretical foundation for illuminating of the pathogenic mechanism of C. graminicola.