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| Cloning and Functional Study of UeCS3.1 Gene in Ustilago esculenta |
| WU Min*, ZHANG Ya-Fen*, XIA Wen-Qiang, HU Peng, CHEN Yue, YU Xiao-Ping, YE Zi-Hong** |
| Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Sciences, China Jiliang University, Hangzhou 310018, China |
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Abstract Ustilago esculenta, a basidiomycete fungus, can infect Zizania latifolia and induce the swelling of tissues near the base of the host plant. The formed edible gall called 'Jiaobai', that is the second largest aquatic vegetable in China. Chitin, the β-1,4-linked linear homopolymer of N-acetylglucosamine (GlcNAc), is the major component of the fungal cell wall and is crucial for the morphogenesis and survival of fungi. The synthesis of chitin is highly conserved and involves several enzymes, of which the chitin synthase (CS) is the key enzyme. Previous study revealed that there are at least 2 but maybe up to 20 chitin synthase genes per species, which were classified into 7 classes. They are basically involved in the whole process of fungal growth and pathogenicity. ClassⅢchitin synthases are crucial for hyphal growth and pathogenicity in some filamentous fungi. In this study, ClassⅢchitin synthases UeCS3.1 (GenBank No. KU302676) in U. esculenta was cloned based on the whole genome sequence of U. esculenta. The genomic DNA of UeCS3.1 was 3 054 bp with one intron. The full length of its ORF was 2 751 bp encoding a protein with 916 amino acids. In addition, it was predicted that UeCS3.1 had 2 catalytic domains of Chitin-synth-1(pfam01644) and Chitin-synth-1N (pfam08407), seven deduced transmembrane regions, with the theoretical isoelectric point of 8.78 and the molecular weight of 103.73 kD by the biological information analysis of protein structure. The phylogenetic analysis showed that the UeCS3.1 has the highest homology with Umchs1 of Ustilago maydis. Then qRT-PCR was used to detect the relative expression of UeCS3.1 during haploid growth and mating process. The expression analysis showed that the expression of UeCS3.1 was up-regulated during haploid growth and mating process and showed a different relative expression level in the T and MT strains. The relative expression of UeCS3.1 in MT strains was higher than that in T strains at 24~48 h during haploid growth process. Furthermore, during the mating process, the relative expression of UeCS3.1 in T strains was significantly higher than that in MT strains at 24 h and it was continuously up-regulated during 0~72 h while that in MT strains tended to be stable at 72 h. So it was speculated that UeCS3.1 was involved in the process of haploid and mating. For further verification, UeCS3.1 deletion strains were constructed by homologous recombination and PEG mediated protoplast transformation. The morphology of UeCS3.1 deletion strains was observed by microscope and growth curve of UeCS3.1 deletion strains was drawn by the values of OD600 at 12, 24, 36, 48, 60 and 72 h. The results showed that the morphology and growth curve of UeCS3.1 deletion strains did not change significantly during the haploid phase. During the mating process, the conjugation tubes of UeCS3.1 deletion strains were formed at 36 h while wild type strains were formed at 24 h. Meanwhile, the hypha of UeCS3.1 deletion strains was shorter than that of wild type strains. Thus, the ability of fusion and hyphal growth of UeCS3.1 deletion strains decreased significantly during the mating process. Totally, these results suggest that UeCS3.1 might be involved in the mating process especially for the hyphal growth in U. esculenta. Above all, this study preliminarily explored the function of the classⅢchitin synthases of U. esculenta, and discussed its role in the mating process, which provided basic materials for the pathogenic mechanism of U. esculenta.
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Received: 07 January 2019
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Corresponding Authors:
** , zhye@cjlu.edu.cn
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| About author:: * The authors who contribute equally |
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