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| Full Length Cloning and Expression of Mitogen-Activated Protein Kinase Kinase Homolog Gene UeMkk1 in Ustilago esculenta |
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Abstract Ustilago esculenta is the endophytic fungus of Zizania latifolia, and induces the plant stem swollen which may be related to its dimorphism. MAPK (mitogen activated protein kinase) signaling pathway has been known to play important regulatory role in the fungi dimorphism. In this study, the gene UeMkk1 (GenBank accession number: KR870332), encoding a mitogen activated protein kinase kinase (MAPKK), was cloned through analysis of the differentially protein profile and transcriptome sequencing data from yeast- and mycelium- form of U. esculenta. The full length of UeMkk1 was 2 204 bp, with a 2 043 bp open reading frame. Clustering analysis between UeMkk1 and MAPK signaling pathway proteins of yeast showed that UeMkk1 belonged to MAPKK protein. Blast results from NCBI and phylogenetic analysis results by MEGA 5.0 showed that UeMkk1 had higher homology to Mkk1 proteins in Ustilago, of which the highest homology was MKK1 of Ustilago hordei, reaching to the identify of 84%. Also the typical Serine/threonin dual specific protein kinase catalytic domain in UeMkk1 showed high conservation to other fungi. Meanwhile, UeMkk1 protein was successfully expressed in prokaryotic expression system, and purified from the lysate supernatant with good purity. The best expression system for UeMkk1 was that induced with 0.2 mmol/L isopropyl β-D-thiogalactoside (IPTG) at 37 ℃, and purified by Ni-NTA with 200 mmol/L imidazole elution. In addition, the morphology microscopic observation of U. esculenta cultured on basic medium with different carbon sources was carried out, showing that fungi hyphae formation was induced by sucrose, and the fungi maintained yeast-like growth type on the other culture medium (PDA, glucose or maltose medium). Meanwhile, real-time fluorescence quantitative PCR (qRT-PCR) analysis of UeMkk1 expression in the fungi after induced by different carbon sources was carried out, and the results showed that UeMkk1 was up-regulated at 4 d after cultured on maltose medium and at 6 d after cultured on PDA or glucose medium, showing a cyclical increased expression. Only on sucrose medium, UeMkk1 was earlier up-regulated (after 2 d, when the hyphae started formation) but fell down obviously when cultured after 8 d (when the growth of hyphae became slowly). The results showed that UeMkk1 was up-regulated during active differentiation and proliferation, indicating the involvement of UeMkk1 in cell wall integrity regulation of U. esculenta, which was similar to other fungi. All the work provideds research foundation for further studying the function of UeMkk1 and exploring its relationship with dimorphism of U. esculenta.
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Received: 28 July 2015
Published: 05 February 2016
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