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菰黑粉菌作为茭白植株体内的内生真菌,其二型态转换与茭白孕茭密切相关,而MAPK途径在真菌二型态转换中具有重要调控作用。本研究在菰黑粉菌中克隆得到了一个MAPK基因UeKss1,通过与酵母菌中的MAPK途径蛋白进行聚类分析表明其属于Kss1的同源蛋白。进一步的系统进化分析表明,UeKss1与黑粉菌属真菌的Kss1同源性最高,达80%以上,且它的丝/苏氨酸双特异性蛋白激酶催化结构域高度保守。不同碳源诱导下菰黑粉菌的生长特征及UeKss1表达分析发现,只有蔗糖培养基能诱导菰黑粉菌菌丝形成,而在PDA、葡萄糖和麦芽糖培养基中,该菌都保持酵母型生长;UeKss1基因的表达量在PDA和葡萄糖培养基中变化不显著,但在麦芽糖培养基中,该基因的表达随着培养时间的延长持续上调表达,而在蔗糖培养基中,UeKss1的表达量虽然也呈现上升趋势,但是远小于麦芽糖的诱导表达量。对UeKss1进行的原核表达纯化,经Western blot验证得到纯度较高的UeKss1蛋白。研究结果可为UeKss1基因的功能研究,阐明其在菰黑粉菌二型态转换中的作用机制提供帮助。
As a endophytic fungus, the second type of black powdery mildew is closely related to the second part of the pregnancy. The MAPK pathway plays an important regulatory role in the second type fungal transformation. In this study, a MAPK gene, UeKss1, was cloned from the powdery mildew of Yarlungsi amyloliquefaciens, and homologous proteins belonging to Kss1 were identified by cluster analysis with the MAPK pathway protein in yeast. Further phylogenetic analysis showed that Kes1 shared the highest homology of Kss1 to Ustilago fungi, accounting for more than 80%, and its catalytic domain of silk / threonine bispecific protein kinase was highly conserved. The growth characteristics of Ustilago ocarina and the expression of UeKss1 induced by different carbon sources showed that only sucrose medium could induce the mycelium formation of Ustilago ocarina, while in PDA, glucose and maltose medium, the strain maintained the growth of yeast . The expression of UeKss1 gene did not change significantly in PDA and glucose medium, but in maltose medium, the expression of UeKss1 gene continued to increase with the prolongation of culture time. However, the expression of UeKss1 in sucrose medium Also showed an upward trend, but far less than the induction of maltose expression. The prokaryotic expression of UeKss1 was purified and the high purity UeKss1 protein was obtained by Western blot. The results of this study can be used to study the function of UeKss1 gene and elucidate the mechanism of UeKss1 function in the transformation of Drosophila melanogaster.