菰黑粉菌MAPK同源基因UeKss1的克隆及表达

Cloning and expression of mitogen-activated protein kinase homolog gene UeKss1 in Ustilago esculenta

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。

The dimorphism of the endophytic fungus Ustilago esculenta may be closely related to the stem swol- len of Zizania latifolia. MAPK （ mitogen activated protein kinase） signaling pathway has been known to play im- portant role of regulation in the fungi dimorphism. In this study, we cloned the gene UeKssl encoding a MAPK protein. Clustering analysis showed that UeKssl with high conserved Sedne/threonin dual specific protein kinase catalysis domain, shared more than 80% homology with other fungi in Ustilago. Growth characteristics of U. es- culenta and expression analysis of UeKssl under induction of different carbon resources showed that fungi hyphae formation was only induced by sucrose, with up-regulated expression of UeKssl. While yeast forms were found in PDA, glucose and maltose media. The expression of UeKssl had no significant changes when induced by PDA and glucose. In contrast, it was obviously up-regulated when induced by maltose even more than su- crose. In addition, UeKssl was successfully expressed and purified in prokaryotic expression systems and con- firmed by western blot. All the work provided the basic materials for further functional study of UeKssl and exploring of the mechanism on dimorphism regulation in U. esculenta.