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菰黑粉菌(Ustilago esculenta)是菰(Zizania latifolia)植株体内的内生真菌,其二型态转换与茭白孕茭密切相关,而丝裂原活化蛋白激酶(mitogen activated protein kinase,MAPK)途径在真菌二型态转换中具有重要调控作用。本研究利用酵母型及菌丝型菰黑粉菌的差异蛋白表达及转录组分析数据,克隆了菰黑粉菌中一个丝裂原活化蛋白激酶激酶(mitogen activated protein kinase kinase,MAPKK)基因Ue Mkk1(Gen Bank登录号:KR870332)。该基因c DNA序列全长2 204 bp,开放阅读框2 043 bp。与模式菌酵母的MAPK途径蛋白进行聚类分析表明,Ue Mkk1属于MAPKK蛋白,NCBI中Blast比对结果及进一步的系统发育分析表明,Ue Mkk1与黑粉菌属真菌的Mkk1同源性较高,其中与大麦坚黑穗病菌(Ustilago hordei)的MKK1一致性达到84%,其丝/苏氨酸双特异性蛋白激酶催化结构域在不同真菌中高度保守。同时,本研究通过大肠杆菌(Escherichia coli)原核表达系统成功表达并从蛋白裂解上清液中纯化得到了纯度较高的Ue Mkk1蛋白。另外,对不同碳源诱导后菰黑粉菌的生长特征观察及Ue Mkk1的表达分析表明,蔗糖能诱导菰黑粉菌菌丝的形成及生长,Ue Mkk1的表达量在菌丝生长后期下调表达;PDA、葡萄糖及麦芽糖培养基中菰黑粉菌保持酵母型生长,Ue Mkk1呈周期性上调表达。以上研究工作为进一步开展Ue Mkk1基因的功能研究,阐明其在菰黑粉菌二型态转换及茭白孕茭中的作用提供了基础材料。
Ustilago esculenta is an endophytic fungus in the plant of Zizania latifolia. The second type of the transition is closely related to the pregnant white catfish, and the mitogen activated protein kinase (MAPK) Fungus two types of state transition has an important regulatory role. In this study, differential expression and transcriptome analysis of yeast-type and mycobacterium fulgidus were used to clone the mitogen activated protein kinase kinase (MAPKK) gene Ue Mkk1 (Gen Bank Accession Number: KR870332). The cDNA sequence of this gene is 2 204 bp in length and 2 043 bp in open reading frame. The results of cluster analysis with MAPK pathway protein showed that Ue Mkk1 belonged to MAPKK protein, Blast comparison in NCBI and further phylogenetic analysis showed that Ue Mkk1 shared high homology with Mkk1 of the fungus, Among them, the homology of MKK1 with Ustilago hordei reached 84%, and its filamentous / threonine bispecific protein kinase catalytic domain was highly conserved in different fungi. In the meantime, the present study was successfully expressed in prokaryotic expression system of Escherichia coli and the purified Ue Mkk1 protein was purified from the protein lysis supernatant. In addition, the growth characteristics and the expression of Ue Mkk1 induced by different carbon sources showed that sucrose could induce the formation and growth of the mycelium of Yersinia pseudomallei. The expression of Ue Mkk1 was down-regulated at the later stage of mycelial growth ; PDA, glucose and maltose medium in black yeast to maintain yeast-type growth, Ue Mkk1 was periodically up-regulated expression. The above research work provides the basic materials for further studying the function of Ue Mkk1 gene and elucidating the role of Ue Mkk1 in the second morphogenesis of Yersinia pestis.