桑褐斑壳丰孢菌pmcamk基因的克隆与表达时相分析

Cloning and Expression Analysis of Calcium/Calmodulin-Dependent Protein Kinases Gene of Phloeospora maculans

钙/钙调素依赖性蛋白激酶(calcium/calmodulin-dependent protein kinases, CAMK)作为钙信号途径中的关键蛋白,在多种细胞过程中发挥重要作用。以桑褐斑壳丰孢菌(Phloeospora maculans)为研究对象,利用反转录PCR结合RACE技术克隆桑褐斑壳丰孢菌(Phloeospora maculans)钙/钙调素依赖性蛋白激酶基因pmcamk的序列全长,利用荧光定量PCR技术,研究pmcamk在桑褐斑壳丰孢菌侵染宿主后不同时期以及其分生孢子在水中萌发过程中的表达模式,以期深入了解桑褐斑壳丰孢菌Ca^(2+)信号转导途径与孢子萌发之间的关系。结果表明,pmcamk的cDNA序列全长为1 422 bp,由1 221 bp的开放阅读框和201 bp的3′非翻译区组成,GenBank登录号为MK713976。pmcamk开放阅读框共编码406个氨基酸残基,蛋白质分子质量约为45.5 kD,等电点为6.28。系统进化树分析表明桑褐斑壳丰孢菌与小麦叶枯病菌的CAMK同源性最高,属于CAMKⅠ类群并且聚类在同一进化分支。在桑褐斑壳丰孢菌侵染桑树的早期阶段(0.25、 0.5、1 d),pmcamk的表达量呈上升趋势,在侵染后2、3、4、6 d其表达量逐渐下降。这与不同时间阶段桑褐斑壳丰孢菌在水中萌发过程中pmcamk的表达趋势基本一致,推测PmCAMK在桑褐斑病菌孢子萌发过程中发挥着重要作用。

As a key protein in the calcium signaling pathway,calcium/calmodulin-dependent protein kinase(CAMK)plays an important role in a variety of cellular processes.In this study,reverse transcription PCR combined with RACE technology was used to clone the full-length sequence of the camk gene of Phloeospora maculans.Quantitative PCR technology was used to research the expression pattern of pmcamk at different stages after P.maculans infecting the host and during its conidial germination in water,in order to gain insights into the relationship between Ca^(2+)signal transduction pathway and spore germination.The results showed that the full length of the cDNA sequence of pmcamk was 1422 bp,consisting of an open reading frame of 1221 bp and a 3'-untranslated region of 201 bp,and the gene accession number was MK713976.The open reading frame of pmcamk encodes a total of 406 amino acid residues,the protein molecular weight is about 45.5 kD,and the isoelectric point is 6.28.The phylogenetic tree indicated that the CAMK homology between P.maculans and Zymoseptoria tritici was the highest,belonging to the CAMK I group and clustering in the same evolutionary branch.In the early stage(0.25,0.5,1 d)of P.maculans infection of mulberry tree,the expression of pmcamk showed an upward trend.The expression level decreased gradually at 2,3,4 and 6 d after infection.This is basically consistent with the expression trend of pmcamk during the germination of P.maculans in water in different time periods.It is speculated that PmCAMK plays an important role in the spore germinationprocess.