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钙/钙调素依赖性蛋白激酶(calcium/calmodulin-dependent protein kinases,Ca MKs)是真核生物钙信号途径中钙调素下游的一类重要靶蛋白,能够引起众多代谢的关键酶或转录因子磷酸化,从而完成对细胞代谢活动或某些基因表达的调节。本研究利用简并引物从玉米大斑病菌(Setosphaeria turcica)中克隆到4个Ca MKs基因片段,分别命名为CAK1、CAK2、CAK3和CAK4,通过3’RACE和5’RACE得到全长c DNA,CAK1、CAK2和CAK3序列提交Gen Bank,登录号分别为EU605885、EU605886和EU605887。CAK1基因开放阅读框(open reading frame,ORF)1 218 bp,3’非翻译区(untranslated regions,UTR)317 bp,5’UTR 500 bp,编码405个氨基酸;CAK2基因ORF 1 194 bp,3’UTR 400 bp,5’UTR 563 bp,编码397个氨基酸;CAK3基因ORF 2 304 bp,3’UTR 405 bp,5’UTR 207 bp,编码767个氨基酸。生物信息学分析表明,CAK1、CAK2、CAK3和CAK4属多功能Ca MKs,均含有12个激酶保守基序;4个Ca MKs的激酶保守结构域分属不同类型,其中CAK1和CAK2含有典型的Ca MK类丝/苏氨酸蛋白激酶催化结构域,CAK3含有LKB1/Ca MKK类丝/苏氨酸蛋白激酶催化结构域,而CAK4含有RCK1样丝/苏氨酸蛋白激酶催化结构域,并分别与真菌的Ca MK、Ca MKK和MAPKAPK有较近的亲缘关系。实时定量PCR(Real-time quantitative PCR,q RT-PCR)分析表明,在诱导分生孢子萌发并形成附着胞的过程中,CAK2和CAK3表达较活跃,各时期表达水平均接近或高于对照,诱导6和24 h时,表达显著上调(P<0.05);CAK1和CAK4则在各时期表达水平均显著低于对照(P<0.05)。本研究获得了玉米大斑病菌中4个Ca MKs基因,并初步明确了其在病菌侵染结构发育过程中的表达模式,为深入研究该病菌Ca2+途径的作用机制提供了基础资料。
Calcium / calmodulin-dependent protein kinases (Ca MKs) are important target proteins downstream of calmodulin in the eukaryotic calcium signaling pathway and can cause many metabolic key enzymes or transcription factors Phosphorylation, thereby completing the regulation of cellular metabolic activity or the expression of certain genes. In this study, four Ca MKs gene fragments were cloned from Setosphaeria turcica using degenerate primers and named as CAK1, CAK2, CAK3 and CAK4 respectively, and full-length c DNA was obtained by 3’RACE and 5’RACE. The sequences of CAK1, CAK2 and CAK3 were submitted to Gen Bank with the accession numbers EU605885, EU605886 and EU605887 respectively. The cDNA of CAK1 gene was 1 218 bp in open reading frame (ORF), 317 bp in 3 ’untranslated region (UTR) and 500 bp in 5’UTR encoding 405 amino acids. The ORF of CAK2 gene was 1 194 bp in length and 3’ UTR 400 bp, 563 bp 5’UTR, encoding 397 amino acids; CAK3 ORF 2 304 bp, 3 ’UTR 405 bp, 5’UTR 207 bp encoding 767 amino acids. Bioinformatics analysis showed that CAK1, CAK2, CAK3 and CAK4 belong to multifunctional Ca MKs, which all contain 12 kinase conserved motifs. The conserved domains of 4 Ca MKs belong to different types, of which CAK1 and CAK2 contain typical Ca The MK-like filament / threonine protein kinase catalytic domain, CAK3, contains the LKB1 / Ca MKK serine / threonine protein kinase catalytic domain, whereas CAK4 contains the RCK1-like filament / threonine protein kinase catalytic domain, Fungal Ca MK, Ca MKK and MAPKAPK have a closer genetic relationship. Real-time quantitative PCR (q RT-PCR) analysis showed that CAK2 and CAK3 were more active during the process of inducing conidial germination and adherent cells, and the expression levels of CAK2 and CAK3 were close to or higher than that of the control at each time. The expression of CAK1 and CAK4 at each time point was significantly lower than that of the control (P <0.05). In this study, four Ca MKs genes from S. turcica were obtained and their expression patterns during the development of pathogen infection were preliminarily determined, which provided the basic information for further study on the mechanism of Ca2 + pathway in S. gifuensis.