梨果黑斑病菌AaCaMK基因克隆、生物信息学分析及其在侵染结构分化中的表达分析

背景] 钙/钙调素依赖型蛋白激酶（Calcium/Calmodulin-Dependent Protein Kinase，CaMK）是真核生物细胞钙信号途径中钙调素下游的一类重要靶蛋白，对病原物生长、胁迫响应及致病性等具有重要的调控作用。目的] 对梨果黑斑病菌互隔交链孢（Alternaria alternata）AaCaMK基因进行克隆、生物信息学分析，并对其在侵染结构分化过程中的基因表达情况进行分析，为进一步研究梨果黑斑病菌钙离子信号途径中AaCaMK对A.alternata侵染结构分化调控的分子机制提供一定的理论依据。方法] 采用同源克隆法从A. alternata JT-03中克隆得到3个AaCaMK基因；通过TMHMM、ProtScale、SOPMA等软件对AaCaMK基因进行生物信息学分析；利用实时荧光定量PCR （RT-qPCR）技术分析AaCaMK在梨果黑斑病菌侵染结构分化过程中的表达情况。结果] 克隆得到片段分别为1 212、1 200、2 349 bp的AaCaMK1、AaCaMK2和AaCaMK3基因；生物信息学分析表明，AaCaMK1、AaCaMK2和AaCaMK3均含有典型的蛋白激酶超家族催化结构域（PKC_Like Superfamily），并且AaCaMK1和AaCaMK2共同含有CaMK类丝/苏氨酸蛋白激酶催化结构域（STKc_CaMK），AaCaMK3含有LKB1/CaMKK类丝/苏氨酸蛋白激酶催化结构域（STKc_LKB1_CaMKK）；同源性分析表明，AaCaMK1、AaCaMK2和AaCaMK3分别与玉米大斑病菌CAK1、CAK2和CAK3的相似性高达94.32%、97.49%和86.57%；RT-qPCR分析表明，AaCaMK1、AaCaMK2和AaCaMK3在疏水及果蜡诱导A. alternata侵染结构分化过程中均显著上调表达（P<0.05），而且果蜡诱导作用更显著。其中AaCaMK1和AaCaMK2在附着胞形成时期（6 h）表达量为对照的1.51倍和3.05倍，而AaCaMK3在侵染菌丝形成阶段（8 h）表达量最高，为对照的2.86倍，并且在果蜡诱导下，这3个基因在芽管伸长阶段（4 h）的上调表达量显著高于疏水界面。结论] 钙信号中AaCaMK基因在疏水及果蜡诱导A.alternata侵染结构分化过程中发挥重要的调控作用。

Cloning, bioinformatics and expression analysis of AaCaMK gene on infection structure differentiation of Alternaria alternate, causal agent of pear black spot

Background] Calcium/calmodulin-dependent protein kinase (CaMK), an important downstream target protein of calmodulin in the calcium signaling pathway of eukaryotic cells, plays an important role in pathogen growth, stress response and pathogenicity. Objective] Cloning, bioinformatics and expression analysis of AaCaMK gene on infection structure differentiation of Alternaria alternate, casual agent of pear black spot, for further clarifying the molecular regulatory role of the AaCaMK gene in the calcium signal pathway on the infection structure differentiation of A. alternata. Methods] The AaCaMK gene was cloned from Alternaria alternata JT-03 by homologous cloning; The AaCaMK gene was analyzed by TMHMM, ProtScale, SOPMA and other software; Real-time quantitative PCR (RT-qPCR) was used to analysis of the expression of AaCaMK on infection structure differentiation of A. alternata. Results] Three isotypes of calcium/calmodulin-dependent protein kinase designated AaCaMK1, AaCaMK2 and AaCaMK3 were identified in A. alternata with length of 1 212, 1 200 and 2 349 bp; The bioinformatics analysis revealed that these three CaMK all contain the PKC_like superfamily domains (PKC_Like Superfamily). The conserved kinase domains of both AaCaMK1 and AaCaMK2 belonged to the catalytic domain of CaMK ser/thr protein kinase (STKc_CaMK) while that of AaCaMK3 belonged to the catalytic domain of liver kinase B1 (LKB1) and calmodulin dependent protein kinase kinase (CaMKK)(STKc_LKB1_CaMKK); The homology analysis showed that the homology of AaCaMK1, AaCaMK2 and AaCaMK3 with Setosphaeria turcica CAK1, CAK2 and CAK3 were as high as 94.32%, 97.49% and 86.57%, respectively; RT-qPCR analysis showed that genes expression of AaCaMK1, AaCaMK2 and AaCaMK3 were all significantly upregulated during infection structure differentiation of A. alternata induced by hydrophobic and fruit wax coating surfaces (P<0.05), and fruit wax showed more significant stimulus effects. Among them, the expression of AaCaMK1 and AaCaMK2 during the appressorium formation period (6 h) were 1.51 and 3.05 folds, respectively, while AaCaMK3 had the highest expression in the infection hyphae formation period (8 h), which was 2.86 folds that of the control. Under fruit wax induction, the up-regulated expression of these three genes was significantly higher at the germ tube elongation stage (4 h) on fruit wax coating surfaces than the hydrophobic surface. Conclusion] AaCaMK in calcium signaling pathway play an important regulatory role on the infection structure differentiation of A. alternata induced by hydrophobic and fruit wax.