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

Abstract:

[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.

Key words:Alternaria alternata;calcium/calmodulin-dependent protein kinase (CaMK);bioinformatics analysis;infective structure differentiation