Cloning And Function Analysis Of AISte7Gene From Alternaria Longipes

Mitogen-activated protein kinase (MAPK) cascades are conserved signaling modules in the eukaryotic cells. They are involved in many major cell processes in fungi such as stress responses, vegetative growth, pathogenicity, secondary metabolism and cell wall integrity.Degenerate primers were designed at the conserved domain of other reported Ste7homologs of other fungi, and a209bp-cDNA fragment was obtained through reverse transcription PCR.Based on this fragment, gene special primers were designed and TAIL-PCR was employed to amplify the flanking regions of the known fragmen.These three fragments were assemblied with DNAman software and named AlSte7. The full length of AlSte7cDNA sequence is1356bp, which encodes491amino acids.There are three introns in the DNA sequence, and all introns obey the GT-AG rule.The AlSte7gene targeting vector was constructed according to Cho (2006), also a fragment containing was amplify from the recombinant plasmid with primer pairs M13F/M13R.Protoplasts of A.longipes were transformed,and PCR was used to screen AlSte7gene deletion mutants. Reverse transcription PCR method was used to verify AlSte7mutant.The wild type of A.longipes and AlSte7mutants were compared and contrasted. The AlSte7gene transformants could not produce fully differentiated spores, and the rate of growth on the PDA decreased by32%-48%. Also the dry weight of mycelia of AlSte7mutantin the PD broth was lower than that of the wild type. Pathogenicity assay was conducted with two different treatments-wounded and unwounded host plant leaves.The result showed that the AlSte7mutants could not infected the intact host leaves, but they recovered their pathogenicity on wounded leaves. There was no difference in the size of the lesions caused by the AlSte7mutants and the wild type strain. These results suggested that the reduced penetration capacity maybe,at least partially responsible for the pathogenicity defect in the AlSte7mutants.