| The black spot which happened on cruciferous vegetables and caused by A.brassicicola is a serious problem in the agricultural production, the previous studies showed the Fus-MAPK signaling pathways played an important role in regulating the pathogenic process in A.brassicicola, prior to anchor role in signal transduction specificity has been studied, but not detailed study was made to the scaffold protein function.The paper mainly using A.brassicicola as the object, by constructed and explored the AbSte5 gene deletion mutant in view of the MAPK signal transduction pathway of Ste5 gene fuction. The main results are as follows:Using saccharomyces cerevisiae Ste5 gene sequences to blast in the local A.brassicicola ATCC96836 genome database in order to find the Ste5 homologous gene location and download sequences. Designed specific primers according to the A.brassicicola Ste5 gene sequence and amplificated AbSte5 3327bp-DNA fragment which encodes 1087 amino acids,then obtained a 3276bp-cDNA fragment through RT-PCR technique. So the AbSte5 gene contains one intron that obeys the laws of GT-AG rule.Through SMART protein fuction prediction website analysed the Ste5 protein sequence,and compared with the Ste5 homologous genes from other fungi, then found that the sequence homology is low, but the amino acid sequence on the structure has a very high similarity, the homology Ste5 genes have a RING, PH, vWA fuction domain, so that we predict the Ste5 gene from A.brassicicola is also a scaffold protein.In the paper, we constructed the AbSte5 gene targeting vector pUCATPH-AbSte5 and the PCR fragment amplified from the new constructed plasmid with primers pairs M13F/M13 R,through PEG method transformed the protoplasts of A.brassicicola. Furtherly, the AbSte5 gene deletion mutants was validated and screened by PCR, through the southern hybridization technology draw a single copy gene insertion loss.Used reverse transcription PCR method to verify Ab Ste5 mutant. Compared with the wild type in the colony phenotype, mycelium and conidium morphology, melanin and pathogenic, we found that the growth rate of AbSte5 gene deletion mutant in PDA decreased, and could not produce conidium, when using in vitro lesf inoculation method without artificial stabbed cabbage leaf, AbSte5 gene deletion mutant can’tinfect cabbage leaf, when inoculated stabbed cabbage leaf, also lost in pathogenicity. We amplified the AbSte5 whole gene and used pCB1532 as framework plasmid to build complementary vector to transform AbSte5 mutant strain protoplast to get the complementary,we found the biological character of the complementary returned to the wild type. Presumably,AbSte5 gene was closely related to the mycelial morphology, formation of conidia and pathogenicity and so on. |
