| Apple marssonina blotch is mainly responsible for causing premature defoliation of apple. The complex and diversification symptoms in the field and the difficulty to obtain the pure culture of this fungus makes the research progress of the disease slow. At present, most research in the field are only stay in medicament control , the basic research on pathogeny and molecular biology was little.Calmodulin (CaM) gene plays an important role in Ca2+ signal transduction system, many experiments proved it participate in the pathogenic fungi control process. Multiphasic analysis is a very good tool for diseases diagnosis and classification of pathogen. Some research reported calmodulin gene can was used for phyletic evolution by PCR amplification and sequencing analysis.We amplified a homologous fragment of CaM gene by the RT-PCR from Marssonina coronaria. The specific primers were designed according to the homologous fragment to obtain the full-length cDNA sequence by the RACE (Rapid Amplication cDNA ends) after Blast with the known sequence. The length of 3′RACE and 5′RACE are 629bp and 599bp respectively, it is easy to get a long 887bp full-length cDNA sequence by splicing and matching. Through the analysis on the NCBI ORF Finder we got a length of 450bp ORF sequence which encode 149 amino acid, there is not exist any temination codon at the dowmstream of the ORF and a poly A tail at the end of the 3' directionality. We found 100%,100%,99.3%,98.6%,85.9% identical to that of Setosphaeria turcica, Colletotrichum gloeosporioides, Gibberella zeae, Magnaporthe grisea, Sclerotinia sclerotiorum and Phytophtora sojae respectively, it proved the high conservative of CaM genes. The CaM gene belongs to the EF-hand. BioEdit analysis indicate that there exists four calcium binding domains (EF-hand structures) and the conserved Gly and Glu residues inside these domains. The CaM gene from Marssonina coronaria like C. gloeosporides and S. turcica donnot change both conserved residues and domains.DNA as amplification template, a 833bp fragment was amplified based on the specific primers which designed according to the ORF. The sequence analysis indicated that the ORF contained six exons and five introns, all of them obey the rules of the junctions GT/AG and the lariat consensus sequence CTRAY is found in introns part except the fourth. We found its introns in number and position was changed in some extent after compare with other fungi.The symptom of the apple marssonina blotch is complex and diversity. To analysis the CaM gene and ITS sequence the 16 isolates DNA of Marssonina coronaria as template was amplified respectively. Results showed that four sixteenth isolates (the rate of variation isolates is 25%) of variation degree occurred for CaM gene and 7 variations (the rate of variation site is 0.8%) in which 6 variations are in coding region. Nine sixteenth isolates (the rate of variation isolates is 56.25%) of variation degree occurred for CaM gene and 6 variations (the rate of variation site is 1.0%). There are 4 isolates have the same variation sites in two genes. The variation of ITS has bigger change than CaM whether the number of variation isolates or sites, however both changes are stable on the whole. It is easy to find the variation are not unified with the complicated diversity of the symptoms which indicated that there is no evident relevant between them.Furthermore, we found that CaM gene specificity inhibitor trifluoperazine TFP was able to inhibit conidial germination in certain level and the spore didn′t change whether in size or shape. It proved CaM gene may play an important role in infection process.
|
PDF Full Text Request