A mutant library of Magnaporthe grisea was constructed by restriction enzyme mediated integration (REMI)

作者：Wei S P^*; Wang G; Chi G B; Guo L Y; Peng Y L

来源：Agrochimica, 2008, 52(1): 34-48.

A mutant library with 49,542 individual transformants of Magnaporthe grisea was established using restriction enzyme mediated integration (REMI) procedure with four plasmids, pUCATPH1, pUCATPH2, pV2, and pCB 1004. Each plasmid was used in combination with one of nine restriction endonucleases (ApaI, EcoRI, EcoRV KpnI, HindIII, SacI, SmaI, SalI or XhoI) to transform M. grisea strain P131 and Y34. The factors affecting the transformation frequency were investigated as well. Results showed that the transformation frequencies varied with the different enzymes, the different concentration of each enzyme used, and the amount of plasmid DNA in the transformation mixer. At the same concentration, among 5 enzymes tested for P131, HindIII generated the highest amount of transformants, while SalI generated the lowest. Highest amount of P131 transformants obtained when enzyme concentration used is 100, 150, 100, 50 and 50 units per ml of tansformation mixture for KpnI, HindIII, SacI, SmaI and SalI, respectively. Meanwhile, with the increase of amount of plasmid DNA in the transformation mixture, the transformation frequency increased, but the number of tranformants generated per mu g of plasmid DNA decreased. The amount of plasmid DNA used also affected the integration nature of plasmid DNA on the fungal DNA and its effect appeared to vary with the enzyme used. Southern blot analysis of 102 randomly selected transformants suggested that the obtained mutant library contained diversiforin of gene mutation; 72% of them had single-locus insertion and 49% of them had single-locus, single-copy insertion. Results also revealed that frequency of REMI events varied among transformants generated by different enzymes. Among four enzymes tested, KpnI yielded the highest level of REMI events, whereas HindIII yielded the lowest. Among 26,649 transformants of P131, 16,660 transformants, which mediated by different enzymes were screened for phenotypic mutation. In total, 60 morphological mutants were found, which included 2 whitish, 1 yellowish, 22 slow growing, 17 sporulation-defect mutants, and 18 mutants with elongated or chubby spores. Pathogenicity test of these transformants on four rice cultivars revealed 31 pathogenicity defects, and 2 cultivar specificity mutants. Therefore, the established mutant library provides us with a large number of tagged, diversiform mutants, from which interested genes can be isolated and their function can be studied.

• 出版日期2008-2
• 单位中国地质大学（北京）; 中国农业大学