产黄青霉突变体库的构建及鉴定植物学专业论文.docxVIP

河北科技大学硕士学位论文 11 万方数据 AbstractAbstract Abstract Abstract Penicillin is one of the important strategic antibiotics．Penicillium chrysogenum， penicillin producing strain，is an important industrial microorganism which has great industrial value．Current industrial strains are derived from a single natural isolate of P chrysogenum NRRL 1 95 1．The pennicillin production increased by more than 1 000 times． So it is difficult to improve pennicillin production by traditional breeding and fermentaion technology．It is necessary to obtain a pennicillin hi曲一producing strain by modem molecular biology technology．This study is intended to improve the industrial P chrysogenum strain by genetic engineering technology to increase the yield of penicillin． The wildtype strain in our research is industrial P chrysogenum．The exogenous bleomycin resistant gene，linked谢th T-DNA which Was synthesized by PCR，was transformed into P chrysogenum protoplast mediated by PEG．A large number of transformants were screened by antibiotic．The mutants obtained in this way laid the foundation for screening penicillin high-producing mutant strains．To identify if the exogenous gene Was transfomed into P chrysogenum，a simple and high throughput genomic DNA extraction method was developed．The method is based on enzyme lysis combining、Ⅳitll heating and ultrasonic treatment．The genomic DNA isolated by the method Can be used as PCR template．Two enzymes treatment，6 mg／mL snailase and 4000 U／mL cellulase，is the best way to lyze the mycelia．The mycelia is treated by the following steps，ultrasonic treatment 10—15 min，lyses at 28。C for 14 h，heating at 90-98。C for 10-15 min，then add 50—100 p．LddH20，mix until the layers separated，The supematant could be used as the PCR template directlyand the PCR bands are clear and accurate． The mutants were cultivated with 24一well plates screen the pennicillin production based the high-throughput detection techniques．The results showed that 324 transforments were identified as mutant