Pichia membranefaciens, which was isolated from the surface of peach fruits, showed effective biocontrol capabilityagainst rhizopus rot of peach fruits. Aminoglycoside antibiotic G418 can inhibit the growth of P. memb...Pichia membranefaciens, which was isolated from the surface of peach fruits, showed effective biocontrol capabilityagainst rhizopus rot of peach fruits. Aminoglycoside antibiotic G418 can inhibit the growth of P. membranefaciens. Theminimal inhibitory concentration of G418 to P. membranefaciens in YPD medium was 100g mL-1. We constructed aphosphoglycerate kinase (PGK) promoter-driven neoR expression cassette, which was called pFL61-neo. The biocontrolyeast P. membranefaciens was transformed with pFL61-neo by lithium acetate method. Expression vector pFL61-neoconferred P. membranefaciens drug resistance to 100g mL-1 G418. The transformant could keep a high percentage ofplasmid-containing of transformant with 67.87% after 50 generations in non-selective medium. The result showed that P.membranefaciens could recognize the promoter and terminator of PGK and direct the expression of heterologous neoRgene. Expression vector pFL61-neo could exist stably in P. membranefaciens. Therefore, it is feasible to utilize G418-resistance as a dominant selectable marker for heterogenous gene expression in antagonist P. membranefaciens.展开更多
[Objective] The study aimed to establish a protoplast transformation system in Alternaria tenuissima. [Method] The protoplast of A.tenuissima was firstly prepared by enzymolysis method; then the yielded protoplast was...[Objective] The study aimed to establish a protoplast transformation system in Alternaria tenuissima. [Method] The protoplast of A.tenuissima was firstly prepared by enzymolysis method; then the yielded protoplast was transformed by G418 resistant DNA plasmid using PEG/CaCl2 method. [Result] The growth phenotype and PCR detection showed that resistance gene had integrated into A.tenuissima genome. The transformation efficiency of this method reached per μg DNA 3-4 transformants. After subculture thrice under nonselective condition, G418 resistance could still inherit stably. [Conclusion] The transformation system of A.tenuissima was successfully established, which laid basis for studying of the gene function of Alternaria tenuissima.展开更多
基金supported by the National Science Fund for Distinguished Young Scholars of China(30225030)the National Natural Science Foundation of China(30430480).
文摘Pichia membranefaciens, which was isolated from the surface of peach fruits, showed effective biocontrol capabilityagainst rhizopus rot of peach fruits. Aminoglycoside antibiotic G418 can inhibit the growth of P. membranefaciens. Theminimal inhibitory concentration of G418 to P. membranefaciens in YPD medium was 100g mL-1. We constructed aphosphoglycerate kinase (PGK) promoter-driven neoR expression cassette, which was called pFL61-neo. The biocontrolyeast P. membranefaciens was transformed with pFL61-neo by lithium acetate method. Expression vector pFL61-neoconferred P. membranefaciens drug resistance to 100g mL-1 G418. The transformant could keep a high percentage ofplasmid-containing of transformant with 67.87% after 50 generations in non-selective medium. The result showed that P.membranefaciens could recognize the promoter and terminator of PGK and direct the expression of heterologous neoRgene. Expression vector pFL61-neo could exist stably in P. membranefaciens. Therefore, it is feasible to utilize G418-resistance as a dominant selectable marker for heterogenous gene expression in antagonist P. membranefaciens.
文摘[Objective] The study aimed to establish a protoplast transformation system in Alternaria tenuissima. [Method] The protoplast of A.tenuissima was firstly prepared by enzymolysis method; then the yielded protoplast was transformed by G418 resistant DNA plasmid using PEG/CaCl2 method. [Result] The growth phenotype and PCR detection showed that resistance gene had integrated into A.tenuissima genome. The transformation efficiency of this method reached per μg DNA 3-4 transformants. After subculture thrice under nonselective condition, G418 resistance could still inherit stably. [Conclusion] The transformation system of A.tenuissima was successfully established, which laid basis for studying of the gene function of Alternaria tenuissima.
