Establishment of restriction enzyme mediated integration and construction of transformants library in Lasiodiplodia theobromae

Abstract:

[Objective] To establish the Restriction enzyme mediated integration (REMI) transformation system and a REMI transformants library of Lasiodiplodia theobromae. [Methods] REMI method was used to obtain transformants of L. theobromae. Linearized DNA of plasmid pUCATPH containing the hygromycin phosphotransferase gene (Hyg) was integrated into chromosomes of wild strain CSS-01s. Sensitive concentration of L. theobromae to hygromycin B was determined. Different enzymolysis systems and digest time on preparation of protoplasts were performed and effect of restriction enzyme quantity on percent conversion was also carried out. The REMI transformants library of CSS-01s was constructed with the optimal condition, and the transformants was confirmed by Southern blot. [Results] This study established REMI method of L. theobromae for the first time and constructed REMI transformants library containing about 6 000 transformants. Southern blot analysis revealed that the plasmid inserted into the corresponding restriction endonuclease sites located on the genomic DNA of five random transformants. [Conclusion] The results suggested that the optimal condition of protoplasts preparation was adding 2% Drislase+2% Snailase enzyme mixture and digested for 4 hours. The transformation efficiency reached to the highest when 30 U Hind Ⅲ was added into per transformation reaction. Transfotmants with differernt phenotype can be obtained using the above optimal conditions.