| Kloechera apiculata strain34-9is an effective biocontrol agent which was isolated from soil of a citrus grove, having the action modes with postharvest diseases in citrus of competition for nutrient resource and the antibacterial substances phenylethyl alcohol procured. However, as for wild-type strain, it exist some problems when it is used for commercial application, such as low biomass and unstable control effect In order to improve the biocontrol activity, we mainly explored the methods of34-9transformation, including spheroplasting and Agrobacterium tumefaciens-medisated transformation (ATMT). We initially established A.tumefaciens-mediated transformation system of34-9, which can help us to the molecular genetic analysis of34-9subsequently.The main results were as follows:1. The optimal conditions for spheroplast formation and regeneration were as follows: yeast cells were in the ogarithmic growth phase after12h, the yeast concentration was108CFU/ml,0.7mol/L NaCl as the osmotic stabilizer,0.3%β-mercaptoethanol was added into EDTA-Na2as the pretreatment, the enzyme preparation was used for1.5%snail enzyme and0.4%cellulase enzyme,28℃shaker during the enzymatic process.2. We primarily set up a genetic transformation system of34-9by ATMT. Various transferring factors, such as co-cultivation time, concentration of yeast and A. tumefaciens, types of membrane for co-cultivation were optimized in this study. The transformation system includes A. tumefaciens strain AGL-1harboring binary vector pTFcm as DNA donor, concentration of AGL-1was OD6000.3,34-9suspension at107CFU/mL, add Acetosyringone200μmol/L into cocultivation and pre-culture, After co-cultivation for48h at25℃in the dark, we successfully transformed34-9and obtained the transformant strains.3. As a result, some transformants have been obtained by ATMT, many of them exhibited stable inheritance after ten rounds of successive cultures. PCR amplification showed that the hph gene was successfully inserted to genome DNA of34-9. |
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