| Kloeckera apiculata strain 34-9, as a postharvest biocontrol yeast, has been shown to have high antagonistic activity against blue mold and green mold caused by Penicillium italicum and P. digitatum in citrus fruit. However, the problems of poor flocculence, low biomass, narrow antagonistic spectrums and antagonistic activity remained to be improved, have limited its commercial application. Therefore, it is necessary to carry out genetic improvement on the strain 34-9, in order to enhance its comprehensive characters and obtain excellent strains. Meanwhile, it will provide a theoretical basis to explore a safe and non-toxic biological agent.The aim of this paper is to modify the genetic characters of strain 34-9. It mainly included that the fusion was completed by using the inactivated parental protoplasts, the key factors of affecting the protoplast formation and regeneration were studied, the optimum conditions for inactivation were determined and the fusants were selected and identified. It also conducted a preliminary exploration of transformation system of strain 34-9 by using Agrobacterium-mediated transformation, lithium acetate transformation and electroporation transformation. The main results were as follows:1. We further confirmed the genus and species name of strain 34-9 by using rDNA-ITS molecular identification. The strain 34-9 was identified as K. apiculata, which was the asexual generation of Hanseniaspora uvarum.2. The proper protoplast preparation and regeneration conditions of strain 34-9 and Saccharomyces cerevisiae were determined separately. The proper conditions for strain 34-9:pretreatment 10 min with 0.05 mol/L EDTA-Na2 and 0.5%β-mercapto-ethanol at 28℃, cultivation time 16 h, enzymatic concentration 1.5% snailase, enzymatic time 3 h, osmotic stabilizer 0.8 mol/L KCl; The proper conditions for S. cerevisiae:optimum sporulation medium Kleyn, cultivation time 26 h, enzymatic concentration 1.5% snailase, enzymatic time 2 h, osmotic stabilizer 0.8 mol/L sucrose.3. The proper inactivation parameter of strain 34-9 and S. cerevisiae were determined respectively. After strain 34-9 was inactivated with 0.9% iodacetic acid in 10 min and S. cerevisiae was inactivated by heat in 15 min at 55℃, they could be fused by 35% PEG-4000 at 28℃.4. Using strain 34-9 and S. cerevisiae as parent strains, after protoplast inactivation, fusion, we screened one performance markable raised fusant RH-1, by the colony size, flocculation detection and antagonistic experiment. The fusant's flocculation formed faster than strain 34-9, which still had a good antagonistic activity.5. We preliminarily determined the electroporation conditions. The optimal electroprated stage for strain 34-9 was at intermediate logarithmic phage. Under the conditions of voltage of 1.5 kv,1 mol/L sorbitol,20μg/mL plasmid DNA, shock time 5 msec, resistance 200Ω, capacitance 25μF and 0.2 cm cuvettes, we obtained five transformants with high genetic stability. |
