Establishment Of Transformation System For Biocontrol Yeast Kloechera Apiculata (34-9) Against Postharvest Diseases Of Citrus

Kloechera apiculata strains34-9is an effective biocontrol microorganism in resisting postharvest pathogens of citrus. Molecular improvement on the strain34-9is necessary to be carried out to enhance its biocontrol activity and commercial application. In this study, we mainly explored the methods of Kloechera apiculata transformation, including LiAc, electroporation, spheroplasting and other methods. Based on the right transform method, we intend to clone and highly express the disease resistance genes from Kloechera apiculata. Our previous work showed that2-phenylethanol is the main antifungal compound in34-9, which can control the blue and green mould of citrus. On this basis, we isolated key enzyme gene PDC of phenylethanol production and expressed in yeast Pichia pastoris. This experiment would provide a experimental base for large-scale expression of PDC in34-9and enhancing the antifungal effect. The main results are as follows:1. The sensitivity of34-9to different Hygromycin B concentrations was examined. We showed that Hygromycin B at100μg/mL could serve for the selection of transformants. The recombinant vector containing homologous arm was successfully transformed into the wild-type host strain X-33, implying that the recombinant plasmid can be used to transform into yeast.2. The transformation was carried out by the LiAc method, spheroplasting and other methods. The factors in different concentrations were screened, including yeast cells, undigested circular plasmid, linearized plasmid, transformation reagent and recovery time after transformation, etc. Although processes were repeated many times, colonies were not found on selection plates. We found that there was no transformant when the activity of protoplast-associated DNase was extremely strong as well as it was inactivated. The results suggested that the DNase was not the main inhibitor for transformation.3. In order to optimize electroporation, the cell viability was measured under different applied voltages. We found that the1.5kV was best for transformation. Considering the other factors, the conditions which is109cells/mL,1.5kV,50μg plasmid DNA,25μF and200Ω, was adopted in the study. Few transformants were observed though the efficiency was not high.4. A pyruvate devarboxylase PDC gene which is1695bp and encodes594amino acids was isolated from the yeast Kloechera apiculata. Then, the secretory recombinant vector was transformed into X-33strain. After induced by methanol, the postitive transformants expressed successfully and protein worked normally.