Study On The Method Of Improving The Biocontrol Effectiveness Of Yeast By Transforming Technology

Antagonistic yeast,as an important biocontrol microorganism of postharvest fruits,has broad prospects for replacing or reducing the use of chemical fungicides,ensuring human and animal health,and reducing environmental pollution.However,common antagonistic yeasts are difficult to achieve or even close to the antifungal effect of chemical fungicides.The use of genetic transformation technology can effectively improve its biocontrol efficacy.In this research,Rho1,Ace-AMP1,AC-AMP2 and MiAMP1 were studied for their stable inheritance and expression in Saccharomyces cerevisiae and Pichia pastoris GS115 strains.The effects of overexpression of rhol gene in recombinant S.cerevisiae on CWI signaling pathway and the biocontrol efficacy of pear fruit diseases were explored,meanwhile the related antibacterial mechanism was revealed.The efficacy of Ace-AMP1,AC-AMP2 and MiAMP1 recombinants on controlling postharvest diseases of pears were tested.Moreover,the possibility of transforming antimicrobial peptides and other foreign genes into unconventional yeast(Cryptococcus laurentii)was investigated..The main findings are as follows:1.RT-qPCR and Western-blotting results showed that rho1 gene could be stably and efficiently expressed in recombinant strains SC/rhol after induced by galactose.At the same time,the analysis of the recombinant yeast cell wall polysaccharides showed that the content of glucan and chitin in the recombinant yeasts was significantly higher than that in the wild yeast,while the mannose content was decreased,and the total amount remained unchanged.This suggests that Rho1,as a major regulator of CWI signaling pathway,can significantly activate the activity of protein kinase C(Pkc1),trigger the mitogen-activated protein(MAP)kinase cascade,and up-regulate the genes involved in cell wall synthesis and actin rearrangement in the pathway(Pkc1,Fks1,Fks2,Chs3,and Rlm1).2.The four yeast strains(SC,SC/pYES6,SC/rho1,and SC/rhol-induced)were able to effectively induce pear fruit resistance to Penicillium expansum in both living and inactivated states,and under the induction culture,SC/rhol-induced strain had the best antifungal effect.The investigation of antifungal mechanism of the recombinant yeasts showed that compared with SC,SC/pYES6 and SC/rhol,the recombinant yeast SC/rhol-induced could not increase the number under the same growth conditions of fruit wound,thereby increasing nutrition and space competitiveness.At the same time,the antifungal test results of yeast metabolites against R expansum showed that overexpression of rho1 gene does not produce antifungal substances in the culture.The cell wall of the recombinant yeast SC/rhol-induced had a stronger ability to induce pear fruit resistance,and the gene expression of PR4,PRl-like,and CHI of pear fruit were significantly higher than those in the other three groups.The results of cell wall analysis in chapter one showed that introduction of rho1 gene and induction of expression increased the chitin and glucan content of the recombinant yeast cell wall,making the cells more capable of inducing host resistance.Combined with the results of the cell wall analysis in Chapter 1,it can be concluded that introduction of the rhol gene increased the content of chitin and glucan in the cell wall of the recombinant yeast,making the recombinant strain SC/rhol-induced more capable of inducing host resistance.3.Based on the codon bias of Pichia pastoris GS115,the sequence of Ace-AMP1,Ac-AMP2 and MiAMP1 was optimized and the Kex2(AAAAGA)restriction site was added to ensure the natural activity of the N-terminus of the target gene.Constructed into pPICZa A vector and transformed into competent cells of GS115.PCR and sequencing showed that antimicrobial peptide(AMP)genes had been recombined into GS115 chromosome and designated as GS115/Ace-AMP1,GS115/Ac-AMP2,GS115/MiAMP1 and GS115/pPICZ?A,4.After induced by methanol,the AMP genes of recombinant yeasts GS115/Ace-AMP1,GS115/Ac-AMP2,and GS115/MiAMP1 were all significantly up-regulated.Western-blotting analysis showed that only in the recombinant strains transformed with AMP genes can the band be detected that matches the expected molecular weight.Results of the determination on AMPs showed that they have been successfully transformed into GS115 and expressed efficiently.The antifungal test results of AMPs in vitro and in vivo indicated that they can effectively inhibit the growth of spores of P.expansum at a low concentration.The transformation of AMP genes can also significantly increase the biocontrol efficacy of P.pastoris.5.The GAP promoter sequence and 25S rDNA sequence were obtained by PCR amplification of whole genome DNA of P.pastoris and C.laurentii,which were constructed by Clonexpress technology into pPICZa A/Ac-AMP2.The PCR and sequencing of the recombinant expression vector revealed that the GAP promoter has successfully replaced the AOX1 promoter,and 25S rDNA has been successfully constructed.