Construction Of Genetic Transformation System In Agrocybe Aegerita And Functional Analysis Of Lignin Decomposition Enzyme Lac Gene Family

Agrocybe aegerita,an edible and medicinal fungus,was employed in this study.The genetic transformation system was established,and the efficient and appropriate promoters was selceted for constructing a general vector for high expression;through genome sequencing and sequence comparison,nine laccase genes are amplified,and the high expression transformaants of them were obtained by PEG mediated protoplast transformation;the expression level of laccase was determined by q RT-PCR.The crude enzyme was obtained by liquid culture,and the enzyme activity were measured by spectrophotometry,which laid the foundation for the nutritional physiology and genetic research of A.aegerita.The main results were showed below:（1）The genetic transformation system of A.aegerita was established:the protoplast of A.aegerita was used as the receptor with the hygromycin resistance gene（hph）was used as the screening marker,and the egfp gene was used as the reporter gene.The genetic transformation system of A.aegerita was constructed by PEG mediated method.The results showed that 150μg/m L of hygromycin could inhibit the growth of A.aegerita.The yield of protoplasts was the highest when the mycelium was hydrolyzed by enzyme at 30℃for 3 hours.The results showed that the exogenous genes hph and egfp had been successfully transferred into the protoplasts of A.aegerita by the screening of hygromycin,PCR and fluorescence microscopy.（2）The establishment of high-efficiency expression system:5 promoter fragments of actin,gpd and pumgpd with different lengths were selected for replacing the original promoter,and the expression level of target gene was analyzed.A.aegerita is a delicious fungus with high economic value.The genetic characterization and gene function of A.aegerita can be recovered through the construction of highly expressed genetic transformation system.A.aegerita strains YSG was employed in this study,and the constructed plasmid using multi-fragments recombination was transformed to protoplast.The expression levels of targeting gene driving by five promoter fragments of actin,gpd and Pumgpd were analyzed in this study.Among the transformants carrying plasmids p Aa-actin-1 or p Aa-actin-2 constructed by promoter elements of actin,the numbers of the targeting gene expression level more than 3times were 50%and 33.33%with the highest expression levels to 10.45 and 6.23times of the control,respectively.Accordingly,the numbers of the transformants carrying p Aa-gpd-1,p Aa-gpd-2 and p Aa-Pumgpd with expression level more than 3times were 0,28.57%and 25%with the highest expression levels to 2.93,7.75 and4.31times of the control,respectively.The yield of transformants with high expression level of targeting gene driven by actin promoter was higher compared with that of gpd and Pumgpd,which indicated actin promoter was suitable for the construction of genetic transformation system of A.aegerita.（3）Sequence characterization of laccase gene family and enzyme activity analysis of overexpression transformants of YSG strain:Nine laccase genes were obtained by genome annotation information and sequence analysis.The clone was connected to the high efficient expression vector after the gene whole length.After the expression of laccase transformants determined by q RT-PCR,the high-efficiency expression transformants were selected,and ABTS,guaiacol and 2,6-DMP were selected for enzyme activity determination,The effects of p H on the laccase activity of nine laccase overexpression transformants of A.aegerita were investigated in different p H environments of 2.8,3.3,3.8,4.3,4.8,5.3 and 5.8,and the effects of EDTA on the laccase activity of nine laccase overexpression transformants of A.aegerita were investigated.The results showed that Laccase in A.aegerita may be acidic protease,ABTS was the best substrate for enzyme-induced reaction,and Cu²⁺promoted laccase activity,except K⁺、Na⁺、Mn²⁺、Ca²⁺、Fe²⁺、Fe³⁺、Mg²⁺、Zn²⁺metal ions on laccase activity.（4）The effect of overexpression of laccase gene family on substrate decomposition and fruiting development:compared with wild strain YSG,the transformants of lac1 overexpression strain could produce fruit body more earlier.