Construction Of Fungal Immunoregulatory Protein Overexpression Vector And Genetic Transformation Of Flammulina Velutipes

Fungal immunoregulatory proteins?FIPs?are small molecule proteins extracted from higher fungi that have a similar structure to the immunoglobulin re-ligation region and are biologically similar to plant lectins.A variety of fungal immunomodulatory proteins have been found to have biological activities such as agglutinating blood cells,anti-tumor,anti-allergy,and lymphocyte proliferation,which have good clinical application prospects.At present,the functional research of FIP mainly focuses on the biological effects on humans and animals,and its biological function on the fungus itself is still unclear.In this study,Flammulina velutipes was used as the research object to construct FIP-fve and LZ-8overexpression vectors,and transformed into Flammulina velutipes by Agrobacterium-mediated transformation to obtain positive transformants;FIP-fve gene overexpression,FIP-fve gene silencing and wild Flammulina velutipes was cultivated,contrast and analysis of phenotypic changes,protein extraction and protein detection and analysis,to lay the foundation for the study of FIP-fve on the biological function of Flammulina velutipes.Firstly,the FIP-fve gene and LZ-8 gene were cloned and the corresponding expression vector was constructed.Two FIP-fve overexpression vectors were constructed,one was pCAMBIA1301-FIP-fve,which was 35S in pCAMBIA1301 vector.The promoter is ligated into the target gene FIP-fve,and the other is pCAMBIA1301-GPD-FIP-fve.The endogenous promoter GPD gene of the Flammulina velutipes and the FIP-fve gene are inserted into the multiple cloning site of pCAMBIA1301,followed by construction of the LZ-8 gene.The overexpression vector pCAMBIA1303-GPD-LZ-8 was also inserted into the multiple cloning site of the GPD promoter gene and the LZ-8 gene.Secondly,Agrobacterium tumefaciens-mediated transformation was used to establish and improve the over-expression vector of Flammulina velutipes genetic transformation system.The infecting material was 1 cm² of Flammulina velutipes mycelium,and the infective condition was Agrobacterium liquid concentration OD₆₀₀00 of 0.5,Agrobacterium AS was added to the bacterial solution to a final concentration of 200?mol�L^-1,and the infestation time was30 min.The hyphae were cultured for 3 days at 25?in a co-culture medium containing 200?mol�L^-11 AS,and screened with YPG medium containing 9?g�mL^-11 Hyg,and finally FIP-fve overexpressed Flammulina velutipes transformants were obtained.The transformation efficiency of pCAMBIA1301-FIP-fve to Flammulina velutipes was 17.5%,the transformation efficiency of pCAMBIA1301-GPD-FIP-fve to Flammulina velutipes was 22.5%,and the transformation efficiency of pCAMBIA1303-GPD-LZ-8 to Flammulina velutipes was 10%.Finally,the selected FIP-fve gene overexpressing Flammulina velutipes hyphae cultured,and the FIP-fve gene was silenced Flammulina velutipes and Flammulina velutipes was cultured in laboratory,until the fruiting bodies matured,and the phenotypic changes of Flammulina velutipes were observed and extracted.The protein is subjected to protein detection analysis.The number of mushroom primordium of wild type Flammulina velutipes was 46,and the number of mushroom primordium of three strains of FIP-fve overexpressing Flammulina velutipes of pCAMBIA1301-GPD-FIP-fve were 69,55 and 71;the number of mushroom primordium of three strains of FIP-fve overexpressing Flammulina velutipes of pCAMBIA1301-FIP-fve were 92,116 and 51 mushrooms.The total protein content of Flammulina velutipes fruit body was tested by Bradford method.The results showed that the protein concentration of Flammulina velutipes overexpressed by FIP-fve gene was similar to that of wild type Flammulina velutipes,while the total protein concentration of Flammulina velutipes with FIP-fve gene silencing was relatively low.In addition,the content of FIP-fve of the target protein was analyzed by SDS-PAGE.The results showed that the content of FIP-fve in wild-type Flammulina velutipes was 0.73 mg�g^-1,and the FIP-fve content of the three FIP-fve over-expressing Flammulina velutipes of pCAMBIA1301-GPD-FIP-fve was 0.75mg�g^-1,0.76 mg�g^-11 and 0.75 mg�g^-1;the FIP-fve content of three FIP-fve overexpressing Flammulina velutipes of pCAMBIA1301-FIP-fve was 0.80 mg�g^-1,0.71 mg�g^-11 and 0.76mg�g^-1;the FIP-fve content of Flammulina velutipes of FIP-fve gene silencing was 0.71mg�g^-11 and 0.69 mg�g^-1,further tested by Western Blot,and the results showed that the expression of FIP-fve in Flammulina velutipes of FIP-fve overexpressed was increased,and the expression of FIP-fve in Flammulina velutipes of FIP-fve gene silencing was decreased.