Agrobacterium Tumefaciens-mediated Genetic Transformation System Of Gene Silencing Of FIP-fve And Gene Cloning Of A Fungal Immunomodulatory Protein From Chroogomphis Rutilus And Its Expression In Pichia Pastoris

The fungal immunomodulatory proteins(FIPs), extracted and identified from macrofungi, are a kind of bioactive proteins with immunomodulatory activities in recent decades, whose structures are similar to natural immunoglobulins, and their biological functions resemble lectins. Until now, more than 10 fungal immunomodulatory proteins from different species have been discovered, which share a high homology. Besides, these FIPs possess antitumor, anti-allergic, agglutination blood cells and other biological activities in vitro.This study includes two parts. The first one is Agrobacterium tumefaciens-mediated genetic transformation of Flammulina velutipes using silenced FIP-fve, including the establishment RNAi vector of FIP-fve and the agrobacterium-mediated genetic transformantion system establishment. In order to study the native biological function of FIP-fve, we have cloned pros and cons of FIP-fve gene and constructed its RNAi vector. Using Agrobacterium LBA4404, the recombinant RNAi vector was transformed into the mycelium of F. velutipes. After optimizing the transformation, the positive transformants of F. velutipes mycelia were screened by hygromycin resistance. Using PCR and phenotypic contrastive observation to detect and view the transformants, we could preliminarily conclude the effect of FIP-fve on F. velutipes stain. Double enzymes digestion showes that the RNAi vector of pTCK303-fve(F)-fve(R) has been successfully constructed. The liquid-nitrogen transformation method was use to obtain the recombinant Agrobacterium transformants. The optimal concentrations of Hygromycin B were 9 μg·mL-1 and 6 μg·mL-1 in solid and liquid culture media, respectively. And the optimal concentration of Cefotaxime was 600 μg·mL-1. As for the explants, the mycelia of F. velutipes were infected 30 min by Agrobacterium LBA4404 bacteria solution (OD600=0.5), and were co-cultured with AS (200 μmol·L-1)for 3 days at 25℃. Finally, we obtained five positive transformants, which exhibited Hyg resistance and could inhibit the growth of F. velutipes to some extent after subculture. In addition, the transformation rate was 8.33%. Observing the phenotypes, the growing rate of transformants mycelia was apparently slower than the wild type of F. velutipes mycelia. Also, the five transformants differ in the growth. So it can be concluded that FIP-fve could have a potential regulating effect on the growth or development of F. velutipes during the culture period of the mycelium, which also can provide direct evidence and research foundation for the biological function of FIP on fungi themselves.The second part was gene cloning of a novel fungal immunomodulatory protein from Chroogomphis rutilus and its expression in Pichia pastoris. For further studies on active ingredients and biological functions of C. rutilus, we used homology-based cloning to obtain a new gene (named FIP-cru) from the genomic DNA of the edible mushroom C. rutilus. Then FIP-cru was integrated into an expression vector pPIC9-FIP-cru and transformed into P. Pastoris GS115 to obtain the efficient expression of recombinant protein. Finally, the biological activities of rFIP-cru were detected. Nucleotide sequencing showed that FIP-cru was composed of 342 bp, which included 113 amino acids with a calculated MW of 12703 Da. BLAST indicated that FIP-cru shared highest homology with FIP-fve(F velutipes) at an identity of 78% of the amino acid sequence. SDS-PAGE and Western blot analysis were detected that two extra protein bands of approximately 14 and 17 kDa were observed, While Western blot using anti-6xHis antibody indeed confirmed the correctness of expressed rFIP-cru, as a 6×His-tag was added in the C-terminal of rFIP-cru to be purified by the pufication histidine column of HisTrap FF, and the best expressed in P. pastoris GS115 at the fourth day with a yield of 148.5 mg-L-1. In vitro, rFIP-cru was capable of agglutinating mouse and sheep red blood cells with the concentration greater than 2 μg·mL-1. In addition, rFIP-cru could apparently stimulate the cell viability of murine splenocytes with the most effective concentration at 4 μg·mL-1. In this concentration, rFIP-cru could enhance the level of IL-2 release up to 476.2 pg·mL-1. The novel FIP-cru gene was functionally and effectively expressed in P. pastoris, and rFIP-cru displayed good biological activities. Overall, the research provides the theoretical basis and experimental evidence for extensive biological functions of FIP and improves the development and application of Chroogomphis rutilus.