عنوان مقاله [English]
Introduction: Silybum marianum (L) Gaert.) Is a member of Asteraceae family, and silymarin is known as a valua effective substance in the plant, which is used to treat a number of diseases, such as liver disease. Chalcone synthase is one of the key enzymes in the production of silymarin. The fungal endophytes live in the tissues, without infection, and have a beneficial effect on the production of secondary metabolites of plants. Recently, several endophytic fungi from milk thistle (Silybum marianum (L) Gaert) have been isolated and studies were the chemical and molecular on its fungus has been considered. Therefore, this study aimed to isolate endophytic fungi from the seeds of milk thistle plant, and comparing the production of fungi and plant silymarin, as well as investigating the Chalcone synthase amino acid sequences associated with the L Silybum marianum and several other fungi from the NCBI database (http: //www.ncbi.nlm.nih.gov).
Materials and Methods: Therefore, this research was carried out to isolate endophytic fungi from the seeds of milk thistle plant, and to compare the production of fungal and plant silymarin. For this purpose, milk thistle seeds were collected from the area of Sari Agricultural and Natural Resources University and after disinfection, They sterilized in conditions, cultivated on MS medium. After appearance of fungi in the culture medium, the fungi were transferred to the PDA medium under sterile conditions the next for purification to WA medium. Fungal growth isolates were transferred to PDB medium and after 10 days of silymarin extraction Molecular identification of endophyte fungi isolates was performed by amplification of ribosomal areas, then the Alignment of the obtained sequence with the sequences on the NCBI site was compared with BLAST online software and identified with phylogeny tree of endophytic fungus. Investigate the bioinformatics of Chalcone synthase in L. Silybum marianum and several other fungi species,is use the NCBI database, was obtained by comparing domains and similar positions of Chalcone synthase, and was Investigating with the clustalo uniprot database. Then it was analyzed with CLC Genomics Workbench5.5.2 software and their specifications table was drawn up. The phylogeny tree of these sequences was drawn using the Mega7 software, using the Neighbor- joining
Results: The results of high performance liquid chromatography confirmed the silymarin production of fungi. The amount of silymarin for leaf samples of intact plant was 33.079 ppm and for the in vitro grown plant was 17.183 ppm and for the endophyte fungus was 7. 29 ppm. Using ITS, molecular identification of isolates was carried out and its species was identified as Alternaria alternata. Also, analysis phylogenic relationship of chalcone synthase, as a key enzyme for production of silymarin, between milk thistle and a few fungi showed its closest proximity to to Alternaria sp.
Conclusion: The study of the production of silymarin in fungi isolated from the seeds of milk thistle , in vitro and in vivo plants, along with the bioinformatics study of the pathway of production of Chalcone synthase enzyme, determined the role of endophytic fungi in the production of silymarin. The fungal isolate was also able to produce silymarin in laboratory conditions