Screening The Interaction Proteins Of ASF1,STE2 And STE3 In Stemphylium Eturmiunum

The Stemphylium genus is an important group of filamentous ascus fungus.In nature,most of the fungi are present in an amphoteric form,but there is also a small part of the sexual reproduction stage,while the Stemphylium eturmiunum is one of the species which has sexual stage in nature.In order to study the differences in the evolution of S.eturmiunum from other cultivars,we find that the evolution of fungi is regulated by several sex-related factors.In the model fungi Saccharomyces cerevisiae,Neurospora and other fungus,studies have shown that the pheromone receptor genes STE2 and STE3 play a role of regulation of sexual development pathways.In addition,Asf1 also has regulatory effect on sexual development.Studies have shown that ?STE2,?STE3 and ?ASF1 single mutant strains could not produce sexual reproduction stage in the appropriate environment.The fungal heterothallism occurs between two cell types: a and ?,and the cell type is controlled by secreted pheromones.Two types of cells produce two pheromone precursors a and ?,respectively,and two corresponding pheromone receptors,STE2 and STE3.In the sexual reproduction process,the processed pheromone ? factor is recognized by the STE2 receptor on the surface of the a cell to initiate the mating signal.Similarly,the processed pheromone a factor is recognized by the STE3 receptor on the ? cell surface to initiate the mating signal.In order to further explore the effects of knockdown of pheromone receptor genes STE2 and STE3 on the expression of other upstream and downstream genes,we conducted the following study: S.eturmiunum as the experimental material,using Trizol method ?STE2 and ?STE3 single mutant strains were extracted and sequenced.The results showed that knockdown of pheromone receptor gene STE2 or STE3 could affect the normal transcription of a series of genes upstream and downstream.After analysis and prediction,we selected a series of differentially expressed genes,trying to screen the interaction of the pheromone receptor STE2 and STE3.By comparison of gene sequences,we found that the STE2 and STE3 genes each contained an intron.We obtained the gene sequence without introns by Overlap PCR,and we found that pheromone receptors STE2 and ORF07010(glycoside hydrolyzate family 32 protein)were able to interact in yeast during subsequent yeast two-hybrid experiment.ASF1 is very conserved in evolution,and it is a molecular chaperone of histone H3/H4,which plays an important role in the biological processes of multiple chromatin levels,such asgene transcription,DNA replication and repair.By homologous sequence alignment,we found that the Asf1 gene was included in the S.eturmiunum genome.Based on the information of the reported Asf1 gene,the specific primers were designed to clone the anti-silencing factor Asf1 and histone H3 and H4 genes in S.eturmiunum.At the same time,Asf1 does interact with histone H4,but does not interact with histone H3,which may be related to its specific function,using in vivo yeast two-hybrid and in vitro Pull-down experimental techniques.A series of transcriptome sequences of the pheromone receptor ?STE2 and ?STE3monoclonal mutant strains,and the anti-silencing factor Asf1 interaction protein were studied in this paper.In order to study the effects of these three protein structure and function to lay a good foundation for the in-depth exploration of biological origin and differentiation process to provide information accumulation.