Application Of RNAi Technology To Verify The Function Of AAP Gene In Grifola Frondosa

Grifola frondosa is a edible and medicinal fungus that is highly favored by consumers for its rich nutrients and medicinal components in its fruiting bodies.The acidic components in the mycelial fermentation broth have significant effects on the biological control of plant pathogens.In recent years,the whole genome sequencing results and functional annotations of G.frondosa have been published on the NCBI website,providing information resources for molecular level research on the structure and function of G.frondosa functional genes.However,there is a lack of technical means to efficiently improve the genetic system of ash tree flowers,which limits the research on functional genes of ash tree flowers.Based on high-throughput sequencing of G.frondosa,this study preliminarily analyzed gene data and functions in the transcriptome database,and determined the target gene AAP gene(involved in amino acids γ-Aminobutyric acid synthesis and transport);Construct an RNAi expression vector to infect the G.frondosa strain and obtain a transformed strain;Apply fluorescence quantitative PCR to analyze the differential expression of target gene AAP in transformants,and combine it with measurement of relevant metabolic indicators to determine the related functions of target gene silencing effect.The main conclusions are as follows:(1)38189 Unigenes were obtained from transcriptome sequencing of G.frondosa,and 12357 homologous protein sequences were detected in Swissprot database;Compare and annotate 1832 Unigenes with KEGG database,divided into 20 biological pathways;Compare annotations with GO database to 12234 Unigenes;Compare annotations with the COG database to 12248 Unigenes.There are 6261 Unigenes that have not been annotated in the database.Based on amino acid transport and metabolic function,the AAP gene was anchored in the GO database.The product is a stable hydrophilic amino acid transport protein composed of 533 amino acid residues,with 12 transmembrane regions and 49.53% content α-Spiral,17.45%extension chain,2.63% β-Corners and 30.39% irregular curls.By combining subcellular localization and transmembrane structural regions,it is analyzed that the AAP gene product is a transmembrane protein present on the cell membrane,and it is speculated that it is related to γ-Metabolism of aminobutyric acid is related.(2)According to the AAP transcriptome CDs sequence,determine the AAP target sequence,design primers,extract RNA from Grifola frondosa to obtain c DNA,and obtain the target sequence by PCR.The Golden Gate method was used to construct an RNAi expression vector.The RNAi expression vector was transfected into Agrobacterium EHA105 by electric stimulation,and the newly prepared G.frondosa protoplasts were infected.Nine resistant strains were screened through hygromycin selection pressure.Continue to culture and extract DNA,conduct PCR identification,and ultimately screen out 1 transformant.(3)The fluorescence PCR detection of AAP gene was performed on the original strain and transformed strain of G.frondosa.The results showed that the expression level of AAP gene in the transformed strain was only 48.5% of the original strain,and the gene expression was significantly downregulated,with an interference effect of over 50%.Determination of hyphae γ-The content of aminobutyric acid indicates that in the transformant strain γ-The content of aminobutyric acid decreased significantly compared to the original strain,while the p H value of the fermentation broth of the transformant strain increased significantly;The determination of total protein content in mycelium showed that the total protein content in the transformed strain decreased significantly compared to the original strain;The determination of soluble sugar content in the mycelium showed that the soluble sugar content in the transformed strain increased significantly and had a significant difference from the original strain;The changes of metabolic index data were basically consistent with the results of fluorescence quantitative analysis and transcriptome data analysis.In summary,RNAi verified that the AAP gene product of G.frondosa is an amino acid transporter protein,which is functionally related to the synthesis of transporters,γ-The synthesis and transportation of aminobutyric acid are related,which in turn affects the p H value of the fermentation broth of the strain and also affects the content of soluble sugars in the mycelium.It accumulates data for the application of edible polysaccharides,especially acidic edible fungi polysaccharides,and also provides resources for the medicinal value of fungi in the biopharmaceutical reserve of plant disease.