Analysis Of Grifola Frondosa By RNAi Technique Function Of HXT2 Gene In Soluble Sugar Metabolism

Grifola frondosa,also known as Polyporus frondosus and maitake,is a high-grade rare edible/medicinal fungus with great development prospects.G.frondosa polysaccharide can inhibit tumor cells,activate the host immune system,have antiviral,anti-radiation,liver protection,and regulate blood lipid and blood pressure.Based on the transcriptome sequencing data,this study preliminarily analyzed the gene data and function of G.frondosa,anchored the RNAi(RNA interference)target gene(HXT2 gene)based on the glucose metabolism function,and predicted and analyzed the function of HXT2 gene using bioinformatics methods;RNAi binary vectors of gene target sequences were designed by RNAi technology to infect G.frondosa protoplasma to obtain transformed strains.The difference of target gene expression in RNAi transformed strains was verified by relative fluorescence quantitative PCR.The metabolic indexes related to transformed strains were measured,and the effect of targeted gene silencing and its function in the growth of mycelia and polysaccharide synthesis were tested,which provided a reference for the related research of G.frondosa polysaccharides.The main findings are as follows:1、Using the Illumina sequencing platform to sequence the transcriptome of G.frondosa,38189 Unigenes were obtained,which were compared with 7 major databases including KEGG,Nr,GO and COG.A total of 31,928 Unigenes were annotated,accounting for 83.61%of the total Unigenes.During the function prediction of G.frondosa gene,the anchor gene DN8915＿c2＿g1＿i8 carried out enrichment analysis of KEGG pathway,and it was found that G.frondosa HXT2 directly promoted the expression of glucose content,and then affected the activity of Snf1 protein kinase,which played an important role in the accumulation of glycogen metabolism.G.frondosa HXT2 protein is a relatively stable hydrophilic protein without signal peptide;The HXT2 protein of G.frondosa is a relatively stable hydrophobic protein;The results of subcellular localization analysis showed that HXT2 is a transmembrane protein;Protein structure prediction indicates that α Spiral and irregular curls are the main components;There are 4 N-terminal glycosylation sites and 3 O-terminal glycosylation sites;There are 13 proteins that interact with the HXT2 protein of G.frondosa.2、The total length of the c DNA of G.frondosa HXT2 gene was 1871 bp,and specific primers were designed to lock the RNAi target sequence.The c DNA was obtained by reverse transcription using the RNA of the sample of G.frondosa as the template.Then,a highly homologous sequence with the length of 263 bp was determined as the target sequence of RNAi using the slave c DNA as the template.The corresponding restriction sites of the p BWA(V)HS interference vector were linked after double restriction enzyme digestion,and the expression vector was constructed by forward and reverse insertion.After that,it was transferred to the G.frondosa hypha infected by Agrobacterium tumefaciens,and 20 resistant transformation strains were screened under hygromycin selection pressure.The mycelial DNA of transformed strains was extracted and identified by RNAi targeting primers,HXT2-R1 and 35S-1 primers and HXT2-F2 and Nos-5 primers for three times in sequence,and finally two positive silent strains were screened out.3、The G.frondosa HXT2 gene of the original G.frondosa strain and the transformation-positive strain was verified by relative fluorescence PCR.The results showed that the HXT2 gene expression of the two transformation-positive strains was only 28.4%and 35.1% of the original strain,and the gene expression was significantly down-regulated,with a down-regulation of more than 50%.The determination of the total protein content of the mycelium showed that the total protein content of the positive strains H1 and H2 was not significantly different,but it was lower than that of the original strain and the difference was significant.It was basically consistent with the result of gene expression.The results of mycelial soluble sugar content determination: positive strain H1> positive strain H2 >original strain,and the differences were significant.Results of mycelium PH determination:Compared with the original control group,the content of mycelium PH of positive strains H1 and H2 was reduced and significantly different from that of the original control group,but there was no significant difference in PH between the two positive strains.In summary,the RNAi experimental results speculate that the G.frondosa HXT2 gene product is a carbohydrate transporter,which is functionally involved in the metabolism and transport of carbohydrates,while the expression of G.frondosa HXT2 gene is negatively correlated with the total sugar amount in the mycelium,and the sugar content in the mycelium of the resisted strain with gene inhibition was significantly increased.Improved strains of target traits can be obtained through targeted regulation of target genes,increasing the extraction amount of bran in the strains,and providing intentional resource reserves for the application of G.frondosa polysaccharides in improving human immunity and plant disease control.