Isolation And Cultivation Of Belete Strains And Colonization Of Phosphate Transporter Gene

The kinds of Bolete are more abundant in our country. There are more than400species reported in the literature. They not only have an important ecological value, but also have a edible highly and medicinal value. As a wild edible fungus resources in Yunnan(one of the most abundant areas in China), Bolete is extremely exported for economic value. In natural conditions,the majority of Bolete commonly form mutual symbiosis with fine roots of predominantly woody plants. In this symbiosis, plants provide carbohydrates to Bolete, which provides mineral elements and water to plants, especially phosphorus. But, their developments and uses are limited. So, Bolete strains'tissue isolation, their basic biological activities and molecular mechanism of symbiosis are extensively studied by many scholars at home and abroad. In this study. eight edible Bolete material was isolated and cultured from their con-esponding fruiting bodies. The transcribed region of ribosomal DNA gene was used to identify molecularly. Furthermore, we selected one strain (Boletus edulis s.l.), studied its biological characteristics and cloned its phosphate transporter. The main achievements in the thesis are as follows:1. Eight bolete strains were isolated using the method of tissue isolation and culture from fruit bodies purchased at local markets. Molecular Identification was also analysed based on internal transcribed spacer sequences. Eight mycelium ITS sequences were consistent with their corresponding fruiting bodies'. Phylogenetic analysis showed that these eight strains belonged to Boletaceae;2. The basic research of WD-4-9isolate was done. We found the best pH(5), temperature(28℃), carbon source(C12H22O11·H2O), nitrogen source(NH4Cl), phosphorus source(KH2PO4), and the best group of carbon, nitrogen, and phosphorus sources (C12H22O11·H2O, NH4Cl, NaH2PO2·H2O) on the basis of Check's medium;3. The conservative sequence of the phosphate transporter gene from WD-4-9isolate was isolated by homology cloning, and then the full-length DNA and cDNA open reading frame (ORF) sequences were cloned by methods of Genome Walking and RACE, respectively. Prediction and analysis of the chemical properties and secondary structure of the protein by the bioinformatics ways were showed. The phylogenetic analysis was also analysed.The above results showed that the conventional method of tissue isolation and culture was an effective separation from the fruitbodies. Molecular identification techniques were also quick and effective to identify isolates and their corresponding fruiting bodies. We found the best carbon source(C12H22O11H2O), nitrogen source(NH4Cl), phosphorus source(KH2PO4) on the basis of Check's medium when WD-4-9isolate was cultured.We also cloned Phosphate transporter gene from WD-4-9isolate and predicted that the gene is a phosphate transporter protein in the MFS superfamily. The gene might play an important role in P exchange between fungi and plant.