Studies On Molecular Identification And Antioxidant Activity Of Polysaccharides Of Boletus In Yunnan

Boletus is a kind of well-known fungi with edible and medicinal values. There are many different speciecs of Boletus in Yunnan Province of China that can be considered as the natural distribution center of Boletus. However, the germplasm resources and genetic diversity of Boletus tend to decrease due to natural factors and hunman activities in recent years. Although the utilization of Boletus has a long history in our country, the researches on its classification and identification, and its effective protection and comprehensive utilization are very limited. Consequently, this research attempts to make an investigation into the molecular identification and genetic diversity of Boletus samples from different regions in Yunnan by means of r DNA ITS sequences, r DNA IGS1 sequences, ITS-RFLP and IGS1-RFLP analysis. The research is involved in three parts. First of all, 13 samples of Boletus from Yuxi, Chuxiong, Qujing, Wenshan regions in Yunnan were studied under investigation of the morphyological characters and the molecular test for identification and genetic diversity. Secondly, seven strains of fungal endophytes isolated from 4 species of Boletus were investigated for observing their identification and diversity charateristics. Thirdly, the antioxidant activity of polysaccharides compounds from 5 species of Boletus were tested and compared. The main results of the research are highlighted as follows:(1) The morphyological characters of 13 Boletus samples from different regions in Yunnan were observed and the molecular identification and genetic diversity were studied by r DNA ITS sequences, r DNA IGS1 sequences, ITS-RFLP and IGS1-RFLP analysis. The results showed that the identification outcome using several molecular markers was consistent and confirmed each other. 13 samples of Boletus can be divided into seven groups by their ITS and IGS1 sequence clustering analysis. The clustering result from ITS and IGS1 sequence was completely similar. The ITS and IGS1 sequences analysis have the same effect for identifying Boletus. 13 Samples from different regions of Yunnan were identified to genus and even species based on the ITS sequence clustering analysis. Meanwhile, these outcomes were basically con-sistent with the traditional classification outcomes. It can be considered that ITS and IGS sequence analysis is a universal and effective molecular identification method. IGS1 sequence analysis can also be used as a new molecular marker. In addition, the classification result of secondary structures of r DNA ITS region was consistent with that of primary structures of r DNA ITS region. Secondary structures of r DNA ITS can be used as an supplementary means of ITS sequence analysis, which can provide more effective and accurative molecular structure information for identification of Boletus. The classification results of ITS-RFLP and IGS1-RFLP fingerprint were respectively consistent with that of ITS and IGS1 sequence clustering analysis, and the ITS-RFLP and IGS1-RFLP fingerprint can be used as an another means of ITS and IGS1 sequence analysis which are helpful to the correct identification of Boletus. In the research, there are some samples of Boletus that also revealed lower diversity and high conservation degree, therefore, in order to distinguish several varieties that were not classified, other kinds of enzyme was needed to study ITS-RFLP of Boletus. In short, combining the molecular biological investigation with the characteristics of morphyology of Boletus, acturally provided more effective and accurative information about its genetic structure and genetic diversity, which can be regarded as an integrated approach to the classification and identification of Boletus.(2) Seven strains of fungi, named MW5-1, MW5-2, HNG1-6, MW1-7, TS3-6, MW3-7, HL3-8, were isolated from four spieces of Boletus edulis, Blotus magnificus, Boletus aereus, Leccinum rugosiceps which were collected from different regions of Yunnan Province. The morphyological characters of the strains were observed, and their ITS sequences were amplified and sequenced. Phylogenetic tree was constructed by the obtained molecular data. The results showed that MW5-1 was Aspergillus fumigatus., MW5-2, Purpureocillium lilacinum., HNG1-6, Trametes hirsuta., MW1-7, Chaetomium aureum., TS3-6, Candida sp., MW3-7, Mortierella sp., HL3-8, Eurotium sp., respectively. The data indicated that fungal endophytes from Boletaceae were different and diversified.(3)The polysaccharides compounds from Boletus edulis, Boletus aereus, Boletus erythropus, Boletus speciosus and Leccinum crocipodium were obtained by hot water extract and ethanol precipitation. Polysaccharide content was tested after removing of proteins by sevag method. The antioxidant activity of polysaccharides compounds from five species of Boletus was tested and compared for the scavenging abilities for the hydroxyl, superoxide and DPPH anion. The results showed that five different polysaccharides compounds had different scavenging abilities. That is DPPH?>?OH>O2-?. The scavenging effect on ?OH in five species of Boletus from high to low is: Leccinum crocipodium, Boletus erythropus, Boletus aereus, Boletus speciosus and Boletus edulis.The scavenging effect on O2-? from high to low is: Boletus speciosus, Boletus edulis, Leccinum crocipodium, Boletus aereus and Boletus erythropus. The scavenging effect on DPPH? from high to low is: Boletus erythropus, Boletus speciosus, Leccinum crocipodium, Boletus edulis and Boletus aereus.