Studies On The Identification Of The Objective Mice's Differentia Expressing Gene Affected By α-amanitin And The Drug Screening Of α-amanitin Resistence

There are abundant mushroom resources in Sichuan province and its nearby districts because of their unique geographical and ecological environment. In the screened mushrooms of high quantity of a-amanitin, a-amanitin was isolated for the identification and its localization in mushrooms was studied. This may provide the theoretical basis for the further research and the large-scale of a-amanitin production. We also discussed the metabolism of a-amanitin in the animals after analyzing its distribution in the poisoned mouse bodies. Toxicity tests in mice on the mushrooms from the different places where poisoning cases happened proved that there was lethal toxin in the mushrooms. After further studies on its morphology and toxicology, it was confirmed to be Amanita virose. RP-HPLC was applied to isolate a-amanitin, which was the main lethal toxin in the poisonous mushrooms. Under the condition of our experiments, we constructed the standard curve and the equation of linear regression of the a-amanitin (Y=5159+1097.4). The distribution and total content of the a-amanitin from the sporocarps of Amanita virosa were then studied. After calculating with the standard curve and the equation of linear regression, it was showed that the total content of a-amanitin was 2883.8ug/g dry sporocarps. The distribution of a-amanitin in sporocarps varied depending on the environments and tissues. Studies on the content of a-amanitin of Amanita virosa from different places showed that the highest was from the Three River Natural Reserved and the followings were from QiJiang of Chongqing, Ya'an, DaYi and Pengxian. For the content of a-amanitin in different tissues of sporocarps, the gill had the highest value, accounting for about 51.09% of the total a-amanitin, this was also thehighest if expressed as the unit component, about 0.29% of the dry weight of gill. RP-HPLC analysis of the tissue fluid of the mice injected by a semi-lethal dose of a-amanitin through the caudal vein revealed that the distribution and metabolism of a-amanitin in the animal tissues had time dependency, a-amanitin was not detected in blood plasma and spleen, but there was a-amanitin accumulation in the liver and kidney after 48h caudal vein injection. These showed that a-amanitin had some direct toxic effects on the liver and kidney after a period of time of the poisoning.Screening the differentially expressed genes affected by a-amanitin with modern molecular biological technique, would lay a good foundation of the studies on the mechanism of action of a-amanitin, the related genes, clinical diacrisis and the antagonistic medicine screening. This would give a theoretical explanation of a-amanitin function at different angles and levels. SSH was used to construct a cDNA subtractive library (positive) with a-amanitin and a cDNA subtractive library (negative) without a-amanitin. 400 clones in the cDNA library were randomly selected for screening with PCR to get 166 cDNA differentia expressing fragments. 85 positive differentia expressing cDNA fragments were screened by Dot bolt, in which there were 42 positive differentia expressing cDNA fragments and 45 negative ones respectively. 9 cDNA clones were selected for sequencing. The fragments sequenced were searched in GenBank and compared with their homological genes. It was found that the gene fragments from the positive subtractive screening were AK013055, AK013732-, NM₀07645, NM-013520, NM₀16974, NM₀21718, and the gene fragments from the negative subtractive screening were D14716, NM₀07563 and NM₀19946.On the basis of the study on the differentially expressed genes for the mice affected by a-amanitin by the suppression subtractive hybridization (SSH) and DNA chips assay, several differentially expressed genes with functional representation were selected according to our experiment and then the resistent drug of a-amanitin was screened by the semi-quantitative polymerase chainreaction assay to construct the fast screening method for the drug. After intervened by the resistent drugs which were ready to be screened, the clinic symptoms of the mice appeared different changes, especially for the groups of the Silybum marianum(L) Gaertn and Ganoderma lucidum. Compared with the poisoned mice, the mice of these two groups behaved actively and move quickly. The comparison of the variation data for the mouse weight of each group showed that the mice weight appeared distinct decrease after 12 hours of the drug intervention, and the twin cauda test showed great difference. The mice of the groups of Silybum marianum (L) Gaertn, Ganoderma lucidum and Perilla frutescens (L.) britt Var. frutescens clearly resumed their weight after 36 hours of the drug intervention and the difference was not prominent. The results of the semi-quantitative polymerase chain reaction assay indicated that each kind of intervening drug had different influence on the mutative gene caused by a-amanitin. Silybum marianum (L) Gaertn, Ganoderma lucidum and Perilla frutescens Var.criasta /Glucyrrhiza uralensis Fisch obviously resumed to down regulation for the gene of up regulation Catn P , and the ratios of the amount of Catn ^ transcription to inner standard P -actin were 0.71, 0.80 and 0.85 respectively, far below to 1.28 of the positive control group. The intervention of Silybum marianum (L) Gaertn and Ganoderma lucidum had up regulation on the transcription of Flt3L gene. After the intervention of these two drugs, the ratios of the amount of Flt3L transcription to inner standards 3 -actin were 0.83 and 0.79 respectively, close to 0.88 of the negative control. The intervention of other drugs also had some influence on the up regulation for the transcription of Flt3L gene, and their intensity was arranged according to the ration of the amount of Flt3L transcription to inner standard P -actin ,and the order was Silybum marianum (L) Gaertn, Ganoderma lucidum, Ganoderma sinense and Perilla frutescens Var.criasta /Glucyrrhiza uralensis Fisch. The transcription of 117r gene in the intervention group of Silybum marianum (L) Gaertn reverted to up regulation the most obviously, compared with the group injected by a-amanitin, whose ratio of transcription amount to inner standardincreased from 0.36 to 0.78, close to 0.83 of the negative control group. In addition, the intervention of Ganoderma lucidum, Ganoderma sinense and Silybum marianum(L)Gaertn had significant influence on the up regulation on the transcription of Rpo2-4 gene, after the intervention by these drugs, the ratio of the transcription of Rpo2-4 gene to 3 -actin increased to 0.58, 0.56 and 0.50 from 0.23 of the group injected with a-amanitin. From these results, we concluded that Silybum marianum (L) Gaertn and Ganoderma lucidum had strong antagonism to a-amanitin.