Comparison Of Specific Genomic DNA Fragment Between The Two Subspecies Of Microtus Fortis And Screening And Coloning Of Anti-Schistosoma Japonicum-Associated Gene

Microtus Fortis (Taxonomy ID: 100897), also named as reed vole, classified as Microtus, Micotinae, Cricetidae, Rodentia,Mammalia on taxonomy. Microtus Fortis mainly distributes in China. Some areas of Russia, North Korea and Mongolia close to Northeast borderland of China also have a small quantity of Microtus Fortis in distribution. Microtus Fortis in China has principally 4 subspecies, and most of them reside the drainage area of Yangtse River. Schistosoma japonicum (one of commonly parasite in China) can infect about 40 kinds of mammalian animals, including the human being, but could not infect Microtus Fortis. It is known as the only animal in Dongting Lake region of China which has the ability of natural resistance to Schistosoma japonicum. The Microtus Fortis domesticated in laboratory has the same biological characteristics above as the wild one and these characteristics could be inherited to its progeny steadily.We got a specific DNA fragment from genomic library of Microtus Fortis. This DNA fragment in genomic DNA of human beings, Kunming mice, Balb/c mice and C57BL/6J mice could not be detected by dot blot hybridization and PCR, apart from genomic DNA of Microtus Fortis. In this report, the differences of genomic DNA in 34 Microtus Fortis were compared between Microtus Fortis calamorum (Dongting Lake region of southern China) and Microtus Fortis fortis (Ningxia province of northern China). These two subspecies are away from more than about 1 200 kilometers each other. The genomic DNA of Microtus Fortis calamorum and Microtus Fortis fortis were extracted and amplified by PCR according to the specific genomic DNA sequence of Microtus Fortis reported previously (Accession number in Genbank: AF277394). The amplified DNA fragments were inserted into pGEM-T easy Vector and sequenced. The DNA fragment sequencing results from the two subspecies were compared to detect whether there was any difference. 19 alleles were found from Microtus Fortis (20 of Microtus Fortis calamorum and 14 of Microtus Fortis fortis). The results of multiple sequence alignment showed that 25 single nucleotide polymorphism (SNP)IUsites were existed in the different Microtus Fortis individuals, including transition (G-*A, A-G, T~*C, C-T), transversion (G-"T, A-*T, T-*A, C桝), insertion (CA) and deletion (TGTTTT). The difference of genomic DNA from two subspecies were obvious, especially on the sites of 146, 192, 223, 224 and 235. The insertion of CA on the sites of 223 and 224 as well as A?G transition on the site of 235 was only occurred in Microtus Fortis fortis .They are not found in Microtus Fortis calamorum so far. They could be divided into two groups according to phylogenetic tree analysis results, one was the genomic DNA of Microtus Fortis calamorum and the other one was that of Microtus Fortis fortis. However, the homologues reached up to 98% between two subspecies. These results are very important for us to further understand the genetic background, biological characteristics, evolutionary rule and the anti-schistosomajaponicum mechanism of Microtus Fortis at the molecular levels. The specific base changes of the DNA fragment between the two subspecies are probably correlative with the animal immigration, survival conditions, and species evolution.The cDNA library of Microtus Fortis bone marrow cells was transferred in situ to nylon membrane, which was divided into eight equals (gA~-gH). Everyone was a gene pool. The gene pools were cultured in LB medium separately and the plasmid DNA was purified. Using Lipofect-2000 Reagent, the transient transfection of HEK293 was performed with plasmid DNA of gene pool. The supemate of cell culture was collected 48 hours after transfection. This supernate was also called conditional media.Cercariae were collected , cultured in vitro and transformed to Schistosomula in the RPMI 1640 medium with rabbit serum. The Schistosomula were cultured in conditional media up to 96 hours. The number of Schistosomula was counted and the death rate was calculated. The inhibition of S...