Animal Genetic Relationship. Muntjac Subfamily And Hsblys Of Eukaryotic Expression

Muntiacinae include Muntiacus and Elaphodus, they belong to Cervidae. Many researchers pay attention to the cytogenetics character of these animals. Recently, we know the number of muntjac deer chromosomes varies from 6 to 48, and there are karyotypic polymorphism intra-species relationships. These animals are extremely useful for phylogenetic studies. In order to discuss the phylogenetic relationships of Muntiacinae, we have studied the nuclear gene sequence of Muntiacus muntjak, Muntiacus crinifrons, Muntiacus reevesi, and Elaphodus cephalophus.We isolated genomic DNA from the four animals. We designed and synthesized the primers of the human GAPDH gene and the Elaphodus cephalophus potassium channel gene. The fragments of GAPDH gene and the potassium channel gene were amplified using the genomic DNA of the four animals as a template. The fragment sizes of the PCR products were 300bp(GAPDH) and 940bp(potassium channel gene). The target bands were recovered and purified. The PCR products were ligated with the plasmid of pMD 18-T Vector by the method of direct T-A cloning. The mixtures of ligation were transformed into DH-5 a . The positive clones were screened by the white/blue colony, and were identified by colony PCR method. The DNA sequences of the recombinant clones were determined using M13-47/RV-M universal primers and they were aligned by the software of clustalw. The nucleotide divergence was 0%-1.5% among three species of Muntiacus, 1.5%-2.0% between Elaphodus and three species of Muntiacus. The results are agreeable with morphological taxonomy. The software MEGA was used to analyze the molecular phylogeny and phylogenetic trees were constructed with the neighbor-joining method and the maximum parsimony method. Phylogenetic analysis shows that GAPDH is a classical nuclear gene, a house-keeping gene and a function gene. It was also shown that the gene is highly conservative. If we study on the phylogenetic relationship in intra-genus or intra-species using GAPDH as a target gene, it may be make a discrepancy. The phylogenetic tree of the potassium channel gene suggests that Muntiacus crinifrons isoriginal, and the basal split separates Muntiacus and Elaphodus. Within the genus Muntiacus, crinifrons is more closely related to muntjak, with reevesi as its sister species. In the other genus there is only one species Elaphodus cephalophus. When phylogenetic trees of the potassium channel intron were constructed, the result strongly supported the above view. We suggest that: to analyse the molecular phylogenetic relationship of recent evolved groups, we can reflect the relationship more precisely if we select the most evolved sequence, such as mt-DNA and intron-DNA.The plasmid pcDNA3. 1 ( + ) /hsBLyS was trans fected into COS-7 cells with the calcium phosphate method and the lipofection method. We cultured the recombined COS-7 cells at optimal condition and harvested cells every 24h. In order to find out the optimal expressive condition, we lysed the cells, extracted the protein, and measure the content of the hsBLyS. The optimal expressing time with the calcium phosphate method was 72h after transfection, and the transfection method of calcium phosphate is proved to be more effective than the lipofection method.