|Other Abstract||Life science is now facing an accelerated developmental moment profited from Human Genome Project and the analogous projects for model organisms. Compared to other placental mammals, cetacean researches are far droped behaind at molecular level, even cetaceans are the animals with the biggest changes of body-sizes, most world-wide distributions on the earth, and on the top niches in aquatic ecosystems. They have experienced a unique evolutionary process which is remarkably distinct to other mammals. In the present study, a BAC genomic DNA library for baiji, Lipotes vexillifer, was constructed, with some further preliminary investigations and discussions on genome evolution of placental mammals.
The following goals were reached in this work:
1) Using pBeloBAC11 and E. coli DH10B as cloning system, and lung tissue as source of genomic DNA, we had constructed a genomic DNA library for baiji. Each step had been optimized so that they can be used as references for other cetacean’s library-construction.
2) The library consists of 149,000 clones with an average insert size of 83kb. Among 175 random selected BAC clones, 2 are pseudo-positive. Based on the estimated haploid genome size of 3.6×109bp for baiji, the library represents about 3.4 equivalents of its haploid genome.
3) PCR amplification for baiji’s four known nuclear genes, the alpha lactalbumin gene, the interphotoreceptor retinoid-binding protein (IRBP) gene, the SINE flanking sequence Mago22 locus and the Mos (c-mos) gene partial sequence showed that about 2, 4, 2, 3 positive clones were got from the 21 super pools, respectively.
4) By positional cloning we obtained the baiji’s alpha lactalbumin gene which showed that our library is well-suited for positional cloning other genes. The gene has a coding region of 1995bp with 4 exons. Compared to that of other eight mammal-species, the sizes, organization and sequences of exons are highly conserved.
5) 20 random selected BACs were end-sequenced by CBCS. We finally got 33 DNA segments among which there are 28 con-coding DNA, 2 single-exon genes and 3 internal exons of three different genes. The result showed that our library is suited for fishing out random DNA markers that can be used as landmark in future physical mapping.
6) The present study for the first time determined the C-values of seven marine mammal species including baiji, finless porpoise (Neophocaena phocaenoides), Chinese white dolphin (Sousa chinensis), Beluga whale (Delphinapterus leucas), bottlenose dolphin (Tursiops truncatus), Californian Sea lion (Zalophus californianus) and Largha seal (Phoca largha) by flow cytometry with chicken (Gallus domesticus) erythrocyte DNA content as standard. Their C-values are 3.68pg, 3.25pg, 3.24pg, 3.09pg, 3.07pg, 2.96pg and 2.76pg, respectively. Their genome sizes are 3.6×109bp, 3.18×109bp, 3.18×109bp, 3.02×109bp, 3.00×109bp, 2.88×109bp and 2.68×109bp, respectively.
7) Using the nowadays available C-value data for placental, we analyzed the evolutionary correlations of mammalian C-value variation. Results showed that mammals’ genome size variation negatively correlates with their evolutionary consequences presented by biodiversity and positively with their evolutionary process presented by numbers of incipient speciation and radiation events. For during evolution environmental changing is the major force that modulates organism’s genome, and the most majority of eukaryotic genome is non-coding DNA that increases following evolution, it’s assumed that stability of environmental factors can influence the genome DNA content.|