[Cloning of the promoter region of Leuciscus Merzbacheri beta-actin gene and detection of its function].

To utilize the gene resources of Leuciscus merzbacheri, a 2,398 bp (SZ21) DNA fragment including the 5'-flanking region and partial open reading frame of the beta-actin gene was obtained through PCR amplification. SZ21 includes a regulatory sequence which contains the 5'-proximal promoter, the first, the second and the third exons and the partial fourth exon sequence. The 5'-proximal promoter region is critical for transcription activity including the CAAT box, TATA box and CArG box. The analysis of putative transcription binding sites of the promoter by on-line software revealed the presence of the critical transcription binding sites (such as E-box, RU49, ZBPF, CEBP and CREB). CMV promoter for eukaryote vector pEGFP-N1-AFP III was destroyed by Aat II digestion. SZ21 regulatory sequence was inserted into the vector pEGFP-N1-AFP III (with destroyed CMV) that can express green fluorescence protein, and beta2 pEGFP-N1-AFP III recombination vector was constructed. Linearized beta2 pEGFP-N1-AFP III was transfected into BHK-21 cell through lipofectin. EGFP expression of the transgenic cell was observed by micro fluoroscope. The results indicated that the cloned Leuciscus merzbacheri beta-actin gene promoter SZ21 has the activity to switch on the EGFP expression in mammal cell, and has a con-tinued starting expression activity passing on from generation to generation in green fluorescence cell. In addition, the SZ21 target fragment was detected in the BHK-21 green fluorescence cell genomic DNA by PCR. This suggested that the SZ21 promoter of beta-actin gene has effective transcription activity and can promote the expression of foreign gene.

[Divergence and systematical evolution of three Leuciscus species in Xinjiang based on mitochondrial DNA control region sequences].

Nucleotide sequence of fish mitochondria DNA (mtDNA) D-loop region from Leuciscus leuciscus baicalensis, L. merzbacheri, and L. idus in Xinjiang, China, were examined by sequencing 667 - 669 bp length of homological fragments in the D-loop from 24 individuals of the three fish species. DNA divergence ranged from 6.39% - 9.89% among the three fish species in the genius of Leuciscus cuvier. The sequence similarity is high and the variation is low between L. Leuciscus baicalensis and L. idus. In contract, the genetic distance is larger between L. Leuciscus baicalensis and L. merzbacheri. The average nucleotide variation within each of the two geographical populations (Sailimu lake and E' erqis river) of L. leuciscus baicalensis is 1.07% and 1.08%, respectively, and such variation is 1.07% between the two populations. These results demonstrate that the two geographic populations of L. leuciscus baicalensis do not appear to have significant genetic differentiation. Sequencing data showed the existence of sufficient genetic variations among three species of fish, as illustrated by distinct haplotypes for each species. The phylogenetic trees built with MEGA1.02 pointed out that the relationship between L. leuciscus baicalensis and L. idus is close, and L. merzbacheri is ancient among three Leuciscus.

[Polymorphism and original analysis of mtDNA D-loop of three Leuciscus species in Xinjiang].

PCR-RFLP technique was employed to amplify about 827bp of mtDNA D-loop hypervariable region of Leuciscus baicalensis, L.merzbacheri and L.idus in Xinjiang. The PCR products of 56 samples with four restriction enzymes were digested: ScaI,HinfI,AluI,DdeI,and RFLP analysis was done then. The study indicates that all of L. species and populations have six haplotypes, L.merzbacheri has two haplotypes: BDAA,BDBA; L.baicalensis has three:AAAA,ABAA,ACAA; L.idus has one:CAAA. It is primarily considered that L.merzbacheri and L.baicalensis have more intra-population mutations. The UPGMA trees with 6 haplotypes and net genetics distance pointed out that L.merzbacheri might be original species, L.baicalensis and L.idus are evolved from L.merzbacheri.