[Screening of tissues pooled cDNA library using probes by restricted fragments of BAC positive clones of ovine MHC].

Under the premise what we have known bacterial artificial chromosome(BAC)clone sequence information and gene annotation predicted in the Chinese Merino sheep major histocompatibility complex (MHC) region, the digested fragments from 6 BAC clones that were located in the MHC region of the Chinese Merino sheep genome BAC library, which were used to screen the cDNA library using plaque in situ hybridization as probes. The full length of positive cDNA clones (sequences) isolated were completely sequenced, and the sequences obtained were aligned with the corresponding known sequence information and the BAC clones with gene annotation. Meanwhile, the sequence similarity was searched in NCBI Blastn database. This work aimed at verification of accuracy of the gene annotation results and initial analysis of gene (sequence) function. At last, 27 positive cDNA clones (sequences) in total were screened through two runs of hybridization. It was also found that these sequences could be positioned in the corresponding BAC clones, and 25 sequences were located in exon area of the annotated gene. It was verified that 23 sequences had the highest sequence similarity with those in the Bos taurus by searching against the NCBI Blastn database; moreover, the function of these sequences were closely relate to immunology.

[Expression of OPN gene during different lactation stages in mammary gland of dairy goat and its effect on growth of MCF-7 cell line].

To investigate the expression pattern and preliminary function of OPN gene in mammary gland of dairy goat during different lactation stages, using b-actin gene as the internal control, the SYBR Green quantitative real-time PCR (QPCR) analysis was conducted to determine the mRNA expression of OPN gene in mammary gland at the 28th, 60th, 100th, 190th, 270th and 330th day after kidding. Recombinant plasmid of pcDNA3.1-OPN was constructed by inserting the fragment of OPN gene into eukaryotic expression vector pcDNA3.1 and used to transfect the MCF-7 cell line following the restrictive endonuclease cleavage and sequence identification of the target gene segment, the effect of OPN gene on MCF-7 cell proliferation was assessed by MTT analysis. The results indicated that OPN gene exhibited the higher expression level in early (28 d) and late (190 d) lactation stages and the lowest level at dry stage (330 d), which demonstrated a high-low-high-low pattern. There was a significant difference (P < 0. 05) in the proliferation between OPN gene transfected and non-transfected MCF-7 cells, which suggested that the expression of OPN gene could stimulate the proliferation of MCF-7 cells.

[Expression of ER, bcl-2, and p53 mRNA in early hybrid embryos of chicken-quail].

Using artificial insemination, 100 female quails were crossed with 10 male chickens. The eggs were collected and hatched in the same incubator. The sex of live hybrid embryos from 66 to 120 hatch hours was determined using multiply PCR of Wpkci. Total 300 male and female embryos at various hatch times were sampled and the relative mRNA abundance of ER, bcl-2, and p53 in the embryos was detected by RT-PCR using beta-actin as the internal standard. The effects of ER, bcl-2, and p53 on the early embryonic development for hybrids between chicken and quail were analyzed. The results showed ER mRNA expression of female hybrids were higher than male hybrids from 66 to 84 hatch hours with a highly significant difference (P<0.01), which indicated that the sex differentiation of hybrids was perhaps happened between 66 to 84 h of embryo stage. The obvious sequential expression of bcl-2 and p53 in the embryonic development indicated that the bcl-2 and p53 genes had an important effect on the development of the hybrid embryos.

[Polymorphism distributions of 9 microsatellite Loci in Chinese merino sheep].

The polymorphism distributions of nine microsatellite loci, BM6506, BM1824, BM6438, ILSTS004 and OarDB6 on Chinese merino sheep chromosome 1 and BM4621, OarHH55, BM143 and OarJMP8 in sheep chromosome 6 were determined by polymerase chain reaction (PCR) and multiplex gel electrophoresis followed by silver staining. Gene frequency (Pi), power of discrimination (DP), heterozygosity (H), polymorphism information content (PIC) and probability of paternity exclusion (PE) were calculated. All loci obeyed Hardy-Weinberg equilibrium. BM4621 displayed the highest DP, H, PIC and PE values among the nine microsatellite loci. Cumulative DP of the nine microsatellite loci is 0.99999 and cumulative PE is 0.99915. These results showed that the nine microsatellite loci could be used in linkage analysis, individual identification and paternity test in Chinese merino sheep.

Analysis of genetic diversity and phylogenetic relationship of red deer subspecies in XinJiang, China.

Polymorphisms for seven microsatellite loci in three red deer subspecies (9 populations) found in XinJiang were detected by polymerase chain reaction (PCR), 12% nondenaturation polyacrylamide gel electrophoresis and the Sanguinetti silver staining method. Numbers of alleles, average effective numbers of alleles (E) and the average rate of homozygosity, allelic frequencies of seven microsatellite loci, polymorphism information content (PIC), mean heterozygosity (H) and genetic distances among the populations were calculated for each population. Dendrograms were constructed based on genetic distances by the neighbor-joining method (NJ), utilizing molecular evolutionary genetics analysis software PHYLIP (3.6). The phylogenetic tree was constructed based on allelic frequencies using maximum likelihood (ML); the bootstrap value was estimated by bootstrap test in the tree. Lastly, phylogenesis was analyzed. The results showed that four of the seven microsatellite loci were highly polymorphic, but BMS2508 and Celjp0023 showed no polymorphism and BM5004 was a neutral polymorphism. It is our conclusion that the four microsatellite loci are effective DNA markers for the analysis of genetic diversity and phylogenetic relationships among the three red deer subspecies. The mean PIC, H and E-values across the microsatellite loci were 0.5393, 0.5736 and 2.64, which showed that these microsatellite loci are effective DNA markers for the genetic analysis of red deer. C.e. songaricus populations from Regiment 104, 151 and Hami are clustered together. C.e. yarkandensis populations from Regiment 35, Xaya and Alaer are clustered together. These two clusters also cluster together. Lastly, C.e. sibiricus populations from Burqin, Regiment 188 and the first two clusters were clustered together. The phylogenetic relationship among different red deer populations is consistent with the known origin, history of breeding and geographic distributions of populations.