Comparative analysis of the genetic diversity of the neutral microsatellite loci and second exon of the goat MHC-DQB1 gene.

This study compared and analyzed the genetic diversity and population structure of exon 2 of the DQB1 gene and 13 autosomal neutral microsatellite markers from 14 Chinese goat breeds to explore the potential evolutionary mechanism of the major histocompatibility complex (MHC). A total of 287 haplotypes were constructed from MHC-DQB1 exon 2 from 14 populations, and 82 nucleotide polymorphic sites (SNPs, 31.78%) and 172 heterozygous individuals (79.12%) were identified. The FST values of the microsatellites and MHC-DQB ranged between 0.01831-0.26907 and 0.00892-0.38871, respectively. Furthermore, 14 goat populations showed rich genetic diversity in the microsatellite loci and MHC-DQB1 exon 2. However, the population structure and phylogenetic relationship represented by the two markers were different. Positive selection and Tajima's D test results showed the occurrence of a diversified selection mechanism, which was primarily based on a positive and balancing selection in goat DQB. This study also found that the DQB sequences of bovines exhibited trans-species polymorphism (TSP) among species and families. In brief, this study indicated that positive and balancing selection played a major role in maintaining the genetic diversity of DQB, and TSP of MHC in bovines was common, which enhanced the understanding of the MHC evolution.

Progress on genetic mapping and genetic mechanism of cattle and sheep horns.

Horns are cranial appendages, which are unique in ruminants. Cattle (Bos taurus) and sheep (Ovis aries) cranial appendages exhibit various forms of morphology, including wild-type two-horn phenotype, polled phenotype and scur phenotype. These animals provide an ideal model for studies on the underlying relationship between quality and quantitative traits of cattle and sheep horn and the molecular mechanisms of horn phenotype as a polygenic regulation for quality traits. In recent years, some research progresses of cattle and sheep horns are successively reported, which helps us better understand the evolutionary origin of new organ, the effects of natural selection, sex selection and artificial selection on horn phenotypes. In this review, we introduce in details the recent advances on the research of horn traits in cattle and sheep, and summarize the genetic mapping of multi-horned phenotypes, the genetic mapping of polled locus, and studies on scur phenotype. Moreover, we discuss potential problems in such research, thereby providing a reference for investigation on the genetic mechanisms of horn traits in ruminants.

Current genetic diversity in eight local Chinese sheep populations.

China has numerous local domestic sheep breeds. In this study, the genetic diversity of eight sheep populations was estimated using 17 microsatellites. Knowledge of such diversity provides novel insight into the degree of breed protection needed and the prediction of hybrid advantage. In total, 17 microsatellites were genotyped in 186 individuals from eight populations. The mean number of alleles (± SD) ranged from 3.71 ± 1.36 in Zhaotong sheep to 11.94 ± 3.58 in small-tailed Han sheep. The observed heterozygote frequency (± SD) within a population ranged from 0.482 ± 0.025 in Zhaotong sheep to 0.664 ± 0.023 in Tibetan sheep. In addition, using pairwise difference (FST) analysis, the highest within-population diversity was observed in Tibetan sheep (πX = 12.8098) and small-tailed Han (πX = 12.67873), and the lowest diversity was observed in Zhaotong sheep (πX = 7.90337). The results for genetic divergence between populations indicated that the populations were significantly different (P < 0.05) based on the average number of pairwise differences between populations (πXY) and the corrected average pairwise differences. Both phylogenetic networks and structure analysis showed that these eight populations were separated into three clusters in accordance with their geographical habitat, except Tibetan and Hu sheep. In short, we genotyped eight local Chinese sheep populations using 17 microsatellites, and the results indicated that their current genetic diversity is decreasing and that new conservation strategies are needed. In addition, significant genetic differences between populations could be used in cross breeding.

Downregulation of miR-199b promotes the acute spinal cord injury through IKKß-NF-κB signaling pathway activating microglial cells.

Inflammatory response played an important role in the progression of spinal cord injury (SCI). Several miRNAs were associated with the pathology of SCI. However, the molecular mechanism of miRNA involving in inflammatory response in acute SCI (ASCI) was poorly understood. Sprague-Dawley (SD) rats were divided into 2 groups: control group (n=6) and acute SCI (ASCI) group (n=6). The expression of miR-199b and IκB kinase ß-nuclear factor-kappa B (IKKß-NF-κB) signaling pathway were evaluated by quantitative reverse transcription-PCR (qRT-PCR) in rats with ASCI and in primary microglia activated by lipopolysaccharide (LPS). We found that downregulation of miR-199b and activation of IKKß/NF-κB were observed in rats after ASCI and in activated microglia. miR-199b negatively regulated IKKß by targeting its 3'- untranslated regions (UTR) through using luciferase reporter assay. Overexpression of miR-199b reversed the up-regulation of IKKß, p-p65, tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) in LPS-treated BV2 cells assessed by western blotting analysis. In addition, BMS-345541 reversed the up-regulation effects of miR-199b inhibitor on the expression of TNF-α and IL-1ß. In the SCI rats, overexpression of miR-199b attenuated ASCI and decreased the expression of IKKß-NF-κB signaling pathway and TNF-α and IL-1ß. These results indicated that miR-199b attenuated ASCI at least partly through IKKß-NF-κB signaling pathway and affecting the function of microglia. Our findings suggest that miR-199b may be employed as therapeutic for spinal cord injury.

'Ectopic' suprasellar type IIa PRL-secreting pituitary adenoma.

BACKGROUND: Ectopic pituitary adenomas (EPAs) are rare, and the suprasellar cistern seems to be the most common location. At this time, no detailed original classification, diagnosis, or treatment protocols for suprasellar pituitary adenomas (SPAs) have been described. CASE DESCRIPTION: A 19-year-old man showed visual disturbances and lack of libido for 3 years, he suffered a sharp decline in vision with only light perception in the last week. Magnetic resonance imaging scans revealed a large suprasellar cystic lesion with a normal pituitary in the sella turcica. Endocrinological findings showed an extremely high prolactin level of 1250 ng/mL. Because of the sharp decline in vision, the patient underwent total removal of the suprasellar lesion using a transfrontal interhemispheric approach. The tumor pedicle originated in the lower pituitary stalk without any connection to the anterior pituitary gland in the sella turcica, while the diaphragma sellae was incomplete. Clinical and endocrinological cure criteria were fulfilled and postoperative pathology confirmed a prolactin-secreting pituitary adenoma. CONCLUSION: Ectopic suprasellar pituitary adenomas (ESPAs) are extremely rare intracranial extracerebral tumors. SPAs can be classified into three types according to their origin and their relationship with surrounding tissue. Only type III is theoretically a true ectopic, based on previous reports. Thus, ESPAs are uncommon compared to other EPAs. Our case is the first reported case of a type IIa 'E'SPA and the first description of this subtype classification until now. The pars tuberalis may be different from the pars distalis, and each subtype of adenohypophyseal cells may have different migration characteristics, which leads to different proportions of each hormone-secreting subtype in SPAs and EPAs. Transsphenoidal surgery is minimally invasive, but transcranial surgery may remain a universal option for the treatment of suprasellar lesions.

Mitogenomic Meta-Analysis Identifies Two Phases of Migration in the History of Eastern Eurasian Sheep.

Despite much attention, history of sheep (Ovis aries) evolution, including its dating, demographic trajectory and geographic spread, remains controversial. To address these questions, we generated 45 complete and 875 partial mitogenomic sequences, and performed a meta-analysis of these and published ovine mitochondrial DNA sequences (n = 3,229) across Eurasia. We inferred that O. orientalis and O. musimon share the most recent female ancestor with O. aries at approximately 0.790 Ma (95% CI: 0.637-0.934 Ma) during the Middle Pleistocene, substantially predating the domestication event (∼8-11 ka). By reconstructing historical variations in effective population size, we found evidence of a rapid population increase approximately 20-60 ka, immediately before the Last Glacial Maximum. Analyses of lineage expansions showed two sheep migratory waves at approximately 4.5-6.8 ka (lineages A and B: ∼6.4-6.8 ka; C: ∼4.5 ka) across eastern Eurasia, which could have been influenced by prehistoric West-East commercial trade and deliberate mating of domestic and wild sheep, respectively. A continent-scale examination of lineage diversity and approximate Bayesian computation analyses indicated that the Mongolian Plateau region was a secondary center of dispersal, acting as a "transportation hub" in eastern Eurasia: Sheep from the Middle Eastern domestication center were inferred to have migrated through the Caucasus and Central Asia, and arrived in North and Southwest China (lineages A, B, and C) and the Indian subcontinent (lineages B and C) through this region. Our results provide new insights into sheep domestication, particularly with respect to origins and migrations to and from eastern Eurasia.

Primary sellar melanocytic tumor mimicking hemorrhagic pituitary macroadenoma: Case report and literature review.

Primary melanocytic tumors of the central nervous system (CNS) are rare lesions, but primary sellar tumors are rarer. Only 10 cases have been reported, and they are often misdiagnosed as pituitary macroadenoma. We report the case of a 54-year-old Chinese man who developed progressive bitemporal hemianopsia and visual loss. Magnetic resonance imaging (MRI) revealed an intrasellar and suprasellar clouded lesion adhering to the optic chiasm, hypothalamus, and hypophyseal stalk that was suspected of being a hemorrhagic pituitary macroadenoma. Because of the atypically giant, hemorrhagic, and upward-growing lesion, an initial trans-sphenoidal approach failed, and subsequent transfrontal craniotomy was adopted to achieve macroscopically complete resection. Histopathologic findings revealed a benign melanocytic tumor. Despite an extensive search, no other primary or secondary site was found. Considering the relatively benign lesion, effective surgery, and potential significant consequences of radiotherapy, the patient received no further treatment and is still alive at the 7-year follow-up. Primary sellar melanocytic tumors are exceptional lesions that are difficult to diagnose before operating and/or obtaining pathological findings. The pathological classification and extent of surgical resection may play a key role in the prognosis. Once this type of lesion is suspected, the transfrontal approach may achieve preferable exposure and resection. Complete surgical resection may be sufficient for relatively benign lesions; otherwise, stereotactic fractionated radiotherapy is indicated. More cases should be reported to improve the treatment strategy.

Genetic phylogeography and maternal lineages of 18 Chinese black goat breeds.

To understand the origin and genetic phylogeography of Chinese black goats, variations of mitochondrial DNA were characterised with 394 goats from 18 breeds, including 91 new individuals from regions poorly studied until now. Comparison of a 481-bp segment revealed a total of 192 haplotypes with 141 variable sites. The haplotype and nucleotide diversities ranged from 0.782 ± 0.079 to 1.000 ± 0.020 and from 0.009 ± 0.001 to 0.045 ± 0.006, respectively, indicating a relatively high genetic diversity in Chinese black goats. Phylogenetic analyses identified five haplogroups (A, B1, B2, C and D). The dominant haplogroups A, B1 and B2 were distributed in most of breeds, while the haplogroups C and D were only presented in the breeds located in north or northwest of China. Analysis of molecular variance and multidimensional scaling plot of F ST analyses indicated no obvious geographic structure among breeds. Furthermore, the migration rates revealed that a wide range of gene flow or gene exchange occurred among breeds, which may result in the weak geographic structure of Chinese black goats. Population expansion analysis based on mismatch distribution indicated that two expansion events in Chinese black goats occurred at 10 and 28 mutational time units. Finally, our findings indicate the multiple maternal origins of Chinese black goats and more gene flow (female-mediated) which occurred during their domestic and breeding histories.

A 1.1 Mb duplication CNV on chromosome 17 contributes to skeletal muscle development in Boer goats.

The Boer goat is one of the top meat breeds in modern animal husbandry and has attracted widespread attention for its unique growth performance. However, the genetic basis of muscle development in the Boer goat remains obscure. In this study, we identified specific structural variants in the Boer goat based on genome-wide selection signals and analyzed the basis of the molecular heredity of related candidate genes in muscle development. A total of 9 959 autosomal copy number variations (CNVs) were identified through selection signal analysis in 127 goat genomes. Specifically, we confirmed that the highest signal CNV (HSV) was a chromosomal arrangement containing an approximately 1.11 Mb (CHIR17: 60062304-61171840 bp) duplicated fragment inserted in reverse orientation and a 5 362 bp deleted region (CHIR17:60145940-60151302 bp) with overlapping genes (e.g., ARHGAP10, NR3C2, EDNRA, PRMT9, and TMEM184C). The homozygous duplicated HSV genotype (+/+) was found in 96% of Boer goats but was not detected in Eurasian goats and was only detected in 4% of indigenous African goats. The expression network of three candidate genes ( ARHGAP10, NR3C2, and EDNRA) regulating dose transcription was constructed by RNA sequencing. Results indicated that these genes were involved in the proliferation and differentiation of skeletal muscle satellite cells (SMSCs) and their overexpression significantly increased the expression of SAA3. The HSV of the Boer goat contributed to superior skeletal muscle growth via the dose effects of overlapping genes.

Skin transcriptome analysis identifies the key genes underlying fur development in Chinese Tan sheep in the birth and Er-mao periods.

Hair follicle development in Tan sheep differs significantly between the birth and Er-mao periods, but the underlying molecular mechanism is still unclear. We profiled the skin transcriptomes of Tan sheep in the birth and Er-mao periods via RNA-seq technology. The Tan sheep examined consisted of three sheep in the birth period and three sheep in the Er-mao period. A total of 364 differentially expressed genes (DEGs) in the skin of Tan sheep between the birth period and the Er-mao period were identified, among which 168 were upregulated and 196 were downregulated. Interestingly, the FOS proto-oncogene (FOS) (fold change = 22.67, P value = 2.15*10^-44) was the most significantly differentially expressed gene. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis found that the FOS gene was significantly enriched in the signaling pathway related to hair follicle development. Immunohistochemical analysis showed that the FOS gene was expressed in the skin of Chinese Tan sheep at the birth and Er-mao periods, with significantly higher expression in the Er-mao period. Our findings suggest that the FOS gene promotes hair follicle development in Tan sheep.

Directed neural differentiation of duck embryonic germ cells.

Although the avian primordial germ cells (PGCs) have been used to produce transgenic birds, their characteristics largely remain unknown. The isolation, culture, biological characterization, and directed neural differentiation of duck EG cells were assayed in this study. The Results showed that the EG cells were got by isolating embryonic gonad and surrounding tissue from 7-day-old duck embryo. The PGCs co-cultured with their gonadal somatic cells were well grown. After passaging, the EG cells were incubated in medium with cytokines and Mitomycin C on inactivated duck embryonic fibroblasts (DEFs) feeder layers. After several passages, alkaline phosphatase (ALP) and periodic acid-Schiff (PAS) resulted positive, cellular markers detection positive for SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81. Karyotype analysis showed the EG cells kept diploid condition and the hereditary feature was stable in accordance with varietal characteristics of duck. These cells grew continuously for 11 passages on DEFs. Under induction of medium with BME, RA, and IBMX, the EG cells lost undifferentiated state, large amount of neural cells appeared with the formation of neural cells networks. Special Nissl body was found by toluidine blue stain after induced for 7 days. Immunofluorescence staining results indicated that differentiated EG cells expressed Nestin, NSE, and GFAP positive. The expression of Nestin, NSE, and GFAP mRNA were positive by RT-PCR. The results revealed that RA can obviously promote the directed differentiation of duck EG cells into neural lineage. The duck EG cells will be useful for the production of transgenic birds, for cell replacement therapy and for studies of germ cell differentiation.

[Regulatory effect of GDNF on the proliferation and differentiation of mammalian spermatogonial stem cells].

The glial cell-derived neurotrophic factor (GDNF) is a member of the transforming growth factor beta (Tgf-beta) superfamily, which is produced by Sertoli cells and plays an important role in the proliferation and differentiation of spermatogonial stem cells (SSC). The addition of proper amount of GDNF to the culture media can promote SSC proliferation in vitro. Besides, GDNF regulates the self-renewal and differentiation of SSCs through various signaling pathways. This review focuses on the effects of GDNF on the proliferation and differentiation of mammalian SSCs and GDNF-mediated signaling pathways.

[Progress on horse genome project].

There is unique genetic information belonging to various kinds of living beings. Understanding of the formation process of organisms and a variety of vital movement is associated with the achievements of genome study. As horse has a notable health condition and great record of the genealogy in the world, thus it becomes a valuable model animal for studying life science. Despite of a late start, the map of the horse genome has undergone unprecedented expansion during the last few years. The current progresses of the horse genome, including genetic map, physical map, comparative genomic map, and functional genomics, were reviewed in this paper. The maps are currently used worldwide to discover genes associated with various traits of significance in horse including general health, disease resistance, reproduction, fertility, athletic performance, phenotypic characteristics like coat color, etc. The results are believed to provide new ideas and approaches for prevention, diagnostics, and therapeutic for horses, and also better foundation of breed selection and equine genetic breeding.

Evolutionary relationship and population structure of domestic Bovidae animals based on MHC-linked and neutral autosomal microsatellite markers.

Major histocompatibility complex (MHC) genes are critical for disease resistance or susceptibility responsible for host-pathogen interactions determined mainly by extensive polymorphisms in the MHC genes. Here, we examined the diversity and phylogenetic pattern of MHC haplotypes reconstructed using three MHC-linked microsatellite markers in 55 populations of five Bovidae species and compared them with those based on neutral autosomal microsatellite markers (NAMs). Three-hundred-and-forty MHC haplotypes were identified in 1453 Bovidae individuals, suggesting significantly higher polymorphism and heterozygosity compared with those based on NAMs. The ambitious boundaries in population differentiation (phylogenetic network, pairwise FST and STRUCTURE analyses) within and between species assessed using the MHC haplotypes were different from those revealed by NAMs associated closely with speciation, geographical distribution, domestication and management histories. In addition, the mean FST was significantly correlated negatively with the number of observed alleles (NA), observed (HO) and expected (HE) heterozygosity and polymorphism information content (PIC) (P < 0.05) in the MHC haplotype dataset while there was no correction of the mean FST estimates (P> 0.05) between the MHC haplotype and NAMs datasets. Analysis of molecular variance (AMOVA) revealed a lower percentage of total variance (PTV) between species/groups based on the MHC-linked microsatellites than NAMs. Therefore, it was inferred that individuals within populations accumulated as many MHC variants as possible to increase their heterozygosity and thus the survival rate of their affiliated populations and species, which eventually reduced population differentiation and thereby complicated their classification and phylogenetic relationship inference. In summary, host-pathogen coevolution and heterozygote advantage, rather than demographic history, act as key driving forces shaping the MHC diversity within the populations and determining the interspecific MHC diversity.

Convergent genomic signatures of high-altitude adaptation among domestic mammals.

Abundant and diverse domestic mammals living on the Tibetan Plateau provide useful materials for investigating adaptive evolution and genetic convergence. Here, we used 327 genomes from horses, sheep, goats, cattle, pigs and dogs living at both high and low altitudes, including 73 genomes generated for this study, to disentangle the genetic mechanisms underlying local adaptation of domestic mammals. Although molecular convergence is comparatively rare at the DNA sequence level, we found convergent signature of positive selection at the gene level, particularly the EPAS1 gene in these Tibetan domestic mammals. We also reported a potential function in response to hypoxia for the gene C10orf67, which underwent positive selection in three of the domestic mammals. Our data provide an insight into adaptive evolution of high-altitude domestic mammals, and should facilitate the search for additional novel genes involved in the hypoxia response pathway.

Identification of a Goat Intersexuality-Associated Novel Variant Through Genome-Wide Resequencing and Hi-C.

Background: Polled intersex syndrome (PIS) leads to reproductive disorders in goats and exerts a heavy influence on goat breeding. Since 2001, the core variant of an 11.7 kb deletion at ~129 Mb on chromosome 1 (CHI1) has been widely used as a genetic diagnostic criterion. In 2020, a ~0.48 Mb insertion within the PIS deletion was identified by sequencing in XX intersex goats. However, the suitability of this variation for the diagnosis of intersex goats worldwide and its further molecular genetic mechanism need to be clarified. Results: The whole-genome selective sweep of intersex goats from China was performed with whole-genome next-generation sequencing technology for large sample populations and a case-control study on interbreeds. A series of candidate genes related to the goat intersexuality phenotype were found. We further confirmed that a ~0.48 Mb duplicated fragment (including ERG and KCNJ15) downstream of the ~20 Mb PIS region was reversely inserted into the PIS locus in intersex Chinese goats and was consistent with that in European Saanen and Valais black-necked goats. High-throughput chromosome conformation capture (Hi-C) technology was then used to compare the 3D structures of the PIS variant neighborhood in CHI1 between intersex and non-intersex goats. A newly found structure was validated as an intrachromosomal rearrangement. This inserted duplication changed the original spatial structure of goat CHI1 and caused the appearance of several specific loop structures in the adjacent ~20 kb downstream region of FOXL2. Conclusions: Results suggested that the novel complex PIS variant genome was sufficient as a broad-spectrum clinical diagnostic marker of XX intersexuality in goats from Europe and China. A series of private dense loop structures caused by segment insertion into the PIS deletion might affect the expression of FOXL2 or other neighboring novel candidate genes. However, these structures require further in-depth molecular biological experimental verification. In general, this study provided new insights for future research on the molecular genetic mechanism underlying female-to-male sex reversal in goats.

Establishment and characterization of a fibroblast line from Simmental cattle.

A fibroblast line (named SCF36) from ear marginal tissue of Simmental cattle was established successfully by direct culture of explants and cell cryopreservation techniques. Biological analysis showed that the population doubling time of the thawed cells was 42.8h. The average viability of the cells was 96.8% before freezing and 91.5% after thawing. Measurements of lactic dehydrogenase and malic dehydrogenase isoenzymes showed no cross-contamination of this cell line with other species. Karyotyping showed that the frequency of cells with chromosome number 2n=60 was more than 90%. Tests for bacteria, fungi, viruses and mycoplasmas were negative. The efficiencies of expression of enhanced green, yellow and red fluorescent protein genes (pEGFP-N(3), pEYFP-N(1) and pDsRed1-N(1)) were between 11.3% and 28.8% after transfection; fluorescence was well distributed in the cytoplasm and nucleus except for some cryptomeric vesicles. This Simmental cattle fibroblast line not only contains the germline of this important cattle breed, which is preserved at the cellular level, but valuable material has also been provided for genomic, postgenomic and somatic cloning research. Moreover, the establishment of these methods may provide both technical and theoretical support for preserving the genetic resources of other livestock and poultry at the cellular level.

[Polymorphic and linkage analysis of microsatellite BMS2508 and FecB gene in sheep].

Genetic polymorphisms of microsatellite locus BMS2508, which was closely linked to the ovine fecundity gene FecB, were detected in prolific (Small Tail Han sheep) and non-prolific breeds of sheep (Texel, Dorset and Chinese Merino). The linkage disequilibrium between microsatellite locus BMS2508 and FecB gene of Small Tail Han sheep was also analyzed. There was the same mutation (A746G) of BMPR-IB gene in Small Tail Han sheep as that of FecB in Booroola Merino ewes, but the FecB mutation was absent in Texel, Dorset and Chinese Merino sheep. The genotype frequencies of BB, B+ and ++ were 0.485, 0.398 and 0.117 in Small Tail Han sheep, respectively. There were eight alleles varied from 94 bp to 116 bp and 15 genotypes detected at BMS2508 locus in four sheep breeds totally 438 individual. The preponderant allele was 100 bp, 94 bp, 94 bp, 112 bp, 100 bp, 100 bp, 112 bp, and the frequency was 0.453, 0.544, 0.802, 0.475, 0.483, 0.439, 0.389 in Small Tail Han (n=307), Texel (n=45), Dorset (n=46), Chinese Merino (n=40), and BB group (n=149), B+ group (n=122), ++ group (n=36) from Small Tail Han, respectively. In Small Tail Han sheep, linkage analysis indicated that there was certain linkage disequilibrium between 100 bp allele of microsatellite BMS2508 and B allele of FecB gene (D' =0.408), and certain linkage disequilibrium between 110 bp and 114 bp alleles of microsatellite BMS2508 and + allele of FecB gene (D'=0.513).

[Characterization of expression of alpha1-acid glycoprotein gene in Beijing fatty chicken (Gallus gallus)].

The specific expression of alpha1-AGP gene in eight different tissues of Beijing fatty chicken was investigated by RT-PCR. The full-length cDNA of alpha1-AGP was inserted into pEGFP-C1 multi-cloning sites to construct recombinant eukaryotic expression vector pEGFP-alpha1-AGP. The lipofectin method was used to transfect the pEGFP-alpha1-AGP into Beijing fatty chicken fibroblast cells. The open reading frame of Beijing fatty chicken alpha1-AGP gene was 612 base pairs in length, which was expressed higher in liver and lung than in muscle. This gene did not express in heart and kidney. The expression efficiency ranged from 31.3% to 47.6% in 24, 48, and 72 h after transformation. The green fluorescence mainly concentrated in the nucleus. With the increase of the expression of green fluorescence, granula was observed in the nucleus. RT-PCR and Western blotting analyses showed that pEGFP-alpha1-AGP had been integrated into the genome of Beijing chicken fibroblast cell with normal expression level. In optimized condition, there was no significant effect (P>0.05) on apoptosis ratio, positive cell shape, growth and reduplication state comparing with the control group. This research established the foundation for further function research of alpha1-AGP gene and application in transgenic animal cloning.

[Study on single nucleotide polymorphisms of H-FABP gene in sheep].

PCR-SSCP and DNA sequencing approaches were applied to assess the single nucleotide polymorphisms (SNPs) and analyze the genetic polymorphisms at partial exon 2 and intron 2 of H-FABP(Heart fatty acid-binding protein)gene, and five sheep populations that comprised of Small-Tailed Han sheep (SH, 48), Ningxia Tan sheep (Tan, 121), Tan x SH F1 (23), Poll Dorset (48) and Suffolk (24) sheep were screened in this study. The result showed: (1)four SNPs at 981(G/A), 1014(A/C) 1019(T/C) and 1058 (-/G ) and nine genotypes (AA, BB, CC, AB, AC, BC, AD, CD and BD) were detected using primer 2, the AA was the predominant genotype. A chi-square analysis suggested that the allele frequencies and genotype frequencies of H-FABP were in Hardy-Weinberg equilibrium except the Tan and Suffolk populations. Statistical analysis revealed a low polymorphism information content (PIC) in the Suffolk and Tan x SH F1 populations (PIC amp; 0.25) but an intermediate PIC in the remaining three populations (0.25 amp; PIC amp;0.50). It meant that the fragment of H-FABP had polymorphisms, which could be used as a candidate gene associated gene with phenotypic traits like intramuscular fat content in different sheep populations. (2)Three genotypes (HH, Hh and hh) determined by a SNP at 2407(T-C) were detected using primer 4. The genotype frequencies were in the order of HHHhhh. A chi-square analysis suggested that the allele frequencies and genotype frequencies of H-FABP were in Hardy-Weinberg equilibrium in the Tan and Poll Dorset populations, and the PIC values were low (PIC amp; 0.25). However, there was no polymorphisms in SH, Tan x SH F1 and Suffolk populations.