Acta Veterinaria et Zootechnica Sinica

The Genetic Distribution and Population Genetic Structure Analysis of MyoD Gene in Different Pig Breeds

ZHU Li;LI Xue-wei;SHUAI Su-rong;LI Fang-qiong;CHEN Lei;LI Ming-zhou

2007, 38(1): 1-7. doi:

Abstract ( 760 )

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RFLP technique was applied in this study to analyze the distribution of MyoD gene in 10 different pig breeds and pig breed crosses. The population genetic information about genetic distribution, variation, and heterozygosity of MyoD gene in different breed population was analyzed. Based on allele frequency, genetic distance and evolution distance among each breed populations were calculated and UPGMA phylogenetic tree was got based on the evolution distances between populations. The results indicated that the distribution of MyoD genotype keep in HardyWeinberg equilibrium in most testing groups but not in Duroc and DLY population. Generally speaking, the genetic diversity of MyoD gene was abundant and these testing breed groups with high genetic variation. The evolution of MyoD gene was under natural selection pressure. The 10 pig breeds were divided into 4 clusters. The first cluster was consisted of four breeds developed from Landrance, the second cluster was two Chinese indigenous pig breeds, the third cluster was three breeds developed from Duroc and the fourth cluster was Tibetan pig breed. The phylogenetic tree was consistent with breeding facts of each breed. From this experiment, we concluded that some RFLP data from functional gene could be used in the evolution research of some closely related species.

Study on the Polymorphism in Exon2 of Insulinlike Growth Factor 2 Gene and Its Relationship with Several Growth Traits in Nanyang Cattle

ZHANG Zheng-feng;CHEN Hong;LI Qiu-ling;LEI Chu-zhao;XUE Kai;WANG XIN-zhuang;WANG Yi-min;NIU Hui

2007, 38(1): 8-13. doi:

Abstract ( 901 )

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Insulinlike growth factor 2 has profound effects on the growth and differentiation of animal embryo. Some researches indicated that the normal level of IGF2 have positive correlation with the embryo to youth growth of many large size mammals. This study was designed to investigate the polymorphism of exon 2 of IGF2 gene and its relationship with growth and development in 126 Nanyang cattle by PCRRFLPs with BsrⅠ. The results showed that the frequencies of alleles A and B were 0686 5 and 0313 5 respectively. The allele A was the dominant allele in Nanyang cattle. Individuals with homozygote of allele B had a significantly higher average body weight for born six months and twenty four months age, a most significantly higher average body length and heart girth for all experiment age, a significantly higher average hucklebone width for twelve months, eighteen months and two years age than that of allele A. They also had a significantly higher body length for six month age, heart girth for twelve month age, hucklebone width for eighteen and two year age than that of heterozygote. It was concluded that IGF2 gene is the major gene affecting the growth related traits of Nanyang cattle or it links with the major gene, and the mutation could be used as the molecular genetic marker to select the cattle for faster growth.

Molecular Cloning and Evolution Analysis of the Yak Heart Fatty Acid-binding Protein Gene

2007, 38(1): 14-19. doi:

Abstract ( 797 )

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The yak heart fatty acidbinding protein (HFABP) gene was cloned and compared the nucleotide sequence of coding region with the homologous HFABP genes in other species including cow, sheep, goat, pig, human, rat, mouse, chick and zebrafish retrieved from the GenBank, and finally a phylogenetic tree was constructed by NJ, MP, ME methods using the coding region DNA sequences of the HFABP genes in all species. The results showed that the yak HFABP gene had 4 exons and 3 introns. The size of exon 1, exon2, exon3 and exon4 was 73,173,102 and 54 bp respectively; while the size of intron 1, intron2 and intron3 was 3 460,1 892 and 1 495 bp respectively.The size of CDS sequence of the yak HFABP gene was 402 bp which encoding 133 amino acids. The nucleotide sequences among different species were quite conservative. The homologies of the coding region of HFABP genes between the yak and cow, sheep, goat, pig, human, rat, mouse, chick and zebrafish were 998%, 978%, 970%, 928%, 888%, 833%, 831%, 764%, 687% respectively. The molecular phylogenetic tree among species was constructed according to the nucleotide sequence of the coding region of HFABP gene. The result indicated that the molecular phylogenetic tree had two branches, branch 1 contained Zebrafish, branch 2 contained yak and other species. Yak and cow, sheep and goat assembled separately, then assembled to a genus, and then assembled to a genus with pig and human, while the rat and mouse assembled to a genus, then assembled to a genus with human, and then chick. This result of phylogenetic clustering was identical to the zoological classification system, indicating that the HFABP gene was also fit to construct molecular phylogenetic tree among different species.

Analysis of Genetic Diversity of Chinese Six Goat Breeds by Microsatellite Markers

2007, 38(1): 20-24. doi:

Abstract ( 711 )

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Thirty microsatellite loci were used to analyze the genetic diversity of 240 goats of Chinese six populations including Aerbasi, Erlangshan, Wuzhumuqin, Alashan, Liaoning and Shannan white goat. Based on the allele frequency, we calculated that mean heterozygosity（He）were from 0.367 to 0.467; mean polymorphism information contents（PIC）from 0.533 to 0.639 and mean effective number of alleles（Ne）from 2.571 to 4.915. Genetic variation within breed and among breeds were analyzed in the study. We clustered the NJ tree according to the Nei standard genetic distances, the result showed that six goat breeds were divided into two clusters. The study provides some clue for conservation and utilization of goat breed in the future.

Genetic Diversity of Two Sites of FASN Gene and Its Association with Growth and Fat Deposition in Chickens

2007, 38(1): 25-30. doi:

Abstract ( 1356 )

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In this study, two SNPs of chicken FASN gene were chosen to analyze genetic diversity and their associations with chicken growth and carcass traits by using a F2 designed resource population. Results showed that: (1) In the PCRRFLP site of HaeⅢ, allelic frequencies differed much among different chicken breeds, as B frequencies are the highest in Red Jungle Fowl (0.85), and the lowest in Taihe and Yugan Black (both 0.21). In the PCRRFLP site of PstⅠ, A frequencies are the highest in Chongren Partridge (0.5), and the lowest in Naked Neck chicken (0.19), and the difference of gene frequencies was small among chicken breeds. (2) In HaeⅢ site, it was significantly associated with most growth traits, individuals with CC genotype had the lowest body weight; No significantly difference was found at PstⅠsite; When considering combined genotypes of the two SNPs, individuals with (BC/GG) genotype had the highest body weight. (3) In HaeⅢ site, individuals with BB genotype had the higher fat deposition capacity than that of others, however no significant differences in fat deposition were observed among different genotypes in Pst Ⅰsite; And, when considering combined genotypes of the two SNPs, individuals with (BC/GG) genotype had the highest fat deposition capacity. (4) There were certain interactional effects within the two SNPs including bodyweight and fat deposition traits.

Genetic Effects of GNRHR and IGF-1 Genes on the Reproductive Traits in Wenchang Chicken

2007, 38(1): 31-35. doi:

Abstract ( 752 )

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Gonadotropinreleasing hormone receptor (GNRHR) gene and insulinlike growh factor 1 (IGF-1) gene which control the reproductive traits were studied as candidate genes on the Wenchang chicken（Chinese indigenous breed）in the present study. The single nucleotide polymorphism of GNRHR and IGF-1 was detected for the association with the total egg production (NE)，average days of continual egglaying (ADCE) and number of doubleyolked eggs (DYE) in Wenchang chicken. PCRRFLP method was used for identification of genotypes. The frequency of restriction enzyme A/a alleles in the population was for GNRHR 0.69 (Bpu1102I A) and 0.31 (Bpu1102I a) and for IGF1 0.53 (PstI B) and 0.47 (PstI b). χ2 analysis of the two genes indicated that the frequencies of GNRHR、IGF-1 fit HardyWeinberg equilibrium. The result showed that GNRHR Bpu1102I Aa genotype had greater number of eggs (300d) at 87.91 compared to 81.20 for Bpu1102I AA (P<0.01). The GNRHR Bpu1102I Aa genotype had greater number of eggs (400 d) at 13547 compared to 125.47 for Bpu1102I AA (P<001). The IGF-1 PstI bb genotype had greater number of eggs (300 d) at 89.03 compared to 82.61 for PstⅠ BB (P<0.05) and PstⅠ bb genotype also had greater number of eggs (400 d) at 137.84 compared to 127.82 for PstⅠ BB (P<0.05). The IGF-1 PstⅠ bb genotype had greater ADCE at 3.38 compared to 2.96、2.78 for PstⅠ BB and Bb (P<0.05) .Two significant effects of genes’ marker were found: GNRHR of dominant effect on number of eggs and IGF-1 of additive effect on that. The current research supports the effects of GNRHR and IGF-1 genes on reproductive traits of chickens.

Effects of EGF or bFGF on the Development of Porcine Parthenogenetic Embryos in vitro

2007, 38(1): 36-39. doi:

Abstract ( 762 )

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EGF and bFGF were added into the culture medium in the different stages. We studied the effects of epidermal growth factor or bisac fibroblast growth factor on the development of porcine parthenogenetic embryos in vitro. The results as follows: The addition of EGF significantly enhanced the cleavage rate of porcine parthenogenetic embryos (P<0.05); The addition of EGF or bFGF significantly enhanced the rates of blastocysts formation of 2-4cell porcine parthenogenetic embryos (P<0.05); Additionally, bFGF had more numbers of blastocysts and higher rates of blastocysts formation than the EGF’s and the control. In conclusion, EGF and bFGF are propitious to the development of porcine parthenogenetic embryos in vitro. And bFGF increased the quality of blastocysts by increasing total cell numbers in porcine parthenogenetic embryos.

Effect of Monochromatic Light on the Egg Performance of Laying Hens

2007, 38(1): 40-45. doi:

Abstract ( 1695 )

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Nineteen to fiftytwo weeks old HyLine Brown hens were exposed to red (660 nm, R), green (560 nm, G) and blue (480 nm, B) light from light emitting diode (LED) lamps and incandescent light (400～770 nm, W) in this study. There were 45 hens in each treatment. All light sources were equalized under 15 lx light intensity for 16 hr daily. The results show: 1) From 19 to 36 weeks, the laying rate in B light (86.8%±0.5%) was significantly higher (P<0.05) than those in other lights; the daily egg yield (47.7±0.34g) in B light was the highest and the feed conversion rate (2.75±0.07) in B light was the lowest among the four light treatments; the daily egg yield and the feed conversion rate in B light were significantly higher (P<0.05) than those in R and G lights. 2) From 37 to 52 weeks, the laying rate in R light was significantly higher (P<0.05) than those in B and G lights, but the egg mass (59.0±0.74 g) in W light was significantly higher (P<0.05) than those in other lights, and its feed conversion rate (2.1±0.04) was the lowest among the four light treatments. 3) There were no significant differences (P>0.05) in cracked egg percentage among all light treatments and the softshell percentage in W light (1.3%±0.3%) was significantly lower (P<0.05) than those in other lights from 19 to 36 weeks. The cracked egg percentage and the softshell percentage were not significant difference in all light groups from 37 to 52 weeks respectively. These results showed that the egg production and feed conversion rate would be increased in laying hens when who was illuminated with either blue light in early experimental stage or W light in latter experimental stage under 15 lx light intensity.

Effect of Serum-Deprivation on Transcription Expression of Lipid MetabolicRelated Gene in Primary Cultured Rat Adipocytes

2007, 38(1): 46-52. doi:

Abstract ( 1012 )

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Rat preadipocytes were isolated by collagenase treatment of epididymal adipose tissue from male SD rats (20-day-old), and differentiated into adipocytes. After induced with experiment model of serumdeprivation/reserum culture, the lipogenesis, lipolysis and relative levels of lipid metabolicrelated gene mRNAs in mature adipocytes were measured through Oil Red O staining, glycerol reagent kit and semiquantitative RTPCR, respectively. The results showed that mRNA levels of hormonesensitive lipase (HSL) were increased according to lasting time of serumdeprivation (P<0.05), and significantly decreased with reserum culture (P<0.05), which coincided with the changes of lipolytic activity in adipocytes. Lipogenesis and mRNA levels of lipogenic genes such as fatty acid synthase (FAS), acetylCoA carboxylase α(ACC1) and carbohydrate response element binding protein (ChREBP) were coordinately reduced according to lasting time of serumdeprivation (P<0.05), and increasingly increased during reserum medium culture (P<0.05). Sterol regulatory element binding protein (SREBP)1c mRNA levels were reduced by serumdeprivation (P<0.05), but did not have significant difference between 36 h and 72 h of serumdeprivation (P>0.05). SREBP1c mRNA levels were not changed by reserum medium culture 36-72 h after 72 h serum-deprivation (P>0.05). It can be concluded that lipogenesis and lipogenic genes mRNA levels coincide with mRNA levels of ChREBP, but do not with that of SREBP-1c, suggesting that ChREBP could be relative to transactivation of FAS and ACC1 in adipocytes, which needs to study further in translation level.

Construction of Recombinant Virus Strain TK-/gG-/PrM+ Recombinized by Pseudorabies Virus Ea Mutant Strain TK-/gG-/LacZ+ and PrM Gene of Japanese Encephalitis Virus

2007, 38(1): 53-58. doi:

Abstract ( 763 )

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Based on the nucleotide sequence of JEV SA14-14-2 strain，a pair of primers was designed．The PrM gene of JEV was cloned into general transfer vector pPgG-uni. The sequence analysis showed that the PrM genes of SAl4 and SA14-14-2 strains had 100% homologyA cotransfection experiment was carried out with the purified pPgGPrM and the genome of TK-/gG-/LacZ+ mutant of pseudorabies virus Ea strain in PK-15 cells. By plaque purification and PCR detection, the recombinant virus TK-/gG-/PrM+ was purified. This recombinant virus strain can be used for the study of duel-valence vaccine of JEV and PRV.

Isolation and Characterization of Porcine Encephalomyocarditis Virus

2007, 38(1): 59-65. doi:

Abstract ( 720 )

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Five isolates of encephalomyocarditis virus were isolated from clinical tissue samples originated from suspected cases of intensive pig farms. Two isolates of them (BJC3 and HB1) were systematically identified. The results showed that round virus particles with diameter of 27 nm were observed by immune electron microscopy. The physicochemical character tests indicated that the isolates BJC3 and HB1 were not able to endure acid and insensitive to chloroform. However, the two isolates had different sensitivity to trypsin treatment. Exposed under 60 ℃ for 1 h, the viruses could be inactivated and protection of bivalent cation at temperature of 50 ℃ was negligible. The specific fluorescence was observed in the cytoplasm of BHK21 cells infected with the isolates by indirect fluorescence assay using polyclonal antibody against EMCV. The VP1 gene and partial segment of 3D gene of the two isolates were amplified by RTPCR and sequenced. Sequence analysis revealed that the nucleotide identity of VP1 gene was 99.49% and the predicted amino acid identity was 98.46% among the two isolates. The nucleotide identity of VP1 genes between EMCV strains in GenBank database and the isolates ranged from 81.61% to 99.59%. Correspondingly, the amino acid identity was above 95.5%. The identity of 3D gene amplified of both the nucleotide and amino acid was 100% among the two isolates and other EMCV strains.Phylogenetic analysis based on the nucleotide sequences of VP1 gene of EMCV showed that the two isolates were located on the backbone of the phylogenetic tree, with lesser variation. These results suggest that EMCV infection exist in pig farms in China.

Cloning and Analysis of a 8.5 kb Sequence from the 3′ End of Transmissible Gastroenteritis Virus Genome

2007, 38(1): 66-71. doi:

Abstract ( 1315 )

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Six fragments about 1.5 kb,2.0 kb,1.1 kb,1.4 kb,1.4 kb,1.3 kb of transmissible gastroenteritis virus strain SCY were amplified by PCR , then were cloned into the pGMT vector and sequences were determined in Takara . 8 495 bp sequence was obtained after assembling by CAP program.Bioinformatics analysis showed that the 8 495 bp sequence included S,M,N,E,NSP3a,NSP3b,ORF7 genes. Phylogenetic trees based on S,M,N genes showed that TGEV SCY strain was closed to the TS,HN2002,TO14,pudue strains.TGEV SCY didn’t bias remarkably on any codons,but some of the amino acids biased slightly on the codons which ended by A or T.

Cloning and Sequence Analysis of the Genome of IBV SAIBk Strain Isolated in China

2007, 38(1): 72-77. doi:

Abstract ( 826 )

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According to the published genome sequences of infectious bronchitis virus (IBV） on GenBank, nineteen pairs of primers were designed and the genome of isolated SAIBk strain of IBV was amplified by RTPCR. All the RT-PCR products were cloned into the pMD18-T vector and sequenced. The genome sequence of SAIBk strain was 27 520 bp in length. The ratio of A+T in the genome was 61.74%. Compared with the genome of Beaudette, Mass41, Cal99, BJ and KQ6, the similarity was 87.2%, 87.6%, 87.2%, 85.6% and 87.5%, respectively. The 3′UTR and 5′UTR were the most conservative region in the genome, the similarity was between 90.9%-97.7%. The S1 gene have the most variations, the similarity was between 74.8%-83.2%.The phylogenetic analysis results showed that the SAIBk genome have a close relationship with S14,LX4,BJ strains which were isolated in China.

Construction of Recombinant Adenovirus Expressing Hemagglutinin Gene of Swine Influenza Virus and Its Immunogenicity Analysis

2007, 38(1): 78-83. doi:

Abstract ( 800 )

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A recombinant adenovirus expressing hemagglutinin (HA) gene of swine influenza virus (SIV) was constructed by homologous recombination technique. HA gene was subcloned into the vector pAdenoVator-CMV5-IRES-GFP. Then the shuttle plasmid was cotransformed into E. coli BJ5183 with adenovirus backbone plasmid pAdenoVatorΔE1/E3 to generate a recombinant plasmid pAd-HA-GFP through homologous recombination. The plasmid was transferred into 293 cells for packing a recombinant adenovirus. The recombinant adenovirus expressing HA gene, named rAd-HA-GFP, was confirmed by the expression of green fluorescent protein (GFP) and westernblot analysis of HA protein. A single intraperitoneal injection (i.p.) of rAd-HA-GFP was conducted in Kunming mice, a high titer of HI antibody was detected at two weeks after the injection, and the antibody lasted at least for 12 weeks. The results showed that the HA protein expressed by rAdHAGFP possess a good immunogenicity.

Prokaryotic Expression and Purification of Mammalian Cellentry Proteins 4 E (mce4 E) of Mycobacterium bovis and Analysis of Circular Dichroism Spectrum

2007, 38(1): 84-88. doi:

Abstract ( 1123 )

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The genome DNA of Mycobacterium bovis(C68-2) was extracted by CTAB method.A pair of primers was designed and synthesized according to the sequences of mammalian cellentry proteins 4E(mce4 E) gene of M.bovis in GenBank. The PCR product was inserted into pGEM-T vector. Then the expression plasmid was constructed by inserting the target gene fragment into pET30a(+) vector. By transforming recombinant pET30a-mce4E into BL21,the mce4E protein of M.bovis was expressed in E.coli. The expressed truncated Histagged proteins accumulated in inclusion bodies. Purification was performed on a nitrilotriacetic acid (Ni-NTA) agarose column, and renaturation was performed by dialysis. Final refolded protein was identified by SDSPAGE and western blotting assay. The protein conformation was analyzed by circular dichroism (CD) analysis. The content of secondary structure was αhelix (39.1%) and freedom curl (60.9%), without β-sheet and turn. Our research give some new insights into the pathology for mammalian, and provide good approach for further research for the target site of medicine action.

Study on Ultrastructure of Immune Cells and Natural Apoptotic Cells in Central Immune Organs of Gushi Chickens

2007, 38(1): 89-95. doi:

Abstract ( 789 )

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The ultrastructure of immune cells and natural apoptotic cells in central immune organs of Gushi chickens were studied by using transmission electronic microscopy technology. The results showed that the ultrastructure of immune cells in thymus and bursa was different between 17dayembryo and 4week and 19week Gushi chickens. The natural apoptosis was found in central immune organs of Gushi chickens. The main changes of apoptotic cells appeared in nucleus. The proliferation of mitochondria happened in apoptotic cells. The chromatin were highly condensed. The Cshaped nucleus and crescent nucleus and petallike nucleus and circleshaped nucleus etc were observed. The apoptotic cells broke into many apoptotic bodies, which were phagocytosed by macrophages.

Effects of Several Mucosal Immune Adjuvants on IgA Secreting Cells of Chicken Small Intestine

2007, 38(1): 96-100. doi:

Abstract ( 781 )

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In this paper we investigated effects of five kinds of mucosal immune adjuvant such as lactic acid bacillus(LAB), CpG DNA, recombinant IL-2(rIL-2), sodium fluoride(NaF) and daidzein, on IgA secreting cells of chicken small intestine (duodenum, jejunum and Peyer’s patch).Chickens of five groups were immunized orally with NDV strain LaSota vaccine containing different mucosal immune adjuvant metioned above respectively.And other two groups were given normal saline and LoSota vaccine as control. The results showed that the areas of IgA secreting cells of the Group LAB were increased significantly than that of Group ND (P<0.01) at 3rd and 5th week after vaccination in small intestine. The areas of Group CpG DNA, rIL-2 and daidzein were increased during the whole immune period. No significant changes were found in Group NaF. The results demonstrated that LAB, CpG DNA, rIL-2 and daidzein were effective mucosal immune adjuvants.

Cloning and Expression of HA Gene of Serumtype B of Haemophilus paragallinarum in E.coli and Study on Biological Activity of Recombinant Protein

2007, 38(1): 101-104. doi:

Abstract ( 706 )

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There is a close correlation between HI titre of Haemophilus paragallinarum(Hpg) HA and protection from infectious coryza. According to the published hemagglutinin antigen (HA) gene sequence of Dalian strain of Hpg, a pair of primers was designed and synthesized for amplifing Hpg’s major protecting antigen (HA gene). A DNA fragment about 1 038 bp was obtained through PCR method from Dalian strain of Hpg. The HA gene was cloned into the expression vector pET-32a and overexpressed in E. coli BL21 (DE3). Hemagglutination test indicated that the purified recombinant protein possess hemagglutinating activity. Western blot test showed that the protein was recognized by the chicken antibody against the Hpg of serotype B. This is first study in China to express HA gene and analysis the recombinant protein.

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