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Bai W.L.,ShenYang Agricultural University | Bai W.L.,Jilin University | Yin R.H.,ShenYang Agricultural University | Dou Q.L.,Qinghai University | And 5 more authors.
Molecular Biology Reports | Year: 2011

κ-casein is one of the major proteins in the milk of mammals. It plays an important role in determining the size and specific function of milk micelles. We have previously identified and characterized a genetic variant of yak κ-casein by evaluating genomic DNA. Here, we isolate and characterize a yak κ-casein cDNA harboring the fulllength open reading frame (ORF) from lactating mammary gland. Total RNA was extracted from mammary tissue of lactating female yak, and the κ-casein cDNA were synthesized by RT-PCR technique, then cloned and sequenced. The obtained cDNA of 660-bp contained an ORF sufficient to encode the entire amino acid sequence of κ-casein precursor protein consisting of 190 amino acids with a signal peptide of 21 amino acids. Yak κ-casein has a predicted molecular mass of 19,006.588 Da with a calculated isoelectric point of 7.245. Compared with the corresponding sequences in GenBank of cattle, buffalo, sheep, goat, Arabian camel, horse, and rabbit, yak κ-casein sequence had identity of 64.76-98.78% in cDNA, and identity of 44.79-98.42% and similarity of 53.65-98.42% in deduced amino acids, revealing a high homology with the other livestock species. Based on κ-casein cDNA sequences, the phylogenetic analysis indicated that yak κ-casein had a close relationship with that of cattle. This work might be useful in the genetic engineering researches for yak κ-casein. © Springer Science+Business Media B.V. 2010.


Bai W.L.,ShenYang Agricultural University | Yang R.J.,ShenYang Agricultural University | Yin R.H.,ShenYang Agricultural University | Jiang W.Q.,ShenYang Agricultural University | And 5 more authors.
Molecular Biology Reports | Year: 2012

Osteopontin (OPN) is a secreted phosphorylated glycoprotein. It has an important role in mammary gland development and lactation, as well as, is thought to be a potential candidate gene for lactation traits. In the present work, we isolated and characterized a full-length open reading frame (ORF) of yak OPN cDNA from lactating mammary tissue, and examined its expression pattern in mammary gland during different stages of lactation, as well as, the recombinant OPN protein of yak was expressed successfully in E. coli. The sequencing results indicated that the isolated cDNA was 1132-bp in length containing a complete ORF of 837-bp. It encoded a precursor protein of yak OPN consisting of 278 amino acid with a signal peptide of 16 amino acids. Yak OPN has a predicted molecular mass of 29285.975 Da and an isoelectric point of 4.245. It had an identity of 65.50-99.16% in cDNA, identity of 52.06-98.56% and similarity of 65.40-98.56% in deduced amino acids with the corresponding sequences of cattle, buffalo, sheep, goat, pig, human, and rabbit. The phylogenetic analysis indicated that yak OPN had the closest evolutionary relationship with that of cattle, and next buffalo. In mammary gland, yak OPN was generally transcribed in a declining pattern from colostrum period to dry period with an apparent increase of OPN expression being present in the late period of lactation compared with peak period of lactation. Western blot analysis indicated that His-tagged yak OPN protein expressed in E. coli could be recognized not only by an anti-His-tag antibody but also by an anti-human OPN antibody. These results from the present work provided a foundation for further insight into the role of OPN gene in yak lactation. © Springer Science+Business Media B.V. 2011.


Bai W.L.,ShenYang Agricultural University | Bai W.L.,Jilin University | Yin R.H.,ShenYang Agricultural University | Zhao S.J.,Animal Science Research Academy of Sichuan Province | And 5 more authors.
Food Control | Year: 2010

Yak meat is of great economic importance to the people living in the cold high altitude area. In the present work, a multiplex PCR-based method was proposed for the sexing of yak meat by amplifying the target sequence of male-specific SRY gene. In a single reaction set, two DNA fragments of 121- and 290-bp were amplified from male meat DNA using two pairs of primers, but only one DNA fragment of 290-bp was amplified from female meat DNA. The assay proposed herein was applied to raw and heat-treated yak meat samples, and the identification results obtained were in perfect agreement with the anatomical sex of the yak meat samples. The method is fast, reliable and cheap. It is potentially suitable for the sexing of yak meat in routine analyses. It can be considered a helpful tool in the quality control of yak meat and meat products. © 2009 Elsevier Ltd. All rights reserved.


Bai W.L.,ShenYang Agricultural University | Bai W.L.,Jilin University | Yin R.H.,ShenYang Agricultural University | Dou Q.L.,Qinghai University | And 6 more authors.
Animal Biotechnology | Year: 2010

The aim of this study was to investigate the polymorphism of the CSN1S1 gene promoter region in 4 Chinese yak breeds, and compare the yak CSN1S1 gene promoter region sequences with other ruminants. A Polymerase Chain Reaction-Single Strand Conformation Polymorphism protocol was developed for rapid genotyping of the yak CSN1S1 gene. One hundred fifty-eight animals from 4 Chinese yak breeds were genotyped at the CSN1S1 locus using the protocol developed. A single nucleotide polymorphism of the CSN1S1 gene promoter region has been identified in all yak breeds investigated. The polymorphism consists of a single nucleotide substitution GA at position 386 of the CSN1S1 gene promoter region, resulting in two alleles named, respectively, G386 and A386, based on the nucleotide at position 386. The allele G386 was found to be more common in the animals investigated. The corresponding nucleotide sequences in GenBank of yak (having the same nucleotides as allele G386 in this study), bovine, water buffalo, sheep, and goat had similarity of 99.68%, 99.35%, 97.42%, 95.14%, and 94.19%, respectively, with the yak allele A386.


Bai W.L.,ShenYang Agricultural University | Yin R.H.,ShenYang Agricultural University | Yang R.J.,Jilin University | Khan W.A.,Cornell University | And 7 more authors.
Journal of Dairy Science | Year: 2013

MicroRNA are approximately 18- to 22-nucleotide nonprotein coding molecules that play important roles in the regulation of gene expression at the posttranscriptional level. In the present study, we assessed the suitability of 8 noncoding small RNA as normalizers for microRNA (miR) quantitative analysis in milk somatic cells of lactating yaks, including 3 small nuclear RNA (snRNA; RNU1A, RNU5A, and RNU6B), 3 small nucleolar RNA (snoRNA; SNORA73A, Z30, and SNORA74A), 1 rRNA (5S), and 1 transfer RNA (Met-tRNA). The snRNA RNU1A, RNU5A, and SNORA73A were identified as the most stable references in milk somatic cells of lactating yaks. Also, a minimum of 3 reference RNA (RNU1A, RNU5A, and SNORA73A) were required for the normalization of microRNA expression data in milk somatic cells of the lactating yak. We further evaluated the suitability of the combination of RNU1A, RNU5A, and SNORA73A as reference RNA in milk somatic cells of lactating yaks via detecting the relative expression of miR 16b, miR 21-5p, miR 145, and miR 155 as microRNA of putative interest. In comparison to the colostrum period, on the whole, the expressions of the 4 microRNA were found to be upregulated at an early period and, thereafter, a declining pattern was exhibited from early to final periods in all microRNA investigated. Based on the results from this study, we recommend that the combination of RNU1A, RNU5A, and SNORA73A can be used as normalizers for microRNA quantitative analysis in future longitudinal studies on milk somatic cells of lactating yaks in relation to lactation. © 2013 American Dairy Science Association.


Bai W.L.,ShenYang Agricultural University | Zhou C.Y.,Animal Health Supervision and Management Bureau of Liaoning Province | Ren Y.,Jilin University | Yin R.H.,ShenYang Agricultural University | And 6 more authors.
Molecular Biology Reports | Year: 2011

The aim of the present work was to investigate single nucleotide polymorphism (SNP) of growth hormone receptor (GHR) gene exon 10, characterize the genetic variation in three Chinese indigenous goat breeds, and search for its potential association with cashmere traits. In this study, a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) protocol has been developed for rapid genotyping of the GHR gene in goats. One hundred seventy-eight goats from Liaoning Cashmere (96), Inner Mongolia White Cashmere (40), and Chengdu Grey (42) breeds in China were genotyped at GHR locus using the protocol developed. In all goat breeds investigated, a SNP in exon 10 of GHR gene has been identified by analyzing genomic DNA. The polymorphism consists of a single nucleotide substitution A → G, resulting in two alleles named, respectively, A and G based on the nucleotide at the position. The allele A was found to be more common in the animals investigated, and seems to be more consistent with cattle and zebu at this polymorphic site found in goats. The Hardy-Weinberg equilibrium of genotype distributions of GHR locus was verified in Liaoning Cashmere, and Inner Mongolia White Cashmere breeds. According to the classification of polymorphism information content (PIC), Chengdu Grey was less polymorphic than Liaoning Cashmere and Inner Mongolia White Cashmere breeds at this locus. The phylogenetic tree of different species based on the nucleotide sequences of GHR gene exon 10 is generally in agreement with the known species relationship. No significant association was found between the polymorphism revealed and the cashmere traits analyzed in present work. © 2010 Springer Science+Business Media B.V.


Bai W.L.,ShenYang Agricultural University | Yin R.H.,ShenYang Agricultural University | Zhao S.J.,Animal Science Research Academy of Sichuan Province | Jiang W.Q.,ShenYang Agricultural University | And 7 more authors.
Journal of Dairy Science | Year: 2014

Quantitative real-time PCR is the most sensitive technique for gene expression analysis. Data normalization is essential to correct for potential errors incurred in all steps from RNA isolation to PCR amplification. The commonly accepted approach for normalization is the use of reference gene. Until now, no suitable reference genes have been available for data normalization of gene expression in milk somatic cells of lactating yaks across lactation. In the present study, we evaluated the transcriptional stability of 10 candidate reference genes in milk somatic cells of lactating yak, including ACTB, B2M, GAPDH, GTP, MRPL39, PPP1R11, RPS9, RPS15, UXT, and RN18S1. Four genes, RPS9, PPP1R11, UXT, and MRPL39, were identified as being the most stable genes in milk somatic cells of lactating yak. Using the combination of RPS9, PPP1R11, UXT, and MRPL39 as reference genes, we further assessed the relative expression of 4 genes of interest in milk somatic cells of yak across lactation, including ELF5, ABCG2, SREBF2, and DGAT1. Compared with expression in colostrum, the overall transcription levels of ELF5, ABCG2, and SREBF2 in milk were found to be significantly upregulated in early, peak, and late lactation, and significantly downregulated thereafter, before the dry period. A similar pattern was observed in the relative expression of DGAT1, but no significant difference was revealed in its expression in milk from late lactation compared with colostrum. Based on these results, we suggest that the geometric mean of RPS9, PPP1R11, UXT, and MRPL39 can be used for normalization of real-time PCR data in milk somatic cells of lactating yak, if similar experiments are performed. © 2014 American Dairy Science Association.


Bai W.L.,ShenYang Agricultural University | Bai W.L.,Cornell University | Yin R.H.,ShenYang Agricultural University | Jiang W.Q.,ShenYang Agricultural University | And 5 more authors.
Livestock Science | Year: 2013

Caseins are important for cheese making, and their amounts and relative concentrations have important influence on the nutritional and technological properties of milk. In the present work, we investigated the mRNA relative proportions and translational efficiency of yak casein transcripts and showed that, for the first time, the four casein mRNAs were not transcribed and translated with the same efficiency. The mRNA transcripts for yak αs1-casein (17.5%) and κ-casein (20.9%) appeared to be less abundant than those of Β-casein (31.9%) and αs2-casein (29.7%). The quantitative determination of the four casein fractions of yak milk showed that Β-casein and αs1-casein were the main caseins, accounting for 48.2% (16.5g/L) and 30.8 (10.5g/L), respectively, whereas the αs2-casein (8.7%, 2.9g/L) and κ-casein (12.3%, 4.2g/L) exhibited less abundance. The αs1-casein transcripts had the highest translational efficiency with a value of 1.8, and the next highest was Β-casein transcripts of which the value was 1.5, whereas the αs2-casein transcripts had the lowest translational efficiency with a value of 0.3. The analysis results of the sequence context of translation initiation codon AUG of the casein mRNA might explain, at least in part, the differential transcriptional and translational rate observed among the casein transcripts. The results from the present work would contribute to elucidating the molecular regulatory mechanisms of yak casein translation. © 2012 Elsevier B.V.


PubMed | Jilin University, Animal Science Research Academy of Sichuan Province, Qinghai University, ShenYang Agricultural University and Research Academy of Animal Husbandry and Veterinary Medicine science of Jilin Province
Type: Journal Article | Journal: Journal of dairy science | Year: 2014

Quantitative real-time PCR is the most sensitive technique for gene expression analysis. Data normalization is essential to correct for potential errors incurred in all steps from RNA isolation to PCR amplification. The commonly accepted approach for normalization is the use of reference gene. Until now, no suitable reference genes have been available for data normalization of gene expression in milk somatic cells of lactating yaks across lactation. In the present study, we evaluated the transcriptional stability of 10 candidate reference genes in milk somatic cells of lactating yak, including ACTB, B2M, GAPDH, GTP, MRPL39, PPP1R11, RPS9, RPS15, UXT, and RN18S1. Four genes, RPS9, PPP1R11, UXT, and MRPL39, were identified as being the most stable genes in milk somatic cells of lactating yak. Using the combination of RPS9, PPP1R11, UXT, and MRPL39 as reference genes, we further assessed the relative expression of 4 genes of interest in milk somatic cells of yak across lactation, including ELF5, ABCG2, SREBF2, and DGAT1. Compared with expression in colostrum, the overall transcription levels of ELF5, ABCG2, and SREBF2 in milk were found to be significantly upregulated in early, peak, and late lactation, and significantly downregulated thereafter, before the dry period. A similar pattern was observed in the relative expression of DGAT1, but no significant difference was revealed in its expression in milk from late lactation compared with colostrum. Based on these results, we suggest that the geometric mean of RPS9, PPP1R11, UXT, and MRPL39 can be used for normalization of real-time PCR data in milk somatic cells of lactating yak, if similar experiments are performed.

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