Yucai Zheng

Yak lactate dehydgogenase A4: purification, properties, and cDNA cloning.

Lactate dehydrogenase A4 (LDH-A4) was purified for yak skeletal muscle. Michaelis constant (Km) analysis showed that yak LDH-A4 for pyruvate was significantly higher than that of cattle. cDNA cloning of LDH-A revealed two amino acid substitutions between yak and cattle. We suggest that the higher Km of yak LDH-A4 might be a result of molecular adaptation to a hypoxic environment.

Identification of yak lactate dehydrogenase B gene variants by gene cloning.

Native polyacrylamide gel electrophoresis showed that two types of lactate dehydrogenase (LDH) existed in yaks. Based on the electrophoresis characteristics of LDH isoenzymes, yak LDH variants were speculated to be the gene mutation on H subunit encoded by B gene. According to the mobility in electrophoresis, the fast-band LDH type was named LDH-Hf and the slow-band LDH type LDH-Hs. In order to reveal the gene alteration in yak LDH variants, total RNA was extracted from heart tissues of yaks with different LDH variants, and cDNAs of the two variants were reverse transcripted. Two variants of B genes were cloned by RT-PCR. Sequence analysis revealed that four nucleotides differed between LDH-Bf and LDH-Bs, which resulted in two amino acids alteration. By Deepview software analysis of the conformation of yak LDH1 variants and H subunit, these four nucleotides altered two amino acids that generated new hydrogen bonds to change the hydrogen bonds network, and further caused subtle conformational changes between the two LDH variants.

Cloning and Polymorphisms of Yak Lactate Dehydrogenase b Gene

The main objective of this work was to study the unique polymorphisms of the lactate dehydrogenase-1 (LDH1) gene in yak (Bos grunniens). Native polyacrylamide gel electrophoresis revealed three phenotypes of LDH1 (a tetramer of H subunit) in yak heart and longissimus muscle extracts. The corresponding gene, ldhb, encoding H subunits of three LDH1 phenotypes was obtained by RT-PCR. A total of six nucleotide differences were detected in yak ldhb compared with that of cattle, of which five mutations cause amino acid substitutions. Sequence analysis shows that the G896A and C689A, mutations of ldhb gene, result in alterations of differently charged amino acids, and create the three phenotypes (F, M, and S) of yak LDH1. Molecular modeling of the H subunit of LDH indicates that the substituted amino acids are not located within NAD+ or substrate binding sites. PCR-RFLP examination of G896A mutation demonstrated that most LDH1-F samples are actually heterozygote at this site. These results help to elucidate the molecular basis and genetic characteristic of the three unique LDH1 phenotypes in yak.

Quantitation of alternative splicing variants of lactate dehydrogenase C gene in testes of adult yak, sexually immature yak calf and sterile male hybrid of yak

Huang, L., Jin, S-Y., Xu, Y-O., Li, Y-P., Lin, Y-Q. and Zheng, Y-C. 2012. Quantitation of alternative splicing variants of lactate dehydrogenase C gene in testes of adult yak, sexually immature yak calf and sterile male hybrid of yak. Can. J. Anim. Sci. 92: 291-296. The main objective of the present study was to analyze quantitatively the alternative splicing of the lactate dehydrogenase C (ldhc) gene in the testes of yak (Bos grunniens) and male sterile yak hybrid. RT-PCR amplification of ldhc cDNA in the testes revealed eight splice variants formed by the deletion of one or more exons in the mRNA transcripts. The deleted exons occur mostly in exons 7 and 4. The deletion of exons caused reading frame shift and formation of stop codon in some variants. Quantitative real-time RT-PCR analysis using ldhc variant-specific primers showed that the mRNA level of full length ldhc decreased dramatically in the testes of sexually immature yak calf (n=6) and male sterile hybrid cattle-yak (n=4) compared with that of adult yak (n=14). The proportions of the ldhc variants assayed differed significantly among adult yak, yak calf and cattle-yak; more ldhc transcripts were spliced in immature or sterile testes. Our results suggest that the alternative splicing could play a role in the regulation of ldhc expression in testes, and could be one factor that plays a role in infertility of yak hybrids.

Analysis of Yak MUC1 Protein Polymorphisms and the Corresponding VNTR Structure

The genotypes and protein polymorphisms of milk epithelial mucin (MUC1) were analyzed by touch-down PCR and SDS-PAGE respectively using blood samples and milk collected from 50 lactating yaks. A total of seven alleles were revealed, namely A, B, C, D, E, F, and G, and the corresponding number of repetitive units of variable number tandem repeat (VNTR) in MUC1 gene were 21, 20, 19, 18, 17, 16, and 15, respectively. Fifteen genotypes of MUC1 were observed in yaks. The genotypes of MUC1 gene matched completely to the phenotypes of milk MUC1 in each individual. This study demonstrated that the yak MUC1 exhibits abundant polymorphisms in both its gene and protein, and the polymorphisms are due to the expression of VNTR in MUC1 gene. The possible cluster of the VNTR was also discussed in different ruminants.

Alternative Splicing of Testis-Specific Lactate Dehydrogenase C Gene in Mammals and Pigeon

The objective of the present study was to confirm the widespread existence of alternative splicing of lactate dehydrogenase c (ldhc) gene in mammals. RT-PCR was employed to amplify cDNAs of ldhc from testes of mammals including pig, dog, rabbit, cat, rat, and mouse, as well as pigeon. Two to six kinds of splice variants of ldhc were observed in the seven species as a result of deletion of one or more exons or insertion of partial sequence of an intron in the mature mRNA. The deleted exons occur mostly in exons 5, 4, 6, and 3. The insertion of a partial sequence of introns, which resulted in an abnormal stop codon in the inserted intron sequence, was observed only in dog and rat. The deletion of exons also resulted in a reading frame shift and formation of a stop codon in some variants. No alternative splicing was observed for ldha and ldhb genes in testis of yak. Native polyacrylamide gel electrophoresis and Western blot analysis revealed no obvious LDH-C4 activity derived from expressed ldhc variants. Our results demonstrated the widespread and unique existence of alternative splicing of ldhc genes in mammals.

Comparison of MUC1 variable number tandem repeat polymorphisms in three yak breeds/populations

Gao, J., Jiang, Z.-R., Liu, X., Zhao, Y.-H., Huang, L., Peng, H.-J., Zedan, D., Jin, S.-Y. and Zheng, Y.-C. 2014. Comparison of MUC1 variable number tandem repeat polymorphisms in three yak breeds/populations. Can. J. Anim. Sci. 94: 411-416. The objective of this study was to compare the MUC1 variable number tandem repeat polymorphisms with adjacent distribution regions in three yak (Bos grunniens) breeds/populations. A total of 215 yaks of three yak breeds/populations (Maiwa yak breed, Jiulong yak breed and Changtai yak population) were surveyed by the polymorphisms of Mucin 1 gene (MUC1). Six MUC1 alleles (B, C, D, F, G and H) forming 16 genotypes were identified. Cloning and sequencing of these alleles demonstrated that they differed in the numbers of variable number tandem repeats (VNTR) units ranging from 14 to 20, and allele H (14 VNTR units) was a new allele in yaks and observed only in Maiwa yak with a very low frequency. Cluster analysis based on MUC1 polymorphisms suggested that Changtai yak population has a closer association with Maiwa yak breed than with the Jiulong yak breed, and may represent an independent yak breed/population. These results provided useful genetic information on the three yak breeds/populations and could be used in yak breeding practice.

Purification and properties of a monomeric lactate dehydrogenase from yak Hypoderma sinense larva.

The objective of the present study was to study the characteristics of lactate dehydrogenase (LDH) from Hypoderma sinense larva. H. sinense larvae were collected from yak (Bos grunniens) and identified by a PCR-RFLP method. Analysis of LDH activity showed that the total LDH activity in H. sinense larva was negatively correlated with the length of larva. Polyacrylamide gel electrophoresis of the extracts of H. sinense larvae revealed one band of LDH, which was then purified by affinity chromatography and gel filtration. This enzyme showed an approximately 36 kDa band on SDS-gel under both reducing and non-reducing conditions, in addition, size exclusion chromatography analysis showed that its molecular weight was smaller than bovine serum albumin (67 kDa), indicating that it contains only one subunit. Michaelis constants (Km) values assay revealed that LDH from H. sinense larva showed significantly lower Km for lactate than other animals. LDH of H. sinense larva was stable at 60 °C for 15 min, and also exhibited high catalytic efficiency in a wide range of pH. HgCl₂ at the concentration of 0.1mM significantly decreased the activity of LDH from H. sinense larva but not at the concentration of 0.01 mM. The results of the present study demonstrate that LDH from H. sinense larva is a thermal stable and pH insensitive enzyme suitable for catalyzing both forward and reverse reactions.

Cloning and expression profiles of yak lipoprotein lipase gene

The objective of this study was to reveal the sequence characteristic and expression pattern of lipoprotein lipase (LPL) gene in high altitude yaks (Bos grunniens). A full-length cDNA of LPL was cloned from yak liver by RT-PCR. The cDNA obtained was 1591 nucleotide (nt) long with a 1437 nt open reading frame encoding 478 amino acids. Yak LPL shares 99.5% nucleotide sequence similarity and 100% amino acid sequence similarity with cattle. Quantitative real-time PCR analysis showed that yak heart and adipose tissue contained significantly higher LPL mRNA level than other tissues assayed. Yak calves contained lower level of LPL mRNA in longissimus dorsi than adult yaks (p < 0.01). Besides, LPL mRNA level in longissimus dorsi was markedly lower in adult yaks that in adult cattle living at low altitude region.

Prokaryotic Expression and Immunogenicity Analysis of Yak Recombinant Myostatin

Myostatin (MSTN) is a negative regulator of skeletal muscle growth. The objective of the present study was to express yak (Bos grunniens) recombinant MSTN protein in E. coli and study its characteristics of immunogenicity. cDNA encoding yak MSTN mature peptide was amplified by reverse-transcription PCR, and cloned into pET28a(+) vector and expressed in E. coli. The expressed recombinant MSTN was purified by affinity chromatography and used to prepare rabbit anti yak MSTN antibody. The results showed that yak MSTN mature peptide gene contained 330 bp nucleotides coding 109 amino acids. Content of the target protein accounted for 21% of the total expression products when MSTN-pET28a(+)-BL21(DE3) bacterium was incubated in LB medium with 0.1 mM IPTG for 6 hours. The molecular weight of the purified yak MSTN recombinant protein was 16.5 kDa, exhibiting excellent immunogenicity as shown by ELISA. The obtained recombinant MSTN of yak is suitable for further analysis of yak MSTN functions.