Cloning and polymorphisms of yak lactate dehydrogenase B gene

Abstract

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.

Figure 1

Native PAGE of LDH isozymes in yak heart and skeletal muscles. LDH1-F: lane 1, 8, 10; LDH1-M: lane 2, 3, 4, 6, 9; LDH1-S: lane 5; multiple band phenotype of LDH isozymes: lane 7. Lane 1 to 9: heart tissues, lane 10: longissimus muscle.

Figure 2

PCR-RFLP analysis of ldhb G896A mutation. M: DL2000 DNA marker (bp). PCR products were digested by Taq I and separated by agarose electrophoresis. Genotypes are indicated on the top of each lane.

Figure 3

Charge distribution of H subunit of yak LDH1 variants. Red color represents negative charge, blue represents positive charge; the two mutations are labeled with a bright yellow color (298 Gln/Arg and 229 Glu/Ala). The areas of red color decreased from LDH1-F, LDH1-M, to LDH1-S.