Screening the partial coding region of metallothionein isoform-2 gene in Zebu cattle

Abstract

Metallothionein (MT) is important because it binds tightly to heavy metals to decrease their toxicity. DNA was isolated from 30 toxic metal exposed and 30 toxic metal unexposed Zebu cows. The amplified metallothionein isoform-2 (MT-2) PCR product (489 bp) was further used for PCR-RFLP and DNA sequencing. MT-2 TaqI PCR-RFLP revealed homozygous genotype (AA) except for the E23 animal (AB). The genotype frequency of AA and AB (E23) genotypes in the exposed groups was 0.967 and 0.033 respectively. DNA sequencing was carried out for the toxic metal exposed sample (E23) and the control group sample (C13). Blast comparisons of the sequences were then aligned against a nucleotide database which revealed 150 nucleotide substitutions consisting of 70 transitions and around 80 transversions. DNA sequencing followed by PCR-RFLP for MT-2 revealed a higher number of nucleotide substitutions (150) for the AB genotype of E23 as compared to the AA genotype (38) of E21. The proportions of transversion mutations in the AB genotype were higher as compared to the MT-2 AA genotype. DNA sequencing was carried out based on random sampling for E21 and C13. Alignment analysis of the E21 and C13 sample revealed 38 nucleotide substitutions consisting of equal numbers of transition and transversion. BLAST analysis of the identified partial sequence revealed 89% identity with Bos taurus, 85% identity with sheep, 98% identity with buffalos and 100% identity with goat MT-2 sequences. Overall findings of the present study revealed DNA sequence variation in the coding region of the MT-2 gene of Zebu cattle which can be utilised to characterize and explore markers for heavy metal homeostasis in Zebu cattle.