Molecular characterization of complete coding sequence of the MBL1 gene in the Indian Buffalo (Bubalus bubalis) breed

Abstract

Mannose-binding lectin (MBL) is a member of the collectin protein family that binds a broad range of microorganisms and activates the lectin-complement pathway of innate immunity. A number of mutations have been found in both the coding as well as the non-coding regions of the MBL1 gene in various species, of which several variations affected the assembly of MBL1, thus leading to a low level of plasmic MBL and innate immune dysfunctions. In the present study, we have reported molecular cloning and characterization of the complete coding sequence of the MBL1 gene in the Indian buffalo breed Murrah. A 951 bp fragment of the MBL1 gene was amplified, cloned and sequenced. Multiple sequence alignment with other buffalo and cattle breeds revealed that the Murrah buffalo MBL1 CDS was 98.1-99.6% homologous to other buffalo breeds, and 98.3-98.5% similar to cattle breeds at nucleotide level. It was 96.8-98.8% homologous to buffalo breeds and 96.8-97.2% similar to cattle breeds at amino acid level. The amino acid sequence of the Murrah buffalo MBL1 contained two non-synonymous amino acid substitutions (L204P and S180P). Further, PCR-RFLP was performed to screen 50 Murrah buffalo for the presence of SNPs, g.855G>A in intron I and g.2686T>C, as well as g.2651G>A in exon 2 region of the MBL1 gene. The ApaI/intron I PCR-RFLP assay revealed a polymorphic pattern with three genotypes viz., AG (90%), GG (8%) and AA (2%), with allelic frequencies 0.94 for G and 0.06 for A. HaeIII/exon 2 PCR-RFLP assay exhibited the presence of three genotypes, namely, TC (66%), TT (32%) and CC (2%) with allelic frequencies 0.15 for T and 0.85 for C. StyI/exon 2. PCR-RFLP assay showed a monomorphic pattern for g.2651G>A with GG genotype only. We further examined the association of these SNPs with milk production traits and somatic cell score (SCS), and found no significant difference for any of the traits. Since the present study has formulated the results on the basis of a relatively small sample size, further studies with a larger sample size are required to validate the effects of polymorphisms.