Determination of Dopamine Receptor Gene Polymorphism by BseNI Restriction Enzyme

This research was conducted at the Department of Animal Science in the Ramin Agriculture and Natural Resources University in 2014-2015. The elevation of egg production and the inhibition of incubation behavior are the aims of modern poultry production . Prolactin is postulated to play a critical role in the onset and maintenance of incubation behavior in birds .In avian, dopamine inhibits prolactin secretion in the brain. So far, at least five distinct dopamine receptor subtypes, DRD1-DRD5, have been identified and classically divided into two classes referred to as D1-like (DRD1 and DRD5) and D2-like (DRD2,DRD3, and DR 4) .DRD1 is located on chromosome 13 and contains an open reading frame of 1356 nucleotides encoding a protein of 451 amino acids. Dopamine stimulates prolactin secretion via activating DRD1 at the hypothalamus level by operating through vasoactive intestinal peptide and the inhibition effect of dopamine on Prolactin secretion is mediated through DRD2 receptors at the pituitary level.This study aimed at identification of the variants of dopamine D1 receptor gene and detection of the allelic frequency in the Khuzestan native chicken at Ramin Agriculture and Natural Resources of Khuzestan province.
For this research 100 laying hens from Khuzestan native chicken Breeding Center (Jahed Livestock Input Corporation) were randomly selected. DNA was extracted from whole blood using salting-out procedure. The PCR-RFLP method was used for allelic differentiation. Dopamine D1 receptor gene was amplified by a specific set of primer for this gene to produce 283 bp fragment. The PCR reactions were carried out in a total volume of 25 μL containing 150 ng of genomic DNA, 1 μL of each primer, .5 μL dNTP, 1 μL MgCl2, 2.5× PCR buffer and 1 U of Taq DNA polymerase. The amplification was performed in a Eppendorf Mastercycler under the following conditions: 95°C for 3 min; 35 cycles of 95°C for 30 s, 58/4°C for 45 s and 72°C for 30 s; and 72°C for 10 min. The amplified fragment was digested with BseN I restriction enzyme. The digestion mixture was composed of 10μL PCR products, 2μL digestion buffer, and 1μL of each enzyme, and then subjected to electrophoresis separation in 2.5% Agarose gel.
The results of the enzyme Restrictive BseNI showed only one A allele and AA genotype and polymorphism was not observed. To determine the quality and quantity of DNA, Nanodrop and Agarose gel was used and the results showed that the extracted DNA was suitable to continue the research. The results of this study were not in par with those of the previous research. Such non compliance could be due to the kind of population studied, the sample size and the type of marker based on which polymorphic was examined. Due to the limited number of cutting sites in restriction enzymes, various DNA fragments were not produced. Therefore, RFLP markers may have not been able to identify all mutations in this sequence. Dopamine receptor gene in Khuzestan native chicken was sequenced for the first time in the present study. Hence, alignment sequences of Khuzestan native chicken and alignment sequence in ClastaW2 were saved in the gene bank. The results of sequencing in ClastaW2 recorded two mutations of type A to G in the base 12 and C to T in the base 198.
Considering the results of gene sequencing, it cannot be stated that a dopamine receptor in this research is monomorphic. However, the enzyme used for dopamine gene could not be able to recognize the restriction sites. Sequencing of dopamine D1 receptor gene in the native chicken population of Khuzestan showed mutation which normally causes genetic polymorphism. However, in this study due to the ineffective choice of the enzyme, monomorphism was detected. These results show the importance of restriction enzyme in detecting genetic variation. Since dopamine is one of the main factors known to reduce prolactin and decrease broodiness as well as the reports indicated that mutations in dopamine D1 receptor different genotypes were significantly associated with increased dopamine.
Due to the important role of restriction enzymes in identification of different mutations, selection of the suitable enzyme is recommended.