Cloning Of HO And BVR Gene Fragment And The Study Of MRNA Expression In Duck

Heme oxygenase(HO) is a kind of antioxidative enzymes which is the rate-limiting enzyme of heme degradation,HO is one of the restrict enzymes.It can produces bilirubin,carbon monoxide(CO), and iron. Then biliverdin was reduced to bilirubin by biliverdin reductase, and the free iron binding with the transferring protein. Eggshell color is a important economic trait for duck., which was decided by the composition and contents of the pigment in the eggshell. Heme oxygenase(HO) and Biliverdin reductase A (BLVRA) are key enzymes in the metabolic process of pigment.In this paper,in order to amplify part of th HO and BVR’s cDNA coding sequences by PT-PCR,using total RNA templates extracted from duck’s uterus,then cloning the sequences for study the two genes’ mRNA expression levers.According to the HO and BVR gene conserved sequences of Gallus gallus, Homo sapiens, Mus musculus and Bos Taurus in GenBank, two pairs of primers was designed to amplify from the extracted total RNA of interstitial part of uterine tube by using RT-PCR the HO and BVR coding region.The RT-PCR products were cloned into the pMD19-T plasmid vecto,then transformed into E.coli JM109, and the positive transformant sidentified by PCR were transferred into LB medium for extracting its’plamid. The nucleotide and the putative amino acid of HO and BVR gene were compared with other animals, such as Gallus gallus, Mus musculus, Bos taurus, Xenopus tropicalis and Homo sapiens, HO gene shares93.1%,70.4%,70.1%,63.6%,71.5%and98.4,91.1%,59.3%,86.2%,91.1%homology in nucleotide and putative amino acid sequence respectively,BVR gene shares92.6%,64.9%,62.8%,69.0%,63.6%and96.2%,59.7%,60.7%,68.4%,59.7%homology in nucleotide and putative amino acid sequence respectively. It is proved that HO gene and BVR gene were considerably conserved in the process of evolution.Use Real Time-PCR to study the expression levers of HO and BVR, use GAPDH as a contrast. According to the Ct values obtained from the Real Time-PCR,analysised by SAS software,there is a conclusion that the whitecolorde and the bluecloured eggshells have a great relationship with the BVR gene mRNA expression lever, the HO gene have a great relationship with the bluecloured eggshells.