Structural features of the 5 ’ flanking region of the Yak ( Bos grunniens ) growth hormone receptor ( GHR ) gene

1Academy of Animal Science and Veterinary Medicine, Qinghai University, Xining, People’s Republic of China, 2College of Life Science and Technology, Southwest University for Nationalities, Chengdu, People’s Republic of China, 3College of Agricultural and Animal Husbandry, Qinghai University, Xining, People’s Republic of China, 4Department of Biosciences, Qinghai University, Xining, People’s Republic of China


Background
Yak (Bos grunniens) is a species of the Bovidae family living on the Qinghai-Tibetan Plateau and its adjacent territories at altitudes from 2 000-5 000 m (LUO et al. 2005, MA et al. 2009).As a multi-purpose domestic animal, yaks are indispensable to the local animal husbandry development because it can provide life necessities such as meat, milk, fur, velour manufacturing, transportation and manure for fuel to the local herdsmen.There are twelve yak populations numbered around 13.3 million in China and the Bazhou yak is one of Chinese yak populations (WIENER et al. 2003).The Growth Hormone Receptor (GHR) gene was identified as a candidate gene affecting key quantitative traits, like growth, milk yield and composition in livestock.At present, only investigations were carried out on the genetic variation in Exon-8, Intron-8 and Exon-10 of the yak GHR gene (VARVIO et al. 2008), no other information on the yak GHR gene is available.The purposes of this study were to -analyze the structural features of the 5' flanking region of the yak GHR gene -compare the 5' flanking region of yak GHR gene with that of other species of the Bovidae family and -construct the phylogenetic tree to indicate the evolutionary relationship among them.

Cloning, sequencing and sequence analyses
The amplified PCR products were electrophoresed on a 1.0 % agarose gel and purified using the DNA Agarose Gel Extraction Kit (Omega) according to the manufacturer's instructions.The purified fragments were cloned into pMD18-T vector (TakaRa, Dalian, China) subsequently transformed into Escherichia coli JM109 (TakaRa, Dalian, China).The identified positive clone was sequenced using an ABI 3730 automated sequencer (Applied Biosystems).

Results
The 888 bp of yak GHR gene Exon 1A (205 bp), P1 promotor region (511 bp) and its partial flanking sequence were sequenced (Figure A1).The sequence was deposited in the GenBank database (acc.no.EF202183).The percentage of simple repeats was 5.63 % and no SINEs, LINEs, LTR elements or DNA elements were found, although one (TG)25 microsatellite DNA was found firstly in yak GHR gene P1 promotor region.There are 37 and 20 potential high-scoring binding sites for transcription factors (threshold: 85.0 point), including C/EBP, HFH-2, HNF-3b, SP1,C/EBPb, and Oct-1 etc, were detected in yak GHR gene P1 promotor region and its partial flanking sequence, respectively.
The molecular phylogenetic tree among 6 species of Bovidae constructed by the UPGMA method based on the sequences of P1 promotor and Exon 1A of the GHR gene (Figure A3), placed the three Bovinae species on one branch and the three Caprinae species on the other.The yak and European bison were joined first, followed by the cattle, and then the Caprinae species, including the sheep, mouflons and the goat.The result of phylogenetic clustering was not only identical to the taxonomy, but also to the phylogenetic clustering using the mitochondrial DNA of yak and other species of Bovidae (GUO et al. 2006, LI et al. 2006)..: Indicates sequence identity with that of the yak, -: Indicates either insertion or deletion Exon 1A sequence is underlined and the 5′ sequence to Exon 1A is promoter P1.The position of microsatellite loci in 6 species of Bovidae family is boxed.
Figure A1 Nucleotide sequence of the 5′ flanking region of the yak GHR gene The promoter P1 is presented in small letters.The Exon 1A is presented in capital and bold letters.The transcription start site is indicated by an arrow and defined as +1.Nucleotide markers are marked on the left side.Upstream nucleotides have negative numbers.Partial potential transcription factor binding sites showed and lined with the arrow noting the orientation.The TATA box is boxed.The (TG)25 microsatellite DNA is indicated by italic small letters.Partial sequence-specific transcription factor, Abbreviation: HNF-3b, hepatic nuclear factor 3 beta; Tst-1, POUfactor; HFH-2, fork head domain factor 2; C/EBP, enhancer binding protein; STATx, signal transducers and activators of transcription; Oct-1, octamer-binding factor 1; Sp1, stimulating protein 1; C/EBPb, enhancer binding protein beta; RREB-1, Ras-responsive element binding protein 1