بررسي ژن BMP 15 در گوسفندان افشاري و آميخته افشاري× برولامرينو

Study of BMP 15 Gene in Afshari and Afshari × Booroola Merino Cross Sheep

افزایش تعداد بره به ازای هر میش به عنوان راهكاری برای افزایش بهره¬وری در پرورش گوسفند مورد توجه زیادی قرار گرفته است. صفات تولید مثلی از وراثت پذیری پائینی برخوردار هستند اما امروزه جهش¬هایی در برخی ژنها از جمله BMP15 یافت شده است كه با چند قلوزایی در ارتباط است. هدف از مطالعه حاضر شناسایی چند شكلی و بررسی بیان ژن BMP15 در تخمدان میش¬های آبستن و غیر آبستن افشاری بود. در این مطالعه از تعداد 35 راس میش افشاری و 45 راس میش آمیخته افشاری× برولا مرینو خون گیری و استخراج DNA به عمل آمد. سه قطعه از ژن BMP 15 به طول¬های 581، 325 و 857 جفت باز به ترتیب برای ناحیه پروموتر، اگزون یك و دو برای وجود چند شكلی از طریق تعیین توالی محصولات PCR مورد بررسی قرار گرفت. نتایج نشان داد كه در پروموتر و اگزون یك هیچ گونه تغییر توالی وجود ندارد اما در اگزون دو، نوكلئوتید 134 دچار تغییر شده و دو ژنوتیپ هموزیگوت CC و هتروزیگوت CA مشاهده گردید. پس از كشتار از 22 راس میش آبستن و 8 راس میش غیر آبستن بافت تخمدان تهیه شد. از نمونه¬ها RNA كامل استخراج و سپس به cDNA تبدیل شد. نتایج بررسی اختلاف معنی¬داری از لحاظ بیان ژن BMP15 بین دو گروه آبستن و غیر آبستن و آلل¬ها نشان نداد. احتمالا این ژن در ارتباط با سایر ژنها و از جمله گیرنده های مربوطه¬اش در ارتباط با تولید مثل ایفای نقش می¬كند و به همین دلیل ممكن است بیان آن در مراحل مختلف تولید مثلی مورد نیاز باشد.

Introduction One of the most important sources of the red meat in Iran is the meat produced from sheep. Increasing lamb per ewe considered as a strategy for improving the efficiency for sheep production, although reproduction traits have low heritability. Several genes associated with reproduction were investigated in the recent years. The BMP 15 gene and its paralog GDF 9 and receptor, BMPR-IB, are related to fecundity in sheep and attracted the interest of breeders recently. All these genes that are members of TGFβ super family are functionally closely related together and they affect expression and secretion of hormones affecting follicle growth and ovulation rate in mammals. BMP15 plays a key role in regulating many processes in granulosa cells and ovulation rate. Mutations in some candidate genes such as BMP 15 proved to affect the lambing rate. Since 2008, introgression of the BMP Receptor IB mutant (FecB) from Booroola Merino (from New Zealand) into Afshari sheep was initiated. Thereafter, several genes that proved to have an effect on reproductive traits were studied in this breed. This study was conducted to identify possible polymorphism(s) in BMP 15 and to compare its expression in ovaries of pregnant and non-pregnant ewes. Materials and Methods To study these, blood samples were collected from 35 and 45 Afshari and Afshari × Booroola Merino ewes, respectively. DNA was extracted from all samples using phenol-chloroform procedure and Total RNA was extracted using the RNA extraction kit, CinnaPure RNA Kit (Cinnagen Inc®, Iran), extraction was performed according to the manufacturer’s instruction. To remove any possible residual DNA contamination, RNA samples were treated with 1 unit of DNase (Vivantis Inc®, Malaysia). The specific primers were designed for three areas of BMP 15, namely promotor (581 bp), exon one (325 bp) and exon two (857 bp) and the targets were amplified using PCR. The PCR products were sequenced using forward and reverse primer for all of the samples. Result and discussion There was no difference among sequences of the promoter and the first exon among samples. But, a nucleotide in position 134 of the second exon, C was replaced by A, was observed in two samples with heterozygote genotypes AC instead of CC. Nonetheless, the codon of amino acid encoding proline is remained unchanged. This mutation occurred in two Afshari × Booroola Merino ewes. This mutation, to our knowledge, was not reported to date. Parents of these ewes were not available and also due to the low frequency of the mutation, detection and identification breed of the origin for the mutation was not possible. To date, such a mutation neither was reported in Afshari nor in Booroola Merino breeds. Obviously, the promoter of the gene is conserved and it shows high similarity amongst related species. Nevertheless, in our study sample size was limited to conclude this well. Considering conservation of the promoter of the gene within the species and closely related species, it appears that the regulatory regions were very protected and required for its sustained action. Given that a number of animals used in the study were twine bearing Afshari × Booroola Merino crosses, but there was no difference between them and Afshari pure breed in terms of BMP15 gene expression and gene sequences. Thereupon, the results of this study indicate that this gene plays no role in litter size of this new genetic component. In order to assess the expression of this gene in ovaries of the ewes, after slaughtering, ovary samples of 22 pregnant ewes and those of 8 none pregnant were collected. Total RNA was extracted from the samples and mRNA converted to cDNA using oligo d (T) primer and reverse transcriptase. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the endogenous control for normalization. Results of real time PCR using designed specific primers showed no difference in BMP 15 gene expression between pregnant and non-pregnant ewes. Conclusion It is possible that this gene plays its role in relation to other genes include their receptors and its expression is needed in different steps of reproduction. This result and reports of other studies suggest that more data on BMP15 gene with a simultaneous expression of other genes in the ovary is needed to clarify the integral role of BMP 15 in reproduction.