بررسی ژن BMP 15 در گوسفندان افشاری و آمیخته افشاری× برولامرینو

نوع مقاله : علمی پژوهشی- ژنتیک و اصلاح دام و طیور

نویسندگان

گروه علوم دامی، دانشکده کشاورزی، دانشگاه زنجان، زنجان، ایران

چکیده

افزایش تعداد بره به ازای هر میش به عنوان راهکاری 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 که با چند قلوزایی در ارتباط است. هدف از مطالعه حاضر شناسایی چند شکلی 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 یک و دو برای وجود چند شکلی از طریق تعیین توالی محصولات PCR مورد بررسی قرار گرفت. نتایج نشان داد که در پروموتر و اگزون یک هیچ گونه تغییر توالی وجود ندارد اما در اگزون دو، نوکلئوتید 134 دچار تغییر شده و دو ژنوتیپ هموزیگوت CC و هتروزیگوت CA مشاهده گردید. پس از کشتار از 22 راس میش آبستن و 8 راس میش غیر آبستن بافت تخمدان تهیه شد. از نمونه ها RNA کامل 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 کند و به همین دلیل ممکن است بیان آن در مراحل مختلف تولید مثلی مورد نیاز باشد.

کلیدواژه‌ها


عنوان مقاله [English]

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

نویسندگان [English]

  • Roghieh Gholipour
  • Leila Danesh Moghadam
  • Mohammad Taher Harkinezhad
Department of Animal Science, Zanjan Faculty of Agriculture, Zanjan, Iran
چکیده [English]

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.

کلیدواژه‌ها [English]

  • Afshari sheep
  • BMP 15 gene
  • multiple lambing
  • Polymorphism
1- Bessa, I. R., R. C. Nishimura., M. M. Franco., and M. H. Dode. 2013. Transcription profile of candidate genes for the acquisition of competence during oocyte growth in cattle. Reproduction in Domestic Animals, 48: 781-789.
2- Barzegari, A., S. Atashpaz., K. Ghabili., Z. Nemati., M. Rustaei., and R. Azarbaijani. 2010. Polymorphisms in GDF9 and BMP15 Associated with fertility and ovulation rate in moghani and ghezel sheep in Iran. Reproduction in Domestic Animals, 45: 666-669.
3- Chang, H. M., J. C. Cheng., E. Taylor., and P. C. Leung. 2014. Oocyte-derived BMP15 butnotGDF9 down-regulates connexin43 expression and decreases gap junction intercellular communication activity in immortalized human granulosa cells. Molecular Human Reproduction, 20 (5): 373-383.
4- Davis, G. H 2005. Major genes affecting ovulation rate in sheep. Genetics Selection Evolution, 37:S11–S23.
5- Davis, G. H. 2004. Fecundity genes in sheep. Animal Reproduction Science, 82: 247-253.
6- Fenwick, M. A., J. M. Mora., Y. T. Mansour., C. Baithun., S. Franks., and K. Hardy. 2013. Investigations of TGF-beta signaling in preantral follicles of female mice reveal differential roles for bone morphogenetic protein 15. Journal of Endocrinology, 154: 3423-3436.
7- Hanrahan, J. P., S. M. Gregan., P. Mulsant., M. Mullen., G. H. Davis., R. Powell., and S. M. Galloway. 2004. Mutations in the genes for oocyte-derived growth factors GDF9 and BMP15 are associated with both increased ovulation rate and sterility in Cambridge and Belclare sheep Ovis aries. Biology of Reproduction, 70: 900-909.
8- Kasiriyan, M. M., H. Hafezian., and N. Hassani. 2011. Genetic polymorphism BMP15 and GDF9 genes in Sangsari sheep of Iran. International Journal of Genetics and Molecular Biology, 3:31-34.
9- Khanahmadi, A., Sh. Gharehveysi., R. Khataminejad., and J. Arab. 2014. Polymorphic Variants of G1 and B4 from GDF9 of Dalagh Sheep. Research on Animal Production, 10: 148-155. (In Persian).
10- Livak, K. J., and T. D. Schmittgen. 2001. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 22-ΔΔCt Method. METHODS, 25: 402–408.
11- Monteagudo, L., R. Ponz., M. Teresa Tejedor., A. Lavina., and I. Sierra. 2009. A 17 bp deletion in the bone morphogenetic protein 15 gene is associated to increased prolificacy in the Rasa Aragonesa sheep breed. Animal Reproduction Science, 110: 139-146.
12- Pirkhezranian, Z., A. Mohammad Hashemi, M. Tahmorthpour and N. Pirani. 2015. Polymorphhism in second exon of BMP 15 gene in Sangsari sheep of Iran. Iranian Journal of Animal Science research, 6 (4): 381-387. (In Persian).
13- Pramod, R. K., S. K. Sharma., A. Singhi., S. Pan., and A. Mitra. 2013. Differential ovarian morphometry and follicular expression of BMP15, GDF9 and BMPR1B influencetheprolificacy in goat. Reproduction in Domestic Animals, 48: 803-809.
14- Solimani, B., M. G. Rahimi., and B. Chaharaein. 2011. The segregation of exon 2 BMP15 gene on twining and traits of weight in Sanjabi sheep. Iranian Jurnal of Biology, 24 (4):487-493. (In Persian).
15- Zhang, C. S., L. Geng., L. Du., Z. Liu., Z. Fu., M. S. Feng., and Y. F. Gong. 2011. Polymorphism study of FecXG , FecXH and FecXB Mutations in four Domestic sheep breeds in the lower yellow River valley of China. Journal of Animal and Veterinary Advances, 10:2198-2201.
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