مقایسه بیان ژن میوستاتین در گوسفند عربی و آمیخته‌های عربی×رومانوف

زمینه مطالعاتی: هدف از پژوهش حاضر، مقایسه بیان ژن میوستاتین در بره های نژاد خالص عربی و آمیخته های آن با رومانوف بود. روش کار: در این مطالعه از 16 راس بره عربی و دورگه در قالب طرح کاملا تصادفی با دو تیمار و هشت تکرار استفاده شد. میش های همسن با میانگین وزنی 2.5±54 کیلوگرم با استفاده از روش لاپاراسکوپی با اسپرم قوچ رومانوف پس از همزمان سازی فحلی تلقیح مصنوعی شدند. بره ها پس از تولد در شرایط یکسان پرورش داده شدند و در سن شش ماهگی کشتار شدند. بلافاصله پس از کشتار، نمونه گیری از قسمت ماهیچه لانگیسموس لومبروم از سمت راست دام انجام شد. پس از استخراج rna کل، کیفیت و کمیت آن بررسی شد و برای تولید cdna مورد استفاده قرار گرفت. ژن گلیسر آلدئید 3 فسفات دهیدروژناز به عنوان ژن خانه دار جهت نرمال نمودن داده ها استفاده شد. در نهایت بیان ژن میوستاتین به کمک realtime qpcr ارزیابی شد. برای تجزیه و تحلیل داده های وزن و بیان ژن بترتیب از نرم افزار sas و rest استفاده شد. نتایج این تحقیق نشان داد که آمیخته گری در این آزمایش سبب کاهش معنی دار بیان ژن میوستاتین در گروه آمیخته های  عربی×رومانوف شده است. میزان بیان ژن میوستاتین در بره های عربی 1.4 برابر بیش تر از گروه آمیخته بود (0.05˂p). با توجه به اینکه بیان ژن بیشتر میوستاتین با جلوگیری از تکثیر میوبلاست ها، رشد عضلانی را کاهش می دهد، می تواند یکی از دلایل اختلاف معنی دار وزن نهایی بره ها در دو گروه باشد. میانگین وزن نهایی بره های عربی و آمیخته ها به ترتیب  1.18±38.52 و 2.62±56.17 کیلوگرم  ثبت شد. وزن نهایی در بره های آمیخته نسبت به بره های عربی افزایش 1.46 برابری داشت. با توجه به کاهش بیان ژن میوستاتین در آمیخته های عربی  رومانوف، می توان نتیجه گرفت که کنترل بیان این ژن می تواند راهکار مناسبی در کنترل رشد بره ها باشد.

Comparison of Myostatin gene expression in Arabi and Arabi ×Romanov cross bred lambs

Introduction Expressed Myostatin gene in the muscles is a member of the TGFβ family. It extends skeletal muscle and regulates the activity of muscle fibers.The Myostatin gene (GDF8) works as a negative regulator of the skeletal muscle growth. Mutation in the Myostatin sequence changes its regulating function and results in the growth and hypertrophy of the muscles. Mutation in Myostatin has been found in various species. This gene has three exons and two introns in all species. Mutant alleles are significantly correlated with the growth rate and desired carcass traits and increase the ratio of muscle to fat and bone. Cross breeding is a common method of exploiting the genetic differences between different breeds and increasing the ability of individuals which is often considered as the quickest way to improve the sheep production efficiency.This study was conducted to investigate the effect of cross breeding on the Myostatin genes in Arabi lambs and its cross with Romanov. Materials and methods: At the beginning of the experiment, the similar ewes were artificially inseminated with Romanove ram sperm by laparoscopy method after estrous synchronization. Born lambs were kept in similar conditions.For this purpose, 16 Arabi and hybrid lambs were used in a completely randomized design with two treatments and eight replicates. After slaughter, the meat samples were immediately collected from right side of  longissimus lumborum muscle and transferred to the lab in liquid nitrogen and stored at 80 °C. RNA extraction was performed using the Trizol (Easy Blue) method. The quantity and quality of the extracted RNA were determined using a spectrophotometer (Thermo Scientific, Nanodrop, 2000C, USA) from 260 to 280 and 1% agarose gel (20 minutes at 100 volts). Total RNA quality was checked after extraction and used to produce cDNA. Synthesis of cDNA was made using the (Gene All) cDNA build kit in accordance with the manufacturer’s instructions. The designing of the forward and reverse primers for the Myostatin gene and the reference one of Glyceraldehyde 3phosphate dehydrogenase was performed using the Primer Quest program on the IDT site (Integrated DNA Technologies). Finally, the expression of Myostatin gene was evaluated by Realtime qPCR. Glyceraldehyde 3phosphate dehydrogenase gene as housekeeping one was used to normalize the data. SAS and REST softwares were used to analyze the final weight and gene expression data, respectively. Results and discussion cDNA synthesis was accomplished after extracting the RNA and measuring its purity. After the cDNA synthesis and dilution, the PCR reaction was performed. Following the successful replication of the desired fragments of Myostatin genes and Glyceraldehyd 3phosphate dehydrogenase, the observation of the products in the agarose gel with the ladder confirmed the correctness of the fragments size. The size of the replicated fragments for the Myostatin and Glyceraldehyd 3phosphate dehydrogenase genes are 81 and 144 bp, respectively. After ensuring the efficiency of the designed primers, the Real Time PCR reaction was performed in two replicates for each cDNA sample. The information obtained was sorted and analyzed. Results indicated that the highest mean of the target gene in Arabi breed and the lowest one in the crossbred group. The difference between the Arabi and crossbred group was significant (p < 0.05). The results indicated that the crossbreeding caused a significant reduction in the Myostatin gene expression. The expression of Myostatin gene in Arabi race was 1.4 fold greater than that in the cross bred group. This was in concordance with the final lamb weights which were significantly different for the two groups. The final weight in the cross bred lambs were higher than the Arabi counterparts. Conclusion Based on the obtained data, the current investigation was the first study in the field of Myostatin gene expression in Arabi×Romanove cross breeding. In overall, the results showed that the cross breeding can significantly affect the expression of the studied gene.  Regarding the effect of Myostatin gene on the muscle function, decreased Myostatin gene expression may influence the final weight gain. As this study showed the effect of crossbreeding on Myostatin gene expression, it is necessary to consider the consequences of crossbreeding on the native sheep breeding programs. This study could help in more understanding the cross breeding effects on the livestock performance