Üreme Mevsimi Dışında Kazak Koyunlarının Üremeleri Üzerine İnhibin-βA Geninin Etkisi

Abstract

Effect of inhibin-βA subunit gene on reproductive performance of Kazakh sheep in non-breeding seasons. Kafkas Univ Vet Fak Derg, 25 (5): 611-618, 2019.Abstract Inhibin-βA (INHBA) could feedback suppress synthesis and secretion of follicle-stimulating hormone (FSH), which correlates with the litter size of sheep. In this study, the inhibin-βA subunit was used as a candidate gene, and Kazakh sheep was used as a test object. The polymorphism of the gene was detected by PCR-SSCP method and its association with litter size was analyzed. The results showed that there were three polymorphisms in the exon 5'UTR region (primer 0-2), for which AA, AB and BB were detected in Kazakh sheep. The correlation analysis showed that genotype AA had 0.13 (P<0.05) lambs and 0.16(P<0.05) lambs more than genotype AB and BB in Kazakh sheep. Vectors that interfering the INHBA expression including PLLU2G-shINHBA-1 (I-1), PLLU2G-shINHBA-2 (I-2), PLLU2G-shINHBA-3 (I-3) and PLLU2G-INHBA-4 (I-4) were constructed by RNA interference (RNAi) technology in the study. After the successfully separated ovarian granulosa cells were transfected with the vector, and the expression level of the gene was detected by quantitative RT-PCR. The results showed that the four vectors suppressed INHBA mRNA levels with a silencing efficiency of 34%, 58%, 39% and 19% respectively, with better interference efficiency of I-2. Then, we determined the contents of INHBA, FSH, luteinizing hormone (LH) and estradiol (E2) in serum by directing intro-ovarian injection of the I-2. The results showed that the INHBA level dropped and the FSH level raised in serum, while LH and E2 levels did not change, indicating the RNAi vector could successfully silence the INHBA expression in vivo. This study sets a good theoretical basis of researching the breeding and estrus properties of sheep, and the short hairpin (shRNA) vector is hopefully used in promoting the fecundity of sheep in practice. Özİnhibin-βA (INHBA), folikül stimule edici hormon (FSH) sentez ve salınımını baskılayabilir ki bu durum koyunlarda yavru sayısı ile ilişkili olabilir. Bu çalışmada, inhibin-βA geni aday gen olarak değerlendirilerek Kazak koyunları çalışma materyali olarak kullanıldı. Genin polimorfizmi PCR-SSCP metodu ile belirlendi ve yavru sayısı ile ilişkisi analiz edildi. Elde edilen sonuçlar, Kazak koyununda ekzon 5'UTR bölgesinde AA, AB ve BB için üç polimorfizmin bulunduğunu gösterdi (primer 0-2). Korelasyon analizi, Kazak koyununda genotip AA'da 0.13 kuzu bulunduğunu (P<0.05) ve AA genotipinde AB ve BB genotiplerinden 0.16 daha fazla (P<0.05) kuzu bulunduğun gösterdi. Bu çalışmada INHBA ekspresyonuna etkiyen PLLU2G-shINHBA-1 (I-1), PLLU2G-shINHBA-2 (I-2), PLLU2G-shINHBA-3 (I-3) ve PLLU2G-INHBA-4 (I-4)'ü içeren vektörler, RNA interferans (RNAi) teknolojisi ile oluşturuldu. Başarıyla ayrılan ovaryum granuloza hücreleri vektörler ile transfekte edildi ve gen ekspresyon seviyesi kantitatif RT-PCR ile belirlendi. Sonuçlar dört vektörün, sırasıyla %34, %58, %39 ve %19 gen susturma etkinlikleri ile INHBA mRN...