Mandalarda IGFBP-3 Geninde Daha Önce Görülmemiş Üç Genotip Tespit Edildi

Abstract

Anadolu su mandası Türkiye’de yetiştirilen tek manda ırkıdır ve sayıları ülke genelinde “Halk Elinde Manda Yetiştiriciliği Projesi” ile artmıştır. Bu çalışmanın amacı, Anadolu Mandalarında et verimi ile ilgili olan insülin benzeri büyüme faktörü bağlayıcı protein - 3 (IGFBP-3) geninin Taq I, Hae III ve Msp I restriksiyon enzimi (RE) ile gen polimorfizmlerinin (İntron 2, Exon 2 / İntron 3, Exon 3) araştırılmasıdır. Toplam 151 kan örneğinden Fenol/Kloroform yöntemi kullanılarak DNA izolasyonu yapılmıştır ve elde edilen DNA örnekleri spesifik primerler kullanılarak Touchdown PZR ile çoğaltılmıştır. Yükseltgenen PZR ürünleri restriksiyon enzimleri ile kesilmiş, %3 agaroz jel elektroforez yönteminde ayrıştırılmış ve genotipler belirlenmiştir. Sonuç olarak, Hae III RE’nde (Exon 2 – İntron 3) AA (%98.68) ve AC (%1.32) 2 genotip ve A (0.99) ve C (0.01) 2 allel tespit edilmiştir. Taq I RE’nde (İntron 2) AA (%7.94), AB (%3.97) ve BB (%88.10) 3 genotip ve A (0.10) ve B (0.90) 2 allel ve Msp I RE’nde (Exon 3) ise sadece AA genotipi ve A alleli tespit edilmiştir. IGFBP-3 geninde Msp I enzimi monomorfik (P>0.05), Hae III enzimi polimorfik fakat önemsiz (P>0.05) bulunurken Taq 1 enzimi yönünden hem polimorfik hem de önemlilik tespit edilmiştir (P<0.001). IGFBP-3 gen bölgelerinin polimorfizmleri Anadolu mandalarında ilk kez araştırılmıştır. Ayrıca IGFBP-3 geninde diğer manda ırklarında yapılan çalışmalardan farklı olarak ilk kez üç yeni genotip (Taq I enzim kesimi sonucunda iki, Hae III enzim kesimi sonucunda bir) tespit edilmiştir. Ayrıca Taq I enzim işlemi sonucu yeni tespit edilen B allelinin frekansı diğer alleline göre çok yüksek bulunmuştur.

Keywords

• Anadolu Mandası
• HaeIII
• IGFBP-3
• PZR-RFLP
• Sivas
• TaqI

Project Number

V-051

Three Previously Unseen Genotypes Detected in IGFBP - 3 Gene in Buffalo Breed

Abstract

Anatolian water buffalo breed is Turkey’s sole water buffalo breed, and their numbers steadily increased with the national “Water Buffalo Breeding by Breeders Project”. This study aimed to investigate of the gene region polymorphisms (Intron-2, Exon-2/Intron-3, Exon-3) of the meat-yield-related Insulin-like Growth Factor Binding Protein-3 (IGFBP-3) gene in Anatolian water buffaloes by TaqI, HaeIII, and MspI restriction endonucleases. The phenol/chloroform method was used for DNA isolation from 151 blood samples, and extracted DNAs were amplified by touchdown PCR using specific primers. Amplified PCR products were digested with restriction endonucleases (REs) and separated in 3% agarose gel electrophoresis (AGE), then genotypes were determined. Results revealed two genotypes [AA (98.68%) and AC (1.32%)] and two alleles [A (0.99) and C (0.01)] for the Exon-2 to Intron-3 region from HaeIII digestion. TaqI digestion of the Intron-2 region revealed three genotypes [AA (7.94%), AB (3.97%), and BB (88.10%)] and two alleles [A (0.10) and B (0.90)]. MspI digestion of the Exon-3 region revealed only the AA genotype and A allele, thus revealing monomorphism. Overall, HaeIII digestion revealed insignificant polymorphism (P>0.05), and TaqI digestion revealed significant polymorphism (P<0.001) for their respective regions. Gene polymorphisms of these regions were investigated for the first time in Anatolian water buffaloes. Additionally, three novel genotypes for the IGFBP-3 gene (one from HaeIII and two from TaqI) were determined for the first time. The novel B allele from TaqI digestion was observed to have a substantial frequency.

Keywords

• Anatolian water buffalo
• HaeIII
• IGFBP-3
• PCR-RFLP
• Sivas
• TaqI

Supporting Institution

Scientific Research Project Fund of Sivas Cumhuriyet University

Project Number

V-051

Thanks

We would like to thank Prof. Dr. Haki KARA for their support in this study

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