Effecient cloning of the rare wheat (Triticum aestivum L.) transcripts

EST (expressed sequence tag) teknolojisi çeşitli organizmalarda genomun işlevsel kısımlarının klonlanmasmı hedeflemede büyük mesafe kat etmeyi sağlamıştır. Bol miktar traskriptlerden (redundant) oluşturulan cDNA kütüphaneleri genelde aralıksız (constitutive) ve yoğun (over-expressed) eksprese olan genleri içermektedir. Ancak, organ spesifik ve çok az eksprese olan genler kolaylıkla işaretlenip klonlanamamaktadır. Bu çalışma, ekmeklik buğdayda düşük seviyede ve nadir sıklıkla eksprese olan gen parçacıklarının klonlanmasmı amaçlamaktadır. Poliakrilamid jel ve motife (farklı baz dizilimli genlerin aynı veya benzer sekansları) dayalı PCR (Polymerase Chain Reaction) amplifikasyonunun birlikte kullanımıyla, 189 buğday gen parçacığı taşıyan 144 bant klonlanmıştır. Gen baz dizilimlerinin analizi yalnızca 22'si gen bankasında kayıtlı olan 74 farklı kontig (contig) ile sonuçlanmıştır. Diğer 52 ise özgün (unique) ve herhangi bir EST veya 'non-redundant' gen bankasında rastlanmamıştır. EST gen bankasının % 5'lik özgün gen klonlama oranına karşın çalışmamızda bu oran yaklaşık % 28 olmuştur. Tanımladığımız bu yaklaşım, buğday da ve belki de farklı genomlarda nadir bulunan ve çok düşük seviyede eksprese edilen genlerin hedeflenmesi ve klonlanması konusunda oldukça ümit vericidir.

Nadir buğday (Triticum aestivum L.) transkript'lerinin etkin klonlanması

Expressed sequence tag (EST) technology has spurred targeting the functional portion of genomes for several organisms. Constructed redundant complementary DNA (cDNA) libraries have targeted a majority of constitutive and over-expressed genes. However, the majority of lowly expressed and un-induced genes have not been able to be easily tagged and cloned. The objective of this study was to clone low and rarely expressed transcripts in wheat. Using a combination of a polyacrylamide gel and a motif-based PCR amplification, we cloned 144 bands containing 189 wheat fragments. Sequence analysis revealed 74 contigs, of which only 22 being present in database. The remaining 52 were unique, and were not present in any public EST or non-redundant database. Compared to 5% efficiency in thejxiblic EST database, the efficiency of cloning of unique sequences was about 28%. Therefore, the approach we describe here is highly promising for targeting the rarely and lowly expressed genes in wheat and, probably, various other genomes.

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