tailieunhanh - Efficient cloning of the rare wheat (Triticum aestivum L.) transcripts

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. | Turk J Biol 29 (2005) 7-13 © TÜB‹TAK Efficient Cloning of the Rare Wheat (Triticum aestivum L.) Transcripts Muharrem D‹LB‹RL‹⁄‹ Central Research Institute for Field Crops, Ankara - TURKEY Mustafa ERAYMAN Mustafa Kemal University, Agricultural College Department of Crop Science, Hatay - TURKEY Received: Abstract: 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 the public 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. Key Words: EST, cDNA, Wheat, Functional Genomics, Cloning Nadir Bugday (Triticum aestivum L.) Transkript’lerinin Etkin Klonlanmas› Özet: EST (expressed sequence tag) teknolojisi çeflitli organizmalarda genomun ifllevsel k›s›mlar›n›n klonlanmas›n› hedeflemede büyük mesafe kat etmeyi sa¤lam›flt›r. Bol miktar traskriptlerden (redundant) oluflturulan cDNA kütüphaneleri genelde aral›ks›z (constitutive) ve yo¤un (over-expressed) eksprese olan genleri içermektedir. Ancak, organ spesifik ve çok az eksprese olan genler kolayl›kla iflaretlenip klonlanamamaktad›r. Bu çal›flma, ekmeklik bu¤dayda düflük seviyede ve nadir s›kl›kla eksprese olan