Int. J. Mol. Sci. 2012, 13(10), 13150-13168; doi:10.3390/ijms131013150
Article

Construction and Evaluation of Normalized cDNA Libraries Enriched with Full-Length Sequences for Rapid Discovery of New Genes from Sisal (Agave sisalana Perr.) Different Developmental Stages

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Received: 4 July 2012; in revised form: 25 September 2012 / Accepted: 26 September 2012 / Published: 12 October 2012
(This article belongs to the Section Biochemistry, Molecular Biology and Biophysics)
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Abstract: To provide a resource of sisal-specific expressed sequence data and facilitate this powerful approach in new gene research, the preparation of normalized cDNA libraries enriched with full-length sequences is necessary. Four libraries were produced with RNA pooled from Agave sisalana multiple tissues to increase efficiency of normalization and maximize the number of independent genes by SMART™ method and the duplex-specific nuclease (DSN). This procedure kept the proportion of full-length cDNAs in the subtracted/normalized libraries and dramatically enhanced the discovery of new genes. Sequencing of 3875 cDNA clones of libraries revealed 3320 unigenes with an average insert length about 1.2 kb, indicating that the non-redundancy of libraries was about 85.7%. These unigene functions were predicted by comparing their sequences to functional domain databases and extensively annotated with Gene Ontology (GO) terms. Comparative analysis of sisal unigenes and other plant genomes revealed that four putative MADS-box genes and knotted-like homeobox (knox) gene were obtained from a total of 1162 full-length transcripts. Furthermore, real-time PCR showed that the characteristics of their transcripts mainly depended on the tight expression regulation of a number of genes during the leaf and flower development. Analysis of individual library sequence data indicated that the pooled-tissue approach was highly effective in discovering new genes and preparing libraries for efficient deep sequencing.
Keywords: sisal; normalization; full-length; cDNA library; knox gene; MADS-box gene; expression
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MDPI and ACS Style

Zhou, W.-Z.; Zhang, Y.-M.; Lu, J.-Y.; Li, J.-F. Construction and Evaluation of Normalized cDNA Libraries Enriched with Full-Length Sequences for Rapid Discovery of New Genes from Sisal (Agave sisalana Perr.) Different Developmental Stages. Int. J. Mol. Sci. 2012, 13, 13150-13168.

AMA Style

Zhou W-Z, Zhang Y-M, Lu J-Y, Li J-F. Construction and Evaluation of Normalized cDNA Libraries Enriched with Full-Length Sequences for Rapid Discovery of New Genes from Sisal (Agave sisalana Perr.) Different Developmental Stages. International Journal of Molecular Sciences. 2012; 13(10):13150-13168.

Chicago/Turabian Style

Zhou, Wen-Zhao; Zhang, Yan-Mei; Lu, Jun-Ying; Li, Jun-Feng. 2012. "Construction and Evaluation of Normalized cDNA Libraries Enriched with Full-Length Sequences for Rapid Discovery of New Genes from Sisal (Agave sisalana Perr.) Different Developmental Stages." Int. J. Mol. Sci. 13, no. 10: 13150-13168.


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