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A rapid and direct approach to find promoters for high-level gene expression in sugarcane (Saccharum spp.)
Abstract
Direct screening of genomic libraries for highly expressed genes is an efficient method to identify the promoters of plant genes expressed at high levels. Eleven genomic clones were isolated after screening a sugarcane genomic library with radioactively labeled first strand cDNA probes synthesized from sugarcane leaf mRNA. Southern analysis of these 11 genomic clones with pooled first strand cDNA indicated that 8 of them probably contain genes with expression levels similar to or higher than ubiquitin. Five different cDNA clones corresponding to these 8 genomic clones were isolated from a sugarcane leaf cDNA library and sequenced. Among the five, one is a novel gene and the other four showed high similarities with elongation factor 1α, α-tubulin, an aquaporin, and a proline-rich protein, respectively. After analyzing expression profiles using Northern blots, and gene copy number using Southern blots, the genomic clones of a sugarcane elongation factor 1α(SEF1α) and a sugarcane proline-rich protein (SPRP) were selected for promoter analysis. Two [] phage genomic clones, which contain both the promoter and coding regions, were subcloned into the plasmid vector pBluescript. The subclones were sequenced and promoter regions were defined by sequence comparison of cDNA and genomic DNA sequences. The SPRP cDNA clone isolated from sugarcane contains the complete coding sequence as well as 99 bp of 5' non-coding sequence, and 184 bp of 3' non-coding sequence. The promoter region of the SPRP gene contains a typical TATA box 172 bp upstream of the ATG translation start site. Analysis of cDNA and genomic clones of SEF1α indicated that in addition to a single intron located within the coding region, the SEF1α contains a second intron located within the 5' untranslated leader region. The transcription start site was estimated by primer extension. There is a 130 bp untranslated leader sequence in the mRNA. The transcription start site of SEF1α is located about 727 bp upstream of the translation start site in the genomic clone. A typical TATA box (5'-TATAAA) and CCAAT box are found at 33 and 77 bp upstream, respectively, of the estimated transcription start site.
Description
Due to the character of the original source materials and the nature of batch digitization, quality control issues may be present in this document. Please report any quality issues you encounter to digital@library.tamu.edu, referencing the URI of the item.Includes bibliographical references (leaves 81-91).
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Citation
Yang, Meizhu (2000). A rapid and direct approach to find promoters for high-level gene expression in sugarcane (Saccharum spp.). Master's thesis, Texas A&M University. Available electronically from https : / /hdl .handle .net /1969 .1 /ETD -TAMU -2000 -THESIS -Y2.
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