Author
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Lingle, Sarah |
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Submitted to: Annual International Plant & Animal Genome Conference
Publication Type: Abstract Only Publication Acceptance Date: 10/31/2004 Publication Date: 1/10/2004 Citation: Lingle, S.E. 2004. Use of real-time PCR to quantify expression and copy number of sucrose metabolism genes in polyploid sugarcane [abstract]. In: Plant and Animal Genome XII Final Abstracts Guide. Plant and Animal Genome XII Conference, January 10-14, 2004, San Diego, California. p. 34. Interpretive Summary: Technical Abstract: Real-time or quantitative PCR is a relatively new technique for quantifying gene expression and gene copy number. This technique was used to determine copy number of Sus2, one gene for sucrose synthase (EC 2.4.1.13), a major enzyme of sucrose metabolism in sugarcane (Saccharum spp. hybrids). Linearized plasmid containing part of the Sus2 gene was used as a standard, and published C-values were used to convert copy/pg to copy/haploid genome. Estimates ranged from 10 copies per haploid genome in S. robustum "Molokai" (2n=80) to 43 copies in S. spontaneum "PIN84-1" (2n=96). RT-PCR was used to determine expression of the Sus2 gene in different tissues from hybrid "Muntok Java" and PIN84-1, using 18s rRNA as a normalizer. Results within genotypes were similar to those from Northern blots: there was low expression in mature leaves and higher expression in immature internodes than in mature internodes. Expression in internodes was generally greater in PIN84-1, in contrast to enzyme activity. Real-time RT-PCR was also used to determine expression of two cell wall invertase genes. Expression of Cwin1 was very low in internodes, but there was no expression of Cwin2. Real-time PCR is a powerful tool for molecular biology in sugarcane, and more quantitative than traditional Northern and Southern analysis. |
