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Comparative expression and characterization of dehydroascorbate reductase cDNA from transformed sesame hairy roots using real-time RT-PCR

한국식물학회지 2006년 49권 6호 p.507 ~ 512
 ( Chun Jae-An ) - Dong-A University Department of Biotechnology

 ( Seo Jee-Young ) - Dong-A University Department of Biotechnology
 ( Han Mi-Ok ) - Dong-A University Department of Biotechnology
이진우 ( Lee Jin-Woo ) - Dong-A University Department of Biotechnology
 ( Yi Young-Byung ) - Dong-A University Department of Biotechnology
 ( Park Gun-Yong ) - National Agricultural Products Quality Management Service
이신우 ( Lee Shin-Woo ) - Jinju National University Department of Biotechnology
 ( Bae Shin-Chul ) - National Institute of Agricultural Biotechnology
조강진 ( Cho Kang-Jin ) - National Institute of Agricultural Biotechnology
 ( Chung Chung-Han ) - Dong-A University Department of Biotechnology

Abstract


The differential transcription activity of dehydroascorbate reductase (DHAR) was scrutinized in the transformed hairy roots, leaves, stems, roots, and developing seeds of sesame (Sesamum indicum L.). Its relative levels of expression were compared via the threshold cycle (CT) method, using real-time RT-PCR. Ubiquitous expression of DHAR in all organs was confirmed with both reai-time and conventional RT-PCR. With the former, DHAR transcript levels were, unexpectedly, 4.7-fold higher in the stem tissue than in the hairy roots; the lowest levels were detected in the seeds. It was possible to determine the transcription activity of hairy root DHAR, with a low amount of total RNA (0.5 ng), using real-time RT-PCR but not with conventional RT-PCR gel analysis. This indicated that the former is more sensitive and efficient than the latter for the detection of gene expression. We also characterized DHAR cDNA cloned from transformed hairy roots, and found that sequence identity for the deduced amino acids of the DHAR enzyme was shared at 60 to 83% among plant species. The algorithm prediction and phylogenetic analysis suggested that the cloned cDNA polypeptide is cytosolic DHAR. Another feature of the cloned cDNA polypeptide was the presence of a CXXS instead of CXXC motif in the active center of the DHAR enzyme.

키워드

Agrobacterium rhizogenes; dehydroascorbate reductase; differential expression; hairy roots; real-time RT-PCR; sesame
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