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Abstract
β-amylase is an enzyme involved in the degradation of transitory starch in photosynthetic tissues of plants and is expressed in high levels in the roots of Medicago sativa L. How the β -amylase gene MsBAM1 is regulated in the roots is not clear. The aims of this study were to: isolate the core promoter of MsBAM1, decipher motifs in silico that are associated with the elevated root specific response, and to find common motifs among core promoters of important starch degradation genes in Medicago sativa MsBAM1 and Arabidopsis (AtGWD, AtPWD, AtMEX1, AtBAM 1 & 3, and AtISA3). Primers were designed from homologous β-amylase genes and used in PCR with M. sativa gDNA. Gel purified PCR band(s) were sequenced, verified and annotated. The starch genes and equal numbers of Arabidopsis non-starch genes promoters were analyzed by the transcription factor binding site database (PLACE). Motif frequencies between starch and non-starch genes were statistically analyzed. A TATA-box motif was identified in the MsBAM1 core promoter. Chi-square and Fisher’s exact test analyses revealed significance of the frequency of the motifs ROOTMOTIFTAPOX1 (RMP1) and GATABOX (GATA) between starch and non-starch genes, but not the DOF motif. The RMP1 and GATA motifs were detected in MsBAM1 and in all the Arabidopsis starch degradation genes but only in a few of the non-starch genes. However, the DOF motif was found in all starch and non-starch degradation genes in similar frequencies. These motifs may work in combination with other known cis-regulatory elements to confer root-enhanced expression in alfalfa roots and co-transcriptional regulation in the starch degradation pathway
Highlights
IntroductionMedicago sativa L. (alfalfa) is a nitrogenfixing legume, and is an important crop in the ecosystem [1]
Grown worldwide, Medicago sativa L. is a nitrogenfixing legume, and is an important crop in the ecosystem [1]
The objectives of this study were to: a) isolate and annotate the Medicago sativa β-amylase1 (MsBAM1) core promoter region, b) determine if any of the motifs predicted in silico are associated with an elevated root specific response, and c) identify motifs from the MsBAM1 core promoter in this study that are common in Arabidopsis key starch degradation genes (AtBAM1&3, Arabidopsis Thaliana Glucan Water Dikinase (AtGWD), AtPWD, AtMEX1 and Arabidopsis Thaliana Isoamylase3 (AtISA3))
Summary
Medicago sativa L. (alfalfa) is a nitrogenfixing legume, and is an important crop in the ecosystem [1]. Evidence of the starch degradation pathway is relatively easy to model in photosynthetic tissues because the process occurs within a diurnal cycle [3]. The transitory starch that accumulates in the chloroplast during the day is subsequently degraded at night to the sugars maltose and glucose that are exported into the cytosol [3,4,5]. In the cytosol, these simple sugars are converted into sucrose for transport throughout the plant in order to maintain cellular metabolism. Sucrose is converted back into starch in non-photosynthetic tissues for storage purposes [3,6]
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