Abstract
BackgroundGlutamine synthetase (GS) acts as a key enzyme in plant nitrogen (N) metabolism. It is important to understand the regulation of GS expression in plant. Promoters can initiate the transcription of its downstream gene. Eichhornia crassipes is a most prominent aquatic invasive plant, which has negative effects on environment and economic development. It also can be used in the bioremediation of pollutants present in water and the production of feeding and energy fuel. So identification and characterization of GS promoter in E. crassipes can help to elucidate its regulation mechanism of GS expression and further to control its N metabolism.ResultsA 1232 bp genomic fragment upstream of EcGS1b sequence from E. crassipes (EcGS1b-P) has been cloned, analyzed and functionally characterized. TSSP-TCM software and PlantCARE analysis showed a TATA-box core element, a CAAT-box, root specific expression element, light regulation elements including chs-CMA1a, Box I, and Sp1 and other cis-acting elements in the sequence. Three 5′-deletion fragments of EcGS1b upstream sequence with 400 bp, 600 bp and 900 bp length and the 1232 bp fragment were used to drive the expression of β-glucuronidase (GUS) in tobacco. The quantitative test revealed that GUS activity decreased with the decreasing of the promoter length, which indicated that there were no negative regulated elements in the EcGS1-P. The GUS expressions of EcGS1b-P in roots were significantly higher than those in leaves and stems, indicating EcGS1b-P to be a root-preferential promoter. Real-time Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) analysis of EcGS1b gene also showed higher expression in the roots of E.crassipes than in stems and leaves.ConclusionsEcGS1b-P is a root-preferential promoter sequence. It can specifically drive the transcription of its downstream gene in root. This study will help to elucidate the regulatory mechanisms of EcGS1b tissue-specific expression and further study its other regulatory mechanisms in order to utilize E.crassipes in remediation of eutrophic water and control its overgrowth from the point of nutrient metabolism.
Highlights
Glutamine synthetase (GS) acts as a key enzyme in plant nitrogen (N) metabolism
Cloning and analysis of EcGS1b-P To clone the regulatory regions of EcGS1b gene, the primer pairs were designed from the corresponding cDNA sequences
Using PCR gene walking on genomic DNA from E. crassipes, the upstream of EcGS1b gene was cloned thrice with the nested-PCR (Fig. 1) and was sequenced
Summary
Glutamine synthetase (GS) acts as a key enzyme in plant nitrogen (N) metabolism. It is important to understand the regulation of GS expression in plant. Eichhornia crassipes is a most prominent aquatic invasive plant, which has negative effects on environment and economic development It can be used in the bioremediation of pollutants present in water and the production of feeding and energy fuel. Eichhornia crassipes is a most prominent aquatic invasive plant [1], with negative effects on environment and economic development [2, 3] It is regarded as a valuable resource with several unique properties, and previous studies have reported that E. crassipes had high absorption efficiency of nitrogen (N), phosphorus (P) and heavy metals [4,5,6]. Studying the biochemical metabolism of E. crassipes from the molecular level assists in further utilizing and controlling this weed
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