Characterization Of Strong And Tissue Specific Promoters Of Soybean Glycine Max L Merr Using Multiple Systems

Characterization of Strong and Tissue-specific Promoters of Soybean (Glycine Max (L.) Merr.) Using Multiple Systems

Each gene in plants is regulated by a unique promoter region, located upstream of the coding region, that directs transcription according to tissue type, developmental stage, or a wide range of inducing factors. Promoters are a large determinant of gene expression at the transcriptional level and are capable of directing complex regulation. In order to study soybean promoters and how they regulate genes, we have employed the use of the green fluorescent protein (GFP) for visualizing expression in three different systems. Four promoters of soybean ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (rbcS) genes were examined using a lima bean transient expression system and a soybean hairy root stable expression system. A soybean glycinin promoter was also studied by monitoring expression in stably-transformed developing seed tissues. The rbcS promoters displayed high expression in the transient lima bean system with the rbcS1 promoter showing expression greater than 2X the expression level of the CaMV35S (35S) promoter. The rbcS2, rbcS3, and rbcS4 promoters all drove expression at greater than half of the expression level of the 35S promoter. In soybean hairy roots, each of the rbcS promoters drove lower expression when compared to the 35S promoter. In stably-transformed soybean seeds excised from greenhouse-grown plants, expression of GFP driven by the glycinin promoter increased at approximately 15 days after flowering from almost no expression, to a very high level of expression at seed maturity. When immature seeds were removed from the pod during early development and cultured in vitro, seed tissues displayed this same increase in GFP expression, but only when the seeds were cultured on media containing certain amino acids. Glutamine, as well as asparagine and proline induced expression of this tissue-specific and developmentally-regulated glycinin promoter. Each promoter analysis system gave insights into regulation of these important genes in soybean, revealing high expression in rbcS promoters and showing induction of the glycinin promoter by certain amino acids. Amino acid induction of seed-specific promoters has not been reported previously. Further studies of promoter regions that drive novel or high-level gene expression may be useful in both basic and applied research.

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