Quantitative analysis of the sucrose synthase gene promoter by transient gene expression in protoplasts

The shrunken gene, encoding the enzyme sucrose synthase, has been cloned and characterized in detail (Werr et al., EMBO J. 4:1373, 1985). The expression of this gene is regulated on the transcriptional level. High transcript levels accumulate in the endosperm of developing kernels. Reduced levels of transcript are detected in roots, leaves and shoots of etiolated seedlings. The transcription rate of this gene can be increased 10-20 fold upon anaerobic stress (Springer et al., Mol. Gen. Genet. 205:461, 1986).

To analyse the shrunken gene promoter for cis-regulatory elements, we established a transient gene expression system in protoplasts of a suspension cell line (BMS, kindly provided by Horst Lorz). By Northern experiments we verified that the internal sucrose synthase gene is expressed in this cell line. The level of transcript can be increased 10-15 fold by anaerobic treatment. Therefore this cell line is suitable to test for the influence of promoter deletions on the transcription rate of the plasmid pSKAN 1 (Werr and Lorz, Mol. Gen. Genet. 202:471, 1986). This plasmid contains a 2 kb promoter fragment of the shrunken gene including the transcription start fused to the bacterial NPTII-coding region of Tn 5. A set of deletion mutants, shortening the 2 kb promoter fragment from its 5'-end towards the transcription start, was constructed and transfected in maize protoplasts. NPTII levels were measured, differences in the efficiency of individual transfections were estimated by cotransfection with a 35s CaMV-CAT gene and used to correct NPTII levels. The functional map so far obtained indicates that multiple regulatory sequences, spread over the entire 2 kb promotor fragment of the sucrose synthase gene, can influence transcription in a positive and negative way.

We will also use this system to identify regulatory element(s) involved in anaerobic induced or tissue specific expression of the sucrose synthase gene.

Christoph Maas and Wolfgang Werr

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