Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 13, Num. 2, 1996

Effect of promoter-stimulatory element combination on transient reporter gene expression in tobacco protoplast using PEG-treatment

Alberto Coego^1 Roberto Vazquez^1 Julio Alfonso^2 Yamilet Coll^2 Merardo Pujol^2 Eduardo Menendez^1 Maria A Lopez^1 Pedro Molina^1 Lazaro Hernandez^1 Beatriz Bencomo^1 Gustavo de la Riva^1 and Guillermo Selman-Housein^1

1 Center for Genetic Engineering and Biotechnology (CIGB), P.O. Box 6162, CP 10600, Havana, Cuba.

2 CIGB of Sancti-Spiritus, P.O. Box 83, CP 60200, S.-Spiritus, Cuba. 7.

Code Number: BA96071
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Introduction

Construction of a novel promoters is very attractive for genetic engineering of any organism. The highest expression level of heterologous genes in dicot plants is conferred by the CaMV 35S promoter (1). Additional increase of its strength have been achieved by the use of 35S promoter upstream activating sequences (35S UAS) (2). The 5' untranslated leader sequences from TMV (Omega fragment) is also reported for enhancing the translation of contiguous genes (3). We previously found that combining the 35S promoter, the TMV fragment, and the 35S UAS in the pBPF Omega5 vector (pFR 14) resulted in a strong enhancement of promotor strenght (4). It have been also shown that the UAS from the Octopine Synthase (OCS) promoter of the A. tumefaciens Ti-plasmid enhance the level of gene expression in plants (5). Here we describe the effect of combining all four mentioned regulatory sequences on transient foreing gene expression in tobacco protoplasts.

Material and Methods

Construction of chimeric gene: Nucleic acid manipulations were done according to (6).

Protoplasts isolation and PEG-treatment: Leaf mesophyll protoplasts of Nicotiana tabacum cv. Petit Havana SR1 were isolated according to (7) from sterile shoot cultures grow on hormone free MS medium (8). PEG-mediated protoplast transformation and culture was done following the Shillito protocol (9).

Quantitative GUS and protein assays: Detection of fluorimetric GUS activity in protoplasts was made according to (10) and protein determined by the Breadford method.

Results and Discussion

We reported previously the construction and use (4) of a promissing vector for achieving high levels of transgene expression in tobacco protoplasts, which yielded a 6 fold increase as compared with the pBI 221 vector (7). In this work a combination of the CaMV 35S promoter, TMVW fragment, the 35S UAS, and the UAS from the OCS promoter repeated 4 times and inserted in the HincII restriction site of the 35S promoter region was fused to the gus gene and compared with the pBPFOmega5 and pBI 221 vectors by transient gene expression in tobacco protoplasts. The vector pBPFOmega 9 exhibited a dramatical 51 fold increase of gus enzyme expression in comparison with pBI221, and more than 15 times the level of expression achieved with the pBPFOmega5 vector.

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10. Shillito R. and Saul M. Plant Mol. Biol A pract. appr1988;161-186.

Copyright 1996 Elfos Scientiae