Analysis of the SHP2 enhancer for the use of tissue specific activation tagging in Arabidopsis thaliana

Antonio Chalfun-Junior,Gerco C Angenent,Marco Busscher,Jurriaan J Mes

doi:10.1590/s1415-47572006000200032

Antonio Chalfun-Junior, Gerco C Angenent + Show 2 more

Open Access

https://doi.org/10.1590/s1415-47572006000200032

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Abstract

Activation tagging is a powerful tool to identify new mutants and to obtain information about possible biological functions of the overexpressed genes. The quadruple cauliflower mosaic virus (CaMV) 35S enhancer fragment is a strong enhancer, which is most commonly used for this purpose. However, the constitutive nature of this enhancer may generate lethal mutations or aberrations in different plant organs by the same overexpressed gene. A tissue-specific activation tagging approach may overcome these drawbacks and may also lead more efficiently to the desired phenotype. For this reason the SHATTERPROOF2 (SHP2) promoter fragment was analysed for enhancer activity. The SHP2 gene is involved in dehiscence zone development and expressed during silique development. The aim of the experiments described here was to identify a dehiscence zone specific enhancer that could be used for tissue-specific activation tagging. The chosen SHP2 enhancer fragment was found to be expressed predominantly in the dehiscence zone and showed enhancer activity as well as ectopic expression activity. This activity was not influenced by its orientation towards the promoter and it was still functional at the largest tested distance of 2.0 kb. Based on these results, the SHP2 enhancer fragment can potentially be used in a tissue-specific activation tagging approach to identify new Arabidopsis mutants with an altered dehiscence zone formation.

Highlights

Activation tagging has become an upcoming tool to generate mutant plants
The SalI-SalI fragment was introduced in the SalI digested pFBP12E vector, resulting in a sense fusion of the SHP2 enhancer to the ‘long’ 1040 bp FBP1 promoter fused to the GUS reporter gene
It has been shown previously that the SHP2 gene is expressed in the dehiscence zone of Arabidopsis siliques (Savidge et al, 1995; Liljegren et al, 1998; Liljegren et al, 2000)

Summary

Introduction

Activation tagging has become an upcoming tool to generate mutant plants. It is an alternative approach for gene function analysis, because loss-of-function mutations has its limitation in cases of functional gene redundancy (Arabidopsis Genome Initiative, 2000). Walden et al (1994) designed a T-DNA based activation tagging approach to identify and isolate novel genes from tobacco, and since it has been largely applied using either T-DNA insertion strategies (Borevitz et al, 2000; Ito and Meyerowitz, 2000; Lee et al, 2000; van der Graaff et al, 2000; Weigel et al, 2000; Huang et al, 2001) or transposon based approaches (Wilson et al, 1996; Marsch-Martinez et al, 2002) This technology has been applied successfully to many plant species like Arabidopsis, rice, tomato, petunia and tobacco We describe the identification of a SHP2 enhancer taken from its natural promoter, which contains all necessary characteristics required for a tissue-specific activation approach

Material and Methods

Results

Discussion