Expression of Two Tissue-Specific Promoters in Transgenic Cotton Plants

In order to evaluate the functions of putative transcriptional regulatory regions of cotton genes, the upstream regulatory sequences (promoters) of two cotton genes, Gh-sp (seed protein) and Gh-rbcS (ribulose-1,5-bisphosphate carboxylase small subunit), were fused with a bb-glucuronidase (GUS) reporter gene and the cauliflower mosaic virus (CaMV) 35S terminator. These two gene constructs were transferred into cotton (Gossypium hirsutum L. cv. Coker 312) by Agrobacterium-mediated transformation. Transgenic plants from the T0 generation were analyzed for expression of the GUS reporter gene in different tissues and developmental stages of cotton. Qualitative and quantitative analyses indicated the GUS gene driven by the Gh-sp promoter were expressed only during seed maturation, beginning approximately 25 d postanthesis. Expression of the GUS reporter gene driven by the Gh-rbcS promoter was detected primarily in leaf tissue from transgenic plants. Levels of GUS expression in leaves of Gh-rbcS/GUS transgenic plants was comparable to that in transgenic cotton plants containing a GUS gene construct controlled by the CaMV 35S promoter. These results indicate that these two tissue-specific promoters have the potential to be used to differentially express interesting genes in developing seeds or leaves of transgenic cotton. In the past few years, genetically modified cotton carrying insectand herbicide-resistant genes has been commercialized successfully. Transgenic cottons are likely to play an increasingly important role in worldwide cotton production by conferring useful agronomic and fiber traits. Typically, the beneficial gene is expressed in transgenic plants under control of a regulatory DNA sequence known as a promoter. Although the strict definition of the term promoter refers only to the core DNA sequences necessary for basal transcription, for the purposes of this report, a promoter is defined as the DNA sequences required to determine the appropriate spatial and temporal expression pattern. The primary regulatory sequences generally are located within 1000 base pairs upstream (5') of the transcription start site in plant genes, although there are cases where regulatory sequences are found P. Song, J.L. Heinen, T.H. Burns, and R.D. Allen, Dep. Biological Sciences, Texas Tech Univ., Lubbock, TX 794093131. Received 3 Mar. 2000. *Corresponding author ([email protected]). Abbreviations: CaMV, cauliflower mosaic virus; EDTA, ethylenediaminetetraacetic acid; GUS, ß-glucuronidase; PCR, polymerase chain reaction. 218 SONG ET AL: EXPRESSION OF TWO TISSUE-SPECIFIC PROMOTERS IN TRANSGENIC COTTON PLANTS further upstream or are downstream (3') of the coding sequences (Dietrich et al., 1992; Zhang et al., 1996). Currently, the most widely used promoter for expression of foreign gene constructs (transgenes) in dicot plants is the cauliflower mosaic virus (CaMV) 35S promoter (Ow et al., 1986). The CaMV 35S promoter provides strong constitutive expression in most dicot plants, including cotton. However, to develop transgenic cottons with specialized agronomic traits such as fiber quality and seed nutrition components, a larger arsenal of constitutive and tissue-specific promoters will be required. The characteristic expression patterns provided by these promoters must be analyzed to determine whether they can be used to express beneficial genes in specific target tissues or developmental stages at maximum levels. Although such promoter tests can be conducted with transient expression assays or in model plant systems such as transgenic tobacco and Arabidopsis, gene expression analysis in stable transgenic cotton plants provides confirmation that these promoters can be used for development of transgenic cotton for commercial production. Although several fiber-specific promoters have been tested in transgenic cotton plants (Dang et al., 1995; John and Crow, 1992; Rinehart et al., 1996), the isolation of constitutive and tissue-specific promoters (Song and Allen, 1997; Song et al., 1998) is also of interest. Here we report the expression patterns of two promoters from cotton that direct seed-specific and leaf-specific expression in transgenic cotton plants. We anticipate that these promoters can be used to direct expression of transgenes in cotton and other plants. MATERIALS AND METHODS