Citrus sinensis constitutive gene promoters: isolation and evaluation of its efficiency on uidA gene expression in Solanum lycopersicum and Citrus sinensis

• Grant number: 13/19700-2
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: December 01, 2013
• End date: February 28, 2017
• Field of knowledge: Agronomical Sciences - Agronomy - Crop Science
• Principal Investigator: Beatriz Madalena Januzzi Mendes
• Grantee: Lígia Erpen
• Host Institution: Centro de Energia Nuclear na Agricultura (CENA). Universidade de São Paulo (USP). Piracicaba , SP, Brazil
• Associated scholarship(s): 14/25077-9 - Characterization of regulatory elements in citrus derived promoters, BE.EP.DR

Abstract

Genetic transformation is an alternative to conventional breeding allowing the introduction of genes that can influence the resistance of plants to diseases. The most used method for genetic transformation of citrus is Agrobacterium tumefaciens. In this method, the T-DNA containing the gene of interest driven by a promoter, is transferred to the plant. Among the promoters usually employed, there is the constitutive promoter CaMV35S isolated from the Cauliflower Mosaic Virus, characterized by the ability to induce strong expression of the transgene. However, new approaches for plant transformation (cisgenesis and intragenesis) requires the use of genetic material derived from the species itself or related species. In this way, there is a demand for citrus native constitutive promoters. Thus, the aim of this work is to identify and isolate constitutive promoters of Citrus sinensis as an alternative to the CaMV35S promoter. For this, genes with a strong constitutive expression will be identified in the Citrus sinensis genome. The promoter region of these genes will be isolated and inserted into an expression cassette for controlling the reporter gene uidA (GUS). The efficiency of the isolated promoters will be assessed after genetic transformation of the model plant Solanum lycopersicum cv. Micro-Tom and C. sinensis cv. Hamlin. The transformation will be via A. tumefaciens containing the plasmid pCambia 2201 with expression cassette containing C. sinensis constitutive promoters or CaMV35S promoter, linked to the uidA (GUS) reporter gene. The identification of transgenic plants will be performed by GUS histochemical assay and PCR analysis and the confirmation of integration of the transgene will be done by Southern blot analysis. The cis-regulatory regions present in each promoter will be characterized in silico. The efficiency of C. sinensis constitutive promoters in the expression of the reporter gene uidA (GUS) will be evaluated and compared to the efficiency of the CaMV35S promoter by RT-qPCR analysis.

Academic Publications

(References retrieved automatically from State of São Paulo Research Institutions)

ERPEN, Lígia. Isolation and characterization of constitutive gene promoters from Citrus sinensis. 2017. Doctoral Thesis - Universidade de São Paulo (USP). Escola Superior de Agricultura Luiz de Queiroz (ESALA/BC) Piracicaba.