Isolation and cloning of fruit-specific E8 promoter from local small fruit tomato (Lycopersicon esculentum L.) and analysis its transient expression

Abstract

Isolation and characterization of specific promoter is important for efficient expression of recombinant proteins in plants. E8 is a fruit specific promoter with 2.2 kb length. To isolate E8 fruit specific promoter, seeds of local small fruit Sardasht genotype were sown in greenhouse and genomic DNA was extracted from young leaves using CTAB method. The promoter region was amplified using specific primers designed based on conserved regions of the sequences available at NCBI database. The amplified fragments ligated into PTZ57R/T vector and transferred into competence cells of DH5α strain of E. coli for cloning. Plasmid was extracted after confirming the presence of the inserted segments in plasmid based on colony PCR test and enzyme digestion. The extracted plasmids were used for sequencing the fragments. Sequence analysis revealed successful isolation of E8 promoter segment. To analyze the activity promoter in tomato tissues, the CaMV35S promoter in pBI121 binary vector replaced by E8 promoter and transferred to GV3101 strain of Agrobacterium. Transient expression was assessed using gus gene under E8 promoter with aim of transgenic Agrobacterium agro-infiltrated into fruit tissue. Presence of indigo color indicated successful expression of gus reporter gene under E8 promoter at the cells receipt the gene construct.