Cloning And Analysis Of Green Tissue-specific Promoter From Citrus

Promoter the light-harvesting chlorophyll a/b binding protein gene has been verified in a variety of plants with green tissue specificity.In this study, we cloned the full-length sequence of the citrus sinensis Cab(chlorophyll a/b binding protein) gene. On this basis.using the genomic walking technique cloned the gene upstream regulatory sequences.and to study the expression of regulatory sequences in transgenic tomato. The main results are as follows:1.Chlorophyll a/b the binding protein gene as the key words search in NCBI,found part lemon Cab amino acid sequence. and Masson pine, jatropha and other16kinds of plants Cab full-length amino acid sequence information. Using DNAman compared the above sequence, and designed the specific primers in the conserved region.Tatal RNA of citrus sinensis was reversed transcription as a template to PCR. We got part of the Cab gene cDNA sequence of284bp. Using RACE amplified the full-length999bp sequence of the citrus sinensis Cab gene, discovered through bioinformatics molecule citrus sinensis Cab full length cDNA of795bp, encoding264amino acids. Primers designed according to the both ends of the coding region of Cab full length cDNA sequence, citrus sinensis genomic DNA as template cloned the full-length genes of Cab.2. Using genomic walking techniques, citrus sinensis genomic DNA as a template, three thermal asymmetric PCR cloning the citrus sinensis Cab gene transcription start sites upstream,708bp. Obtained sequences were submitted to the PLACE and Plant Care. Sequence analyses revealed that it contained the TATA-box and CAAT-box that is a general promoter of conservative elements.and5copies of GATA-box, a known cis-acting element related to green tissue-specific expression,and some light responses homeopathic components.3. To investigate the tissue expression pattern of the cloned regulatory sequence. an expression vector containing this sequence fused with GUS was constructed for transformation into tomato by using agrobacterium-mediated method. 4. Histochemical staining of transgenic tomato showed that GUS reporter gene was predominantly expressed in immature green fruits and leaves but not in mature red fruit and flowers, indicating that the promoter display a strong green tissue-specific expression pattern in transformation into tomato.