Abstract: 〖JP2〗Suppressing the expression of gene encoding lycopene -cyclase (LcyB), the key enzyme regulating the metabolism of lycopene to -carotene, is an effective way to increase the content of lycopene. In the present study, a 1 534 bp promoter of 5 upstream of LcyB gene was cloned from Micro-Tom tomato (Lycopersicon esculentum) and bioinformatically analyzed in the database of plant cis-acting regulatory element (PlantCARE). The result showed that several important cis-acting elements were identified in the promoter, including the TATA-box, CAAT-box, Circadian (response to circadian rhythm), Box I (response to light), Box-W1 (response to fungal elicitor), LTR (response to low-temperature), P-box (response to gibberllin), ERE (response to ethylene) and TGA-element (response to auxin). The LcyB promoter was inserted into the plasmid pKannibal between Mcr I and Xho I restriction sites to replace the CaMV 35S promoter, resulting in pK-LcyBp vector. Based on tomato genomic DNA sequence reported in the GenBank (Accession No. AEKE02020044), two pairs of primers containing various restriction sites were designed to amplify the 3 terminal 276 bp fragment of LcyB gene from Micro-Tom tomato. The two 276 bp LcyB fragments were separately cloned into the plasmid pK-LcyBp between Xho I/Kpn I and Hind III/Cla I sites, in sense or antisense directions, generating pK-LcyBp-RNAi-LcyB construct harboring LcyB RNA interfering expression frame driven by LcyB promoter. The RNA interfering expression frame was then cloned into the Not I site of the binary expression vector pART27 to achieve the RNAi binary vector targeting to silencing LcyB gene driven by its native promoter. This investigation lays the foundation for future increasing lycopene content through specific knock-out of LcyB gene by RNAi in tomato fruit. 〖JP〗