Lycopene β-Cyclase Gene Silencing In Tomato

Post-transcriptional gene silencing (PTGS) is a nucleotide sequence-specific process of RNA degradation. It leads to the degradation of homologous RNA from among endogenous genes, transgenes or RNA virus. RNA silencing represents an evolutionarily conserved defense mechanism that plays a key antiviral role in protecting plants against virus infection. Post-transcriptional gene silencing (PTGS) can be used to control the gene expression and study the gene function in transgenic plants.Lycopeneβ-Cyclase is a key enzyme in carotenoid biosythetic pathway and catalyzes the formation ofβ-carotene from lycoene. Tomato lycoeneβ-Cyclase gene was amplified by RT-PCR from tomato leaf mRNA and cloned in pGEM-T vector. A 350 bp lycoeneβ-Cyclase gene fragment was subcloned in pDon221 and pJawoh18 from pGEM-T-Lyc-b. After verification by PCR and restriction analysis, ihpRNA vector pJawohl8-2b containing lycoeneβ-Cyclase gene fragment was constructed successfully by Gateway technology and was transfered into tomato plants by Agtobacterium-mediated transformation. We constructed a efficient gene silencing vector to silence the Lycopeneβ-Cyclase Gene, then investigate the function of Lycopeneβ-Cyclase gene in caroteniod biosynthesis pathway. Transfected tomato got restriction callus and 17 regeneration plants, four of which are positive by PCR and RT-PCR detection. Positive plants are restrictive to 200 mg/L Basta. During ripening process, the fruits of the positive tomatoes become red gradually. That means Lycopeneβ-Cyclase gene may be silent. The bar gene was used as the selective marker gene in transgenic tomato. The transgenic tomato plants were produced in a herbicide tolerence system, so that the transgenic plants can be used in the fields.