Functional Research Of PSY And LCYE Gene In Biosynthesis Of Lycopene

Lycopene is one of the plant natural pigments and the most powerful antioxidants found in plant, belongs to carotenoid. It can quench singlet oxygen or capture peroxylradicals by the way of pHysics and chemistry. It’s ability of scavenging free radicals is better than other carotenoids and vitamin E. Lycopene also can block the tissue cells’ gene mutation under the effect of external mutagen except it’s strong antioxidation. In addition, lycopene is a kind of low cholesterol agent, it can inhibit the biosynthesis of cholesterol.The biosynthesis processes of lycopene include a series of reactions, such as condensation, dehydrogenation, cyclization, hydroxylation, and epoxidation. Among the reactions, the pHytoene synthase(PSY) catalyze 2 GGPP to compound 1 pHytoene, which generates lycopene by pHytoene desaturase(PDS) and ζ-carotene desaturase(ZDS). Lycopene have two major metabolic pathways. One is producing α-carotene through LCYE, and then turning to lutein; The other is producing β-carotene through LCYB, and the β-carotene is precursor of abscisic acid(ABA).Objective: Lycopene is mainly in tomato, watermelon, carrot, pepper and other fruits and vegetables, and tomato fruit is the main source of lycopene,so the research of cultivating tomato fruit with high content lycopene become a hot spot.The purpose of this research is using genetic engineering means to cultivate transgenic tomato germplasm with high lycopene.Methods: Using pBI121 as the vector framework, constructed the fruit specific overexpression vector of lycopene synthesis genePSY by fruit specific promoter E8; Using pCAMBIA1301 as the vector framework, constructed the fruit specific RNAi expression vector of lycopene metabolism gene LCYE according ‘hairpin’ RNA(hpRNA) by fruit specific promoter E8 too; Transformed processing tomato Liger 87-5 by agrobacterium-mediated method; Identified transgenic plants by PCR; Measured these two genes’ transcription and expression level by real-time quantification RT-PCR; Finally measured the content of lycopene in transgenic tomato fruits by spectropHotometer.Results and conclusion: 1. Using DNA/RNA of proceesing tomato as template, PCR amplification obtained 167 bp third intron of LCYE gene, 2207 bp E8 promoter, 1280 bp PSY gene, and 337 bpconservative sequence of LCYE gene; There were four isocaudarnersadded to two sides of Intron, and there was a HA tag sequence added to terminus of PSY gene; The sequencing results indicated the related target stripes were amplified successfully.2. Inserted Intron into vector pBI221, and constructed intermediate vector pRi221 with Intron intervening sequence; Through two isocaudarners, BamHI/Bgl II and XhoI/SalI, which were on two sides of Intron of pRi221, successively Inserted conservative sequence of LCYE gene into two sides of Intron in forward and reverse derection, and constructed vector pRiE8-Lcye; Connected interference fragment to expression vector pCAMBIA1301, and finally constructed the fruit specific RNAi expression vector pCRiE8-Lcye. All of the vectors were detected by restriction enzyme digestion or PCR and obtained expectant stripes. In addition, both of expression vectors were sequenced, the results showed they were constructed correctly.3. Agrobacterium tumefaciens with PSY overexpression vector transformed tomato by leaf disc method and obtained 6 plants. PCR detection of these 6 plants were all obtained expectant stripes, that indicated there were 6 transgenic tomato plants with PSY overexpression obtained successfully; Agrobacterium tumefaciens with LCYE RNAi expression vector transformed tomato by leaf disc method too and obtained 1 plant. PCR detection of this one plant was obtained expectant stripes, that indicated there was 1 transgenic tomato plant with LCYE interference obtained successfully.The genes’ expression level and the change of lycopene content in transgenic tomato need further study after bearing fruits.