Cloning And Characterization Of Phytoene Synthase Encoding Gene From Melon Fruit And Its Transformation To Melon

Melon (Cucumis melo L.) is one of the most important horticultural crops in the world, it contains a lot of carotenoids, especiallyβ-carotene.β-carotene is transformed into the vitamin A after digested and absorbed in the body, so theβ-carotene also known as the original vitamin A. Medical studies have shown that carotenoids have important roles in enhancing human immunity, preventing cardiovascular disease and cancer. Phytoene synthase (PSY) is the key enzyme in the melon carotenoid biosynthesis pathway, and plays a key role in the synthesis of carotenoids in melon. To improve the quality of melon, it has important significance to study melon phytoene synthase activity and its'regulatory mechanism by the use of modern molecular biology methods. In this study, we measuredβ-carotene content at different developmental stages of melon fruit by HPLC. We cloned the full length of melon PSY cDNA by RT-PCR. We also analyzed the Psy expression characteristic and its genetic transformation to melon.The main results are as follows:1.β-carotene content in different genotypes'muskmelon fruit at different development stages were measured by HPLC. The results showed that in the fruit development of three genotypes (M01-3, Homoken and J-01), there are very littleβ-carotene content in early melon fruit development andβ-carotene content rapidly increased in nearly mature fruit and followed by declined after mature. Orange flash Homoken had the highestβ-carotene content in nearly mature fruit and significantly higher than white flash M01-3 and light green flash J-01.2. Primers were designed according to Psy gene conservative sequence in GenBank, A 1443bp cDNA was cloned from M01-3 melon fruit by RT-PCR, it encoded 422 amino acids and the registration number in GenBank is GU361622. Real-time RT-PCR was used to analyze Psy expression characteristic. The results showed that Psy expression level were different among root, stem, leaf, flower and fruit tissues. The Psy expression level was very low in the early development stage of fruit and reached the maximum at nearly mature fallowed decreased after mature. The expression trend of Psy was consist with the trend ofβ-carotene content in the development of fruit. 3. The ORF of Psy was ligated to pET-30a(+), and transformed into E. coli BL21 (DE3) competent cells to construct recombinant plasmid pET-Psy and analyzed by 8% SDS-PAGE. The specific expressed proteins induced by IPTG were about 46.7kDa in molecular weight. The results indicated that the protein was induced in vivo protein expression in the melon.4. Whole ORF of Psy was cloned in to clone vector PMD18-T followed by sequencing determination. The recombinant plasmids PMD18-T-Psy were isolated, double digested with BamHI and Sal1, ligated into BamHI - SalI digested pBI121 and transformed into E.coli competent cells DH5αand obtained sense and antisense expression vector, respectively. Agrobacterium-mediated method was used to genetic transformed sense and antisense expression vector into the melon,respectively. Melon seedlings with Kan resistance have been successfully obtained and they will be identified in a few weeks.