Cloning And Expression Of Lycopene Cyclase-β Gene In Melon Fruit And Its Genetic Transformation To Melon

Melon(Cucumis melo L.) is one of the most important horticultural crops in the world,it contains a lot of carotenoids,especially β-carotene.Medical studies have shown that carotenoids have important roles in enhancing human immunity,preventing cardiovascular disease and cancer.Lcyb is the key enzyme in the melon carotenoid biosynthesis pathway,and plays a key role in the synthesis of carotenoids in melon. In this study,we measured β-carotene content at different developmental stages of melon fruit by HPLC.We cloned the full length of melon Lcyb cDNA.We also analyzed the Lcyb expression characteristic and its genetic transformation to melon.The main results are as follows:(1) β-carotene content in two different genotypes muskmelon fruit at different development stages were measured by HPLC.The results showed that in the fruit development of two genotypes (M01-3,Homoka),there are very little β-carotene content in early melon fruit development and β-carotene content rapidly increased in nearly mature fruit and followed by declined after mature.Orange flesh Homoka had the highest β-carotene content in nearly mature fruit and significantly higher than white flesh MO1-3.(2) Primers were designed according to Lcyb gene conservative sequence of other plants in GenBank, The full-length cDNAs of Lcyb gene from melon fruit were cloned by RT-PCR and3’,5’RACE.The sequence analysis showed that Lcyb gene contained1871bp and coded504amino acid residues and the registration number in GenBank is GU457407.(3) Real-time RT-PCR was used to analyze italic expression characteristic.The results showed that Lcyb transcripts are easily detected in M01-3’s and Homoka’s leaves,stems,mature fruit,roots and flowers. The highest expression level existed in M01-3’s flowers and Homoka’s mature fruit,The expression level of Lcyb increased with the development stage of fruit and reached the maximum at25day fruit in MO1-3and45day afte pollination fruit in ’Homoka’.(4) We constructed the antisense and sense expression vectors of Lcyb successfully by PCR and digested with restricted enzymes. Melon seedlings with Kan resistance have been successfully obtained.