Cloning, Expression And Genetic Transformation Of The Genes In Carotenoid Metabolism Pathway In Watermelon (Citrullus Lanatus)

Carotenoids are aliphatic or alicyclic compounds with numerous conjugated carbon-carbon double bonds as chromophore skeleton. Carotenoids are a kind of natural pigments biosynthesized de novo by plants, algae, fungi and bacteria, which are paid wide attention to owing to their beneficial effects. Watermelon(Citrullus lanatus) is one of the most popular fruits in the world. Carotenoids are responsible for the different flesh colors of watermelon fruit. To investigate carotenogenesis and metabolic regulation in watermelon and to carry out genetic manipulation of carotenoid and to explore the anti-tumor effects of carotenoids will provide basis for the development and utilization of carotenoids and the molecular breeding and germplasm innovation of watermelon.In the present dissertation, the genes in carotenoid metabolism pathway in watermelon were cloned and changes in carotenoid profiles and in the expression pattern of the genes in carotenoid metabolisms during fruit development and ripening were observed in four watermelon cultivars. The factors affecting watermelon cotyledon regeneration and genetic transformation frequency were investigated. Then geranylgeranyl pyrophosphate synthase gene(GGPS) was introduced into watermelon via Agrobacterium-mediated transformation. In addition, the molecular mechanism for anti-tumor effects of lycopene and β-carotene was also investigated. The main results are as follows:The full-length c DNAs of geranylgeranyl pyrophosphate synthase gene(GGPS) and phytoene synthase gene(PSY) were cloned from watermelon fruit using RT-PCR and RACE. Bioinformatics methods were used to analyze the c DNAs obtained and putative amino acid sequences.(1) The full-length c DNA of watermelon GGPS(Gen Bank accession number: KF914758) consists of 1,445 bp including an open-reading frame of 1,092 bp, which is predicted to encode a putative protein of 363 amino acid with a calculated molecular weight of 39 k Da. The deduced amino acid sequence of watermelon GGPS shows 96 % and 95 % identities with those of muskmelon and cucumber, respectively. The watermelon GGPS protein comprises a putative transit peptide in the N-terminal region.(2) The full-length c DNA of watermelon PSY(Gen Bank accession number: KC166870) consists of 1,561 bp including an open-reading frame of 1,266 bp, which is predicted to encode a putative protein of 421 amino acid with a calculated molecular weight of 47.3 k Da. The deduced amino acid sequence of watermelon PSY shows 95 % and 94 % identities with those of muskmelon and pumpkin, respectively. The watermelon PSY protein comprises a putative transit peptide in the N-terminal region and a predicted transmembrane domain.Changes in carotenoid profiles during fruit development and ripening were measured by HPLC in four watermelon cultivars: red-fleshed ??CN66‘‘, pink-fleshed ??CN62‘‘, yellow-fleshed ??ZXG381‘‘ and white-fleshed ??ZXG507‘‘. The expression pattern of twelve genes(GGPS, PSY, PSY-A, PDS, ZDS, CRTISO, LCYB, CHYB, ZEP, NCED1, NCED2 and NCED3) directly involved in carotenoid metabolism was investigated by RT-q PCR. The predominant carotenoid detected in ripening fruit of red-fleshed ??CN66‘‘ and pink-fleshed ??CN62‘‘ was lycopene along with low amounts of phytoene, phytofluene, δ-carotene, β-carotene, lutein and violaxanthin. Lycopene appeared at 12 DAP and became a main carotenoid at the later stages. The transcript levels of carotenogenic genes(include GGPS) in ??CN66‘‘ sharply increased during 18-30 DAP, and concomitantly, fruit accumulated the massive amounts of carotenoids. In yellow-fleshed ??ZXG381‘‘, lutein and violaxanthin contents were positively correlated, respectively, with CHYB and ZEP transcript levels during the fruit development and ripening. The trace amounts of carotenoids in white-fleshed ??ZXG507‘‘ were accompanied with the low transcript levels of most biosynthetic genes(especially GGPS), and NCED1, a carotenoid oxidative cleavage enzyme gene, kept high transcription levels. The results suggest that differential transcriptional regulation of carotenoid metabolic genes is very important in determining the type and amount of carotenoids accumulated during watermelon fruit development and ripening.The plant expression vectors harboring carotenogenic gene(GGPS, PSY and LCYB), directed by watermelon fruit-specific expression AGPL1 promoter, were constructed and transferred into Agrobacterium tumefacien EHA105. The factors affecting cotyledon regeneration and genetic transformation frequency of watermelon cultivar ―Hong Yi Hao‖ were investigated. The GGPS gene was introduced into watermelon via Agrobacterium-mediated transformation.(1) The cotyledon taken from 3 day-old seedlings was used as explants for regeneration and transformation. MS medium containing 1 mg/L 6-BA and 0.2 mg/L IAA was optimum for shoot regeneration. 1/2MS medium containing 0.1 mg/L NAA and 0.2 mg/L IBA was optimum for root regeneration.(2) The optimum concentrations of selective Kanamycin for resistant shoots induction and resistant root regeneration were 75 mg/L and 40 mg/L, respectively. The optimum concentrations of Ampicillin and Cefotaxime to inhibit the growth of EHA105 were 200 mg/L and 300 mg/L, respectively. Pre-culturing explants for 2 days and infection with EHA105(which contained plant expression vector p AGPL1-GGPS) with OD600 0.5 for 10 minutes, then co-culturing for 3 days, could improve transformation frequency. The integration of GGPS gene into the regenerated plantlets was confirmed by PCR. The transformation frequency was 1.33 ‰.The effects of lycopene and β-carotene were investigated on the viability of human esophageal squamous carcinoma cell EC109 by MTT method. The involvement of PPARγ, p21WAF1/CIP1, Cyclin D1 and Cox-2 in effects of lycopene and β-carotene was analysed by Western Blotting. Lycopene and β-carotene(5-40 μM) inhibited the proliferation of EC109 cell in dose- and time-dependent manners. GW9662, a potent antagonist of PPARg, partly attenuated the inhibition of EC109 cell proliferation by these carotenoids at 3 μM. Lycopene and β-carotene(40 μM) could up-regulated the expression of PPARg and p21WAF1/CIP1, and down-regulated the expression of Cyclin D1 and Cox-2. The modulation effects of these carotenoids treatment on the expression of PPARg, p21WAF1/CIP1, Cyclin D1 and Cox-2 were inhibited by GW9662. The results suggest that the inhibition of EC109 cell proliferation by lycopene and β-carotene involve PPARg signaling pathways, and via modulating the expression of p21WAF1/CIP1, Cyclin D1 and Cox-2.