Functional Verification Of The Lycopene Beta-cyclase Gene (Lcyb) From 'Cara Cara' Navel Orange (Citrus Sinensis [L.] Osbeck)

'Cara Cara' navel orange(Citrus sinensis[L.]Osbeck) red-fleshed cultivar,is a bud sport of 'Washington' navel orange.The major pigments in the flesh of 'Cara Cara' navel orange are lycopene and beta-carotene.It is useful for the research of the carotenoid biosynthesis of the citrus fruits,lycopene beta-cyclase(LCYB) plays a very important role in the carotenoid biosynthesis of the citrus fruit.Lycopene is transformed into beta-carotene by the action of lycopene beta-cyclase(LCYB).It's a key point in the transformation from Lycopene to other pigments in the carotenoid metabolic pathway.Based on the cloned lycopene beta-cyclase gene(Lcyb) from 'Cara Cara' navel orange, we transformed the cDNA fragment of this gene into tomato(Lycopersicon esculentum Mill.) plants 'Zhongshu No.5' using the Agrobacterium-mediated transformation to verify its function.We verified that the kanamycin-resistant gene had been integrated into the genome DNA after PCR and Southern blotting hybridization;and analyzed the physiological and biochemical characteristics of the transgenic plants.The main results were as follows:1.Expression vectors which were constructed with cDNA fragments of the Lcyb gene were introduced into the tomato plants via Agrobacterium-mediated transformation. A total of 14 kanamycin-resistant plants were obtained.The PCR and Southern blot analysis of the putative transgenic plants showed that the T-DNA was integrated into the tomato genome with various copy numbers from 1-3.2.The color of the fruits from two transgenic tomato plants were obvious yellow compared with the red fruits from the non-transgenic tomato plants.Measurements of the contents of lycopene and beta-carotene in the transgenic fruits by using high performance liquid chromatography(HPLC),showed that the contents of beta-carotene in yellow transgenic fruits were much higher than that in red fruits of wide-type plants.Oppositely, the contents of lycopene were very low in the yellow transgenic fruits.It's suggested that over-expression of lycopene beta-cyclase gene(Lcyb) in the transgenic plants resulted in accumulation of beta-carotene in the transgenic fruits which was absence in the wide-type plants.3.RT-PCR analysis showed that the activities of target gene in 8 out of 14 different transgenic plants expressed,whereas the expression levels were different in individuals. Real-Time PCR analysis showed the expression of genes had involved in the carotenoid biosynthesis in the transgenic fruits.4.The inheritance pattern of the transgenes in T₁ segregation population were analyzed based on PCR with NPTâ…¡primers.And the results showed that NPTâ…¡gene inherited as a dominant genetic locus with the segregation ratio 3:1.