Increasing Carotenoid Content In Tomato By Over Expressing Cryptochrome

Cryptochrome genes (CRY) are a novel class of plant genes encoding proteins that share sequence similarity with photolyases, a rare class of flavoproteins that absorb light in the blue (B) and UV-A regions of the spectrum and involved in photorepair of UV light–damaged DNA. Cryptochromes play major roles in plant photomorphogenesis, such as inhibition of hypocotyl elongation by blue light, stimulation of leaf expansion by blue light and regulation of floral initiation by day length.Arabidopsis CRY1 and CRY2 are predominantly nuclear proteins that mediate regulation of gene expression and entrainment of the circadian clock in response to light. Overexpressions of the CRY1 and CRY2 photoreceptors result in hypersensitivity to blue/UV-A light, with transgenic seedlings exhibiting unusually short hypocotyls and high levels of chlorophyll, anthocyanin and flavonoid. On fusion withβ-glucuronidase (GUS), both the Arabidopsis CRY1 C-terminal domain (CCT1) and the CRY2 C-terminal domain (CCT2) mediate a constitutive light response. Tomato CRY2 overexpressors show phenotypes similar to but distinct from their Arabidopsis counterparts, including a high-pigment phenotype, resulting in overproduction of anthocyanin and chlorophyll in leaves, increasing content of flavonoid and carotenoid in fruits. CRY2 overexpression causes an increased outgrowth of axillary branches but a shortened hypocotyl under blue light.In order to increase the content of carotenoids in tomato and to examine if the Arabidopsis CRY genes functioning the same way as the tomato CRY genes in tomato, we transformed tomato with a series of constructs carrying the Arabidopsis CRY1/ CRY2 cDNA or their C-terminal domains under the control of the cauliflower mosaic virus 35S promoter. In addition, we cloned polygalacturonase promoter characterized it byβ-glucuronidase (GUS) expression analysis,in order to express exogenous genes including the Arabidopsis CRY genes in fruit specific manner in the future. The main results were shown as follows:(1) Spectrophotometric measurements indicated an increased pigmentation in leaves of transgenic plants. The concentration of anthocyanins of the transgenic plant was up to 3.2-fold of the control plants. The concentration of the chlorophylls and carotenoids was also increased after overexpression of the CRY genes.(2) Carotenoid contents in red-ripe fruits from wild type and different lines of transgenic plants were determined by using HPLC. Lycopene content showed a significant (up to 3.6-fold) increase, and the total carotenoid content was also significantly increase (up to 3.4-fold). Some plants showed modest increase in the content of lycopene and total carotenoids.(3) The effects of different media with various hormone concentrations on tomato adventition bud differentiation were tested in cotyledon. The results showed that MS + 1.0mg/L ZT was the best medium for adventition bud differentiation. The effect of antibiotics on inhibiting Agrobacterium was also studied. Results indicated that the medium with 200 mg/L cefotaxin could inhibit Agrobacterium very well.(4) We cloned polygalacturonase gene promoter and fused withβ-glucuronidase (GUS). The construct was introduced into tomato by Agrobacterium-mediated transformation in order to confirm the friut specificity of this promoter and to use it for friut specific expression of CRY genes. After selection on kanamycin containing medium and PCR screening for the presence of the transgene, seven transformants were obtained.This study optimized the tomato regeneration and genetic transformation system.We also overexpressed the Arabidopsis CRY gene and their C-terminal domains in tomato. In the transgenic tomato's fruits the lycopene and the total carotenoids were increased 3.6-fold and 3.4 fold. This result is of great significance in exploiting new variety of tomato and functional food.