Functional Characterization Of Gentiana Lutea Phytoene Desaturase (PDS) And Lycopene Epsilon-cyclase (LYCE) Gene Promoters

Carotenoids are a large class of yellow, orange and red pigments, which are synthesized within plastids in higher plants through isoprenoid biosynthetic pathway. Carotenoids are important nutritional composition in human diets and have important biological functions in human health. They are not only the precursors of vitamin A, but also playing important roles in protecting vision, decreasing the incidence of varieties of cancers and cardiovascular diseases, enhancing immune ability and so on.At present,"golden rice","golden canola"and"golden potato"have already been abtained through carotenoid genetic engineering. These achievements greatly enhanced the confidence in developing carotenoid genetic engineering in higher plants. However, a major limitation is our poor knowledge of the regulation mechanisms of carotenoid biosynthetic pathway and the expression regulation mechanisms of carotenogenic genes in higher plants.Phytoene desaturase (PDS) and Lycopene epsilon-cyclase (LYCE) are key regulatory enzymes in carotenoid biosynthetic pathway. Characterizing the functions of PDS and LYCE promoters could provide theoretic evidence in elucidating the molecular control mechanisms of carotenogenic gene expression. The abtained tissue or organ specific promoters could be used in the molecule breeding of nutritional improvement in major staple crops and flower colour modification in horticultural plants. Therefore, functional characterization of gentian (Gentiana lutea) PDS and LYCE gene promoters not only has great theoretical significance, but also has potential practical application value.The promoter fragments for gentian PDS and LYCE genes were cloned from genomic DNA by the inverse polymerase chain reaction (Zhu C., unpublished data). Using PLACE and PlantCare databases to analysis promoter cis-acting elements indicated that the full-length of PDS promoter (1077 bp) contained a putative TATA box located in the up-stream (-68 bp) of 3′end of promoter, the full-length of LYCE promoter (938 bp) contained a putative TATA box located in the up-stream (-69 bp) of 3′end of promoter. Furthermore, both of these two promoters had CAAT boxes whose function was enhancing gene transcription, light regulation cis-acting element box I, G box, I box, ATC motif, ABA regulation cis-acting element ABRE and so on. The similar cis-acting elements existed in PDS and LYCE gene promoters indicated that these two promoters might have similar functions in regulating gene expression.The full-length promoter fragments were fused to a gene encoding beta-glucuronidase (GUS) and the fusion constructs were introduced into tomato fruits and leaves by Agrobacterium-mediated transient transformation. The results of GUS transient expression indicated that PDS and LYCE gene promoters did not drive the GUS gene expression in chloroplasts-containing leaves and immature green fruits of tomato. During the development of tomato fruit, the transition from chloroplasts to chromoplasts occur, expression levels of the GUS gene driven by PDS and LYCE gene promoters gradually increased, the highest expression levels of GUS were found in mature chromoplast-containing fruits. This revealed that the expression of PDS and LYCE gene promoters was associated with chromoplast formation and the development of fruits of tomato. In order to extensively study the function of PDS gene promoter in tomato, fusion gene of GlPDSPro:GUS was introduced into tomato by Agrobacterium-mediated stable transformation. We extracted genomic DNA from transgenic tomato plants. Genomic DNA PCR analysis confirmed that stable transgenic tomato plants were obtained.