Studies on production of petunia with deep yellow flower by genetic engineering of carotenoid and betalain-related genes

概要

Petunia (Petunia × hybrida) is a popular commercial bedding plant that comes in many colors, but there are no petunia cultivars with deep yellow petals. Here the expression levels of genes related to carotenoid biosynthesis and degradation were compared in three white- and two pale yellow-flowered petunia cultivars. The carotenoid biosynthetic genes; phytoene synthase (PSY), phytoene desaturase (PDS), ζ-carotene desaturase (ZDS), carotenoid isomerase (CRTISO), lycopene β-ring cyclase (LCYB), chromoplast-specific lycopene β-ring cyclase (CYCB), lycopene ε-ring cyclase (LCYE), β-ring hydroxylase (CHYB), and zeaxanthin epoxidase (ZEP), and the carotenoid degradation gene, carotenoid cleavage dioxygenase (CCD4) were isolated from petunia. Expression analysis revealed that CHYB and ZEP might be critical to enable yellow-flowered petunia to produce higher lutein, violaxanthin, β-carotene, and zeaxanthin in corollas. Moreover, the 226-bp insertion of a non-autonomous transposable element tourist-like miniature-inverted repeat (MITE) was found in the coding region of CCD4 only in yellow cultivars. The insertion interrupts gene expression because of a premature stop codon after the transposon sequence and could form secondary structures in hairpin RNA that could then be processed into a mature miRNA. Besides, the normal function of CCD4 isolated from a white cultivar was introduced to a yellow cultivar. The transformants showed more fading pale-yellow flowers, and carotenoid content was decreased. These results indicate that the lack of CCD4 expression and high CHYB and ZEP expression result in more carotenoid accumulation in the corolla of yellow-flowered petunia.

Furthermore, genetic engineering by Agrobacterium-mediated gene transfer method was used for altering corolla color. Modification of carotenoid biosynthetic pathway, β-carotene ketolase (crtW) gene from marine bacteria Brevundimonas sp. strain SD212 with transit peptide (tp) sequence of the pea (Pisum sativum) was introduced under various promoters; the cauliflower mosaic virus 35S, the 2 kbp upstream promoter region of a β- ring hydroxylase gene of Lotus japonicus (LjCHYB), and the 1.4 kbp upstream promoter region of an α-expansin gene from a cotton (Gossypium hirsutum) with the 1 kbp upstream promoter region of an RDL1 gene from a cotton (Gossypium arboreum) (GhEXPA/GaRDL1). The nine transformed plants of each three lines under various promoters of 35S, LjCHYB, and GhEXPA/GaRDL1 which showed color alteration, were generated in this study. The presence of the introduced gene in transformed plants was detected using RT-PCR and Southern blot analysis. RT-PCR analysis revealed that all transformed plants showed the 423-bp band of crtW while wild type showed only the 155-bp band of EFlα as a reference gene. In southern blot analysis, hybridization of EcoRI digested genomic DNA and the integration of the introduced gene cassette was detected. Transformed plants showed different insertion sites and copy numbers. The HPLC analysis was accomplished to identify each carotenoid composition and content in corolla and leaf comparing between wild type and transformed plants. In wild type and transformed plants, violaxanthin, lutein and β-carotene were mainly detected in corolla and leaf. Surprisingly, some unique peaks of ketocarotenoid were detected and identified as astaxanthin, adonixanthin, 3'-hydroxyechinenone, canthaxanthin, 3-hydroxyechinenone and echinenone in the transformed plants. These ketocarotenoid compositions were found both corollas and leaves in 35Sp while found specifically in corollas of transformed LjCHYBp and GhEXPA/GaRDL1p plants. These results suggest that the LjCHYB and GhEXPA/GaRDL1 promoters can be used as a flower-specific promoter. The corolla of transformed crtW petunia was successfully altered from yellow to orange color by ketocarotenoid accumulation, but it was accumulated particularly in the tube and inner limb except in the outer limb which showed more slightly accumulation of carotenoid pigment. In overcome this limitation, betalain-related genes, cytochrome P450-type enzyme CYP76AD6 from Beta vulgaris and DOPA 4,5-dioxygenase (DOD) from Mirabilis jalapa were introduced under 35S promoter. The corolla of transformants successfully turned to deep yellow which was caused by betaxanthin accumulation. In conclusion, this study provides how different in the expression of the carotenoid biosynthetic pathway and CCD4 genes comparing between white- and yellow- flowered petunia. With the limitation of carotenoid accumulation in the outer limb, transformation with the betalain biosynthetic gene was successfully turned corolla to a deep yellow color.