The Mechanism Of Carotenoid Accumulation And Key Gene Expression In Orange-Heading Chinese Cabbages

Heading colour breeding has become an important research area in chinese cabbage breeding. Orange-leaf-head (OLH) varity of chinese cabbage has come out for several years. We have known that The color of OLH Chinese cabbage was resulting from owing to much more carotenoid than the white-leaf-head(WLH) one, but the mechanism of carotenoid accumulation is still unknown. In this paper, using the WLH Chinese cabbage as a control, we try to discover the reason of carotenoid accumulation in three fields. First, we compared the chromoplasmid in cell and subcelluar level between OLH and WLH by microscope and electron microscope. Second, comparing the difference of genes transcription in heading leaf by cDNA-AFLP technology. Third, comparing the expression of seven enzyme genes related in carotenoid metabolism by real-time PCR. The main results are shown as below:1. Using the heading leaf and apical bud of OLH and WLH Chinese cabbage as meterials, we compaired their cell and subcelluar structure, especially plasmids by optical microscopy and electron microscopy. It was found that the chromoplasmid of OLH is mostly distribute in parenchyma cell of heading leaf, and mainly in the vascular bundles, and less in parenchyma cells of the petiole. There were about 6 chromoplasmids in parenchyma cell of OLH heading leafs, and the carotenoids was stored as the crystalline in chromoplasmid. but there was no carotenoids accumulatied in the apical bud cells.2. Using the heading leaves of OLH and WLH Chinese cabbage from the same F1 as materials, the gene expression differences was analysised by cDNA-AFLP technology with 256 pairs of primers.A total of 12 differential expression fragments were obtained; their size were from 100 to 500bp; half fragments were special expressed in OLH or WLH leaf respectively. 10 fragments of which were cloned successfully. As a result, four different fragments related to energy metabolism; one fragment related to signal transduction; one fragment related to resistance; four fragments function was still unclear.3. Using differenr part of leaves from OLH and WLH Chinese cabbage as materials, the transcriptional difference of seven enzyme genes in carotenoid metabolism were compared by real-time PCR with GAPDH as a reference gene. It was found that:(1) The transcriptions of PSY, PDS, ZDS in the upstream of lycopene were significantly up-regulated in OLH leaf than in WLH leaf, especially, the expression of PSY gene was about two times higher in OLH leaf than in WLH leaf;(2)The transcriptions of LYCB, LYCE, BCH, ZEP in the downstream of Lycopene were no obvious differentce between OLH and WLH leaf, it may not the key genes for carotenoid accumulation in OLH leaforange cabbage;(3) The transcription of LYCB gene was significantly up-regulated in heading leaves and the outer leaves , especially, about 2.1 times higher in outer leaves of WLH than that of OLH ,At the same time, the transcription in the inner leaves showed a consistent between the two materials.(4) The transcription of LYCE gene was significantly higher in outer leaves and inner leaves of OLH material, and its expression in outer leaves was higher than in inner leaves; while the expression shows a consistent in heading leaves between the two materials(5) The transcription of BCH gene was significantly higher in outer leaves and lower in inner leaves of WLH than that of OLH, while the expression shows a consistent in heading leaves between the two materials;(6) The transcription of ZEP gene was no significant difference in outer leaf,heading leaf and inner leaves between the two materials.