Fine Mapping And Gene Expression Of Wax Gene In Chinese Cabbage (Brassica Rapa L. Ssp. Pekinensis)

The cuticular wax covering the epidermal cells in plants which contains the lipophilic compounds is the hydrophobic layer. The main compounds include fatty acids, alkanes, primary alcohols, secondary alcohols, aldehydes, ketones, and so on. Cruciferous vegetable crops generally covered with white frosted. The function of the cuticular wax is to reduce non-stomatal water loss, protect the plant from external mechanical damage, pest invasion, and solar radiation. In this study, the inbred lines of waxy and glossy in Chinese cabbage were the test materials, using a scanning electron microscope to observe the epidermal wax structure and gas chromatography-mass spectrometry to detect chemical composition. Finding out the genetic regular of waxy trait, using SSR markers to locate the wax gene and analysis of the predicted genes using real-time PCR technology, further validation of candidate genes is the target gene. The main results are as follows:1. The material of waxy and glossy was dried directly and then using scanning electron microscopy to observe the samples. Scanning electron microscope found out that Chinese cabbage epidermal wax was the rodlike structure with top slightly papillae, and glossy in a magnification of5000times, compared with the waxy material, the performance of a relatively smooth. The scanning electron microscope observation results are consistent with the results with the naked eye observations, the rod-like structure that is observed under scanning electron microscope is the the white frosted phenotype seen to the naked eye, glossy has not such a structure of the wax layer.2. By the chloroform extraction, the Chinese cabbage epidermal wax was extracted and used gas chromatography-mass spectrometry to detect the wax composition. It contains saturated alkanes with an even number of carbon atoms and nine indeterminate alcohols, which is the main ingredient in the wax compounds.3.08A161and08A235-2inbred were selected as the parents. Genetic analysis was doing in the Fi, F2and BC1generation. The results showed that the waxy character was qualitative character, and the glossy trait is controlled by a single recessive genetic locus.4. Selecting308F2homozygous recessive individual as the wax gene for mapping population, screening181pairs of SSR primers and the wax gene located on A01linkage group, designing SSR primers in the nearby area of nia-m086a, two markers which linked wax genes closely were found: SSRzx45, and SSRzx54, genetic distances were2.3cM and0.8cM, respectively.5. These two markers will be join in the larger F2recessive individual to determine the recombinant, the analysis results showed that wax gene was located between SSRzx79and SSRzx107, genetic distance of0.25cM and0.2cM, respectively. The physical distance between SSRzx79SSRzx107about86.4kb and15candidate genes within this zone, Bra013809by comparison found Arabidopsis CER2has a similar function, and CER2involved in wax metabolic pathway, therefore predict that Bra013809is a gene associated with wax metabolism.6. The analysis of Bra013809protein showed that it may be a non-secreted protein and the protein does not exist in the N-terminal signal peptide for the gene sequence Bra013809characterized. The protein contained in a variety of modification sites including the N-myristoylation sites, cAMP-and cGMP-dependent protein kinase phosphorylation sites, and the tyrosine kinase phosphorylation sites, casein kinase2phosphorylation sites, N-glycosylation sites, protein kinase C phosphorylation sites in protein5-286transferase activity domain. Therefore we speculated that the protein was transferase enzyme. The cluster analysis showed that Bra013809protein had a high homology with Arabidopsis CER2.7. In order to proof Bra013809wax metabolism, we analysis the the Bra013809gene expression pattern at the transcriptional level. The results showed that a high level of expression in Bra013809waxy plants, glossy plants is suppressed and have higher levels of expression Bra013809on wax plant stems and leaves, which shows that Bra013809was a wax metabolism-related gene.