Cuticular Waxes Analysis And Screening Of Genes Involved In Cuticular Wax Biosynthesis In Wheat Leaf And Spike

Cuticular wax, covering the above-ground parts of plants, is the outmost layer of them. It directly interacts with the environment thus crucial for plants’ growth, development and adaptation. Wheat is one of the most important staple in the world. Its stress resistance and yield is closely related to the cuticular wax. In order to unravel the cuticular wax compositions of wheat and obtain wax biosynthetic related genes, we utilized GC-MS to analyze their leaf cuticular waxes at different growth stages. The cuticular waxes of different organs were also analyzed. After acquiring the changes of wax compositions within the whole growth stage and the feature of wax compositions of different organs, samples from different organs of 5 cultivars were collected for RNA-seq analysis. Based on the annotation and the expression levels of the Unigenes in each sample, the candidates that responsible for the biosynthesis of primary alcohols and alkanes were obtained. With wheat nulli-tetrasomics, we identified a potential gene locus that participates in the synthesis of primary alcohols which also verified the result of RNA-seq analysis. Finally, the wheat CER1 candidates, which are supposed to be involved in alkanes synthesis, were cloned and the plant expression vectors were constructed. The results are as follows:1. The leaf cuticular wax of Chinese Spring at different growth stages was analyzed, primary alcohols and alkanes are the main components. The total wax load increases rapidly before heading stage and peaks at heading to filling stage, then decreases. At all growth stages, primary alcohols are the dominant components. Before heading stage, alkanes and primary alcohols accumulate swiftly, and then the proportion of primary alcohols reduces while that of alkanes increases.2. The cuticular waxes of seedling leaves, flag leaves, spikes and sheaths of 8 cultivars were analyzed. Primary alcohols are the predominant components in seedling leaves. It’s still the dominant components in flag leaves; while alkanes and diketones are the second or third important components which are different within the cultivars. The diketones and alkanes are predominant in cuticular wax of spikes. In sheaths, diketones are the dominant components.3. The seedling leaves, flag leaves and spikes from different cultivars were collected as samples for RNA-seq analysis. Through GO and KEGG annotation and the expression levels of Unigenes in different samples, 7 reliable candidates of FAR and 12 reliable candidates of CER1 were obtained. In addition, 53 FAR and 28 CER1 candidates that are highly expressed in spikes were detected. The wheat genome database was used to predict their chromosome positions.4. The nulli-tetrasomic lines were used to verify the functional FAR candidates. Through the leaf cuticular wax analysis of CS, N7AT7 D and N7DT7 A, the significant decrease of primary alcohols in N7DT7 A at jointing and heading were detected. The dramatic reduction of C28 alcohol was observed after carbon-chain length analysis. Based on the result, a gene locus on 7D chromosome that responsible for the synthesis of primary alcohols, especially C28 alcohol, is detected, which is in accordance with RNA-seq analysis result.5. Based on the RNA-seq analysis and wheat genome database, 5 wheat CER1 candidates were cloned and the plant expression vectors were constructed. Further plant transformation is required to analyze their function.In this study, the changes of wax compositions at wheat different growth stages and the feature of wax compositions of different organs were acquired; genes that involved in wheat cuticular wax biosynthesis were obtained. These results have paved a solid foundation for the further study of wheat cuticular wax synthesis.