| A hydrophobic cuticle that serves as a protective barrier against non-stomatal water loss and environmental damage covers the surfaces of terrestrial plants. Cuticular waxes are complex mixtures of hydrophobic lipids, consisting mostly of very-long-chain fatty acids and their derivatives. KCS is the key enzyme in the enlongation of VLCFA and VLCFA is the precursors of cuticular wax.In the present study, we dectected the composition and and observed the ultrastructure of cuticular wax on the leaves of Triticum monococcum and Aegilops tauschii during different developmental growth stages including seedling stage, jointing, heading and filling stage using GC-FID and SEM. Finally we found the main difference of wax components between this two species.We cloned the homologous genes of TaKCS1, TaKCS2 and TaKCS4 from the cDNA of Triticum monococcum and Aegilops tauschii. After the difference analysis of their amino acids,we transformed the homologous genes of TaKCS2 and TaKCS4 fromTriticum monococcum and Aegilops tauschii into Micro Tom to elaborate the overexpression impact of KCS towards the cuticular wax composition of tomato, which can be used to validate the function of KCSs and lay foundation for revealing molecular synthetic mechanism of wax on wheat.The results of the present research were as follows:1.The results of GC detection showed that with developmental growth of wheat, the total wax load accumulated, the proportion of primary alcohol reduced and the proportion of alkanes increased. The amount of diketone on Aegilops tauschii was constant at different developing stages, however there was little increase of diketone on Triticum monococcum. The changes of fatty acids and aldehydes were not obvious. The main difference of wax composition between this two species was the carbon chain length of the primary alcohol. In Triticum monococcum C28 alcohol accounted for absolute advantage dominance(45.75%-78.58%), while in Aegilops tauschii C26 alcohol accounted for the absolute dominance(62.16-80.09%).2.The results of SEM showed that the wax crystal structures of adaxial and abaxial surface were in similar shape in both Triticum monococcum and Aegilops tauschii. In Triticum monococcum, it was in the shape of platelets in seedling stage. Rod-like crystals began to appear and much more appeared in heading stage, but the shape of platelets was the main form.Wax crystals on heading leaves were sparse compared with waxes on seedling leaves and became much sparser on filling stage leaves.It is always the shape of platelets in different developmental stages of Aegilops tauschii, the intensity of wax crystals became sparser during this developmental stage.3.Based on the bioinformatics analysis we successfully cloned three genes from Triticum monococcum and Aegilops tauschii respectively.Then we make a statistic analysis of their chromosome preliminary positioning, the length of their genes, the length of their ORF and the amount of their amino acids.We also make alignments of nucleotide and amino acid sequences between Triticum monococcum and Aegilops tauschii.Finally the different genes TmKCS2, TmKCS4, AtaKCS2 and AtaKCS4 were successfully introduced into tomato "Micro Tom" by Agrobacterium-mediated transformation. After resistance screening and PCR identification,we successfully got several positive plants respectively.GC detection results of transgenic plants showed that: Compared with the control, the carbon chain length distribution of wax components did not change. However, the amount of total wax, total primary alcohols increased. Among them the content of C24:0-OH, C26:0-OH, C28:0-OH and most alkanes elevated at different levels. |
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