Cloning And Characterization Of The ThCER6 Gene From Thellungiella Halophila

The plant epicuticular wax covers the aerial organs of almost all land plants. It is the collective term of the lipid components covering the outmost surfaces of aerial plant tissues. And it also includes the suberin matrix in underground and wound tissues, as wel as lipids of the pollen and seed coat. The plant wax is predominantly comprised of very long chain aliphatic lipids, such as alkanes, ketones, primary alcohols, esters and secondary alcohols. But wax also includes triterpenoids and minor secondary metabolites, such as sterols and flavonoids. These wax components are synthesized in the plant epidermal cells, then secreted out of the cells to form the diverse crystalline structures spontaneously, including columnar-shaped, rods, tubes, vertical plates, dentritic-, and umbrella-like structures. The chemical and physical characteristics of the plant wax are so complexed, indicating that wax may play important roles in plants: such as the regulation of nonstomatal water loss; the protection against UV radiation; the resistance to bacterial and fungal pathogens; the intermediate of the plant-insect interactions and the signal transduction required for fertilization.The synthesis and secretion pathway of the plant wax are conservative in all plants. First, the de novo synthesis of the saturated fatty acid to the Cl 6 or CIS occurs in the plastid. Then the C16 or CIS saturated fatty acid are transported out of the plastid for the elongation to the very long chain fatty acid (VLCFAs). The derivatives are produced from VLCFAs on the endoplasmic reticulum (ER) catalysed by membrane-associated multienzyme complexes. Up to date, only several enzymes about the wax synthesis are separated and we can use them to predict the wax synthesis and secretion pathway. Not only in the plastid, but also on the ER, the fatty acid sybthesis pathway is composed of a series of four-step reactions cycle: condensation reduction dehydration and second reduction. The acyl chain will be added C2 unit from malonyl-CoA (ACP) every cycle. There are two pathways for the production of the fatty acid derivatives: the acyl reduction pathway to produce primary alcohol esters and maybe aldehydes and the decarbonylation pathway to aldehydes, alkanes, secondary alcoholsand ketones.The CER6 of Arabidopsis thaliana is a key condensing enzyme catalyzing the synthesis of the C20-C24 fatty acid. The cer6 mutants showed severely waxless. We cloned a CER6 homologue, ThCER6, from Thellungiella halophila, and its ORF is 1494bp encoding a protein with 497 deduced amino acids. ThCER6 shares 90% identity at NA level and 95% identity at AA level with CER6. From the Southern analysis, we knew there was only one copy of the ThCER6 in the T.halophila genome. Drought, salt and ABA all promote the expression of the ThCER6, but high temperature (38), suppresses its expression.T.halophila, a close relative of A.thaliana, have similar heredity characteristics and growth habits with A.thaliana: small genome, short life cycle, self-fetilization, abundance seeds, and have several prominennces to A.thaliana: higher tolerance and more waxes amounts, etc.PTGS (Post Transcription Gene Silence) exists in almost all species. PTGS in plants, RNAi in animal and quelling in Neurospora have the similar mechanisms of PTGS. dsRNA is digested to siRNA, which induces the degradation of mature mRNA, thus suppresses the expression of specific gene. PTGS has been used as a new technique for the functional genomics.We used a shuttle vector, pHANNIBAL, and a binary expression vector, pART27, to specificly silence the ThCER6 in T. halophila and A. thaliana. The transgenic lines showed severely waxless phynotype. The results indicate the ThCER6 plays important roles in the synthesis pathway of epicuticular waxes in T. halophila.