Cloning Of WIN1 And Functional Research Of RNAi In Thellungiella Halophila

Waxes form a continuous lipid membrance covering the epidermal cells of all aerial plant organs. It mainly comprised of long-chain aliphatic compounds derived from very long chain fatty acids that are enlonged from C16 or C18 long fatty acids by many fatty acid elongation (FAE) complexs. Waxes are most evident in the form of epicuticular crystals that are exposed on the surface of certain organs and species. Cutin is the major component of the cuticle that overlays the cell walls of epidermal cells and forms the protective tissue on primary aerial organs. The chemical and physical properties of cuticular waxes indicate that they have vital functions for plant life. including the regulation of epidermal permeability and nonstomatal water loss and protection against insects, pathogens, UV light and the prevention of postgenital organ fusion and pollen–pistil interactions.We report here the isolation of an ethylene response factor(ERF)-type transcription factor through a systematic functional genomics approach in Arabidopsis thaliana. The transcription factor, belonging to the plant-specific family of AP2/EREBP transcription factors, was termed WIN1?SHN1(At1g15360), for wax inducer 1, its homologous gene SHN2and SHN3 have a similar functional role in Arabidopsis. It can activate wax deposition, enhance drought tolerance, and modulate cuticular permeability in WIN1?SHN1 overexpressing plants. and that the wax accumulation phenotype is companied by an induction of several wax biosynthesis pathway genes. WIN1 overexpression induces compositional changes and an overall increase in cutin production in vegetative and reproductive organs, while its downregulation has the opposite effect.The new salt tolerance model plant Thellungiella halophila, a close relative of Arabidopsis 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 wax amounts, etc. At the cDNA sequence level, T. halophila are predicted to be 90% to 95% identical to A. thaliana All of these have revealed the potential of new genomics-based approaches with T. halophila to identify and clone valuable new genes conferring traits not apparent in A. thaliana, especially the genes involved in the biosynthesis of plant epicuticular wax.For further study the function of WIN1, we reported the cloning of WIN1 homolog from Thellungiella halophila (ThWIN1), anlysing the expression pattern and its functions using different expression systems.The results were shown as follows:1. cDNA cloning and sequence analysis of ThWIN1We obtained the flower total RNA in Thellungiella halophila According to the the deduced amino acid sequences of WIN1 from GeneBank, degenerate PCR primers were designed. Reverse transcriptase (RT)-PCR amplification and 5'and 3'RACE amplification were used to obtain the full-length transcript sequences of ThWIN1 The ThWIN1 cDNA was 846 nucleotides long including 579 bp ORF for a 192 amino acids peptide. The deduced amino acid sequence of ThWIN1 was 84% identical to AtWIN1.2. Expression analysis of ThWIN1Total RNA was extracted from seedlings, leaves, roots, flowers ,stems, siliques. RT-PCR analysis showed that ThWIN1 was mainly expressed in flowers tissues and to a lesser extent, in stems and siliques tissues.ThWIN1 transcript was not detactable in roots and leaves by using our approach.3. Preparation of gene silencing expression constructsThe ThWIN1 gene fragment of 114 bp was PCR amplified from flower cDNA and constructed into the plant expression vector pCAMBIA-3301H, then transferred into Agrobacterium GV3101; Using Floral Dip method, the flower of Thellungiella halophila have been transformed. Screening the transformed seeds by using herbicide Finale and have gotten about 14 Basta-resistance plants of the gene silence of ThWIN1. T1 transformants were obtained.4. Preparation of gene overexpression constructsThe full length of ThWIN1 ORF was constructed into the plant expression vector pCAMBIA-3301H and transformed into Agrobacterium GV3101. Using Floral Dip method , the flower of Thellungiella halophila have been transformed. Screening the transformed seeds by using herbicide, T0 transformants were obtained.5. Studied the protein interaction with ThWIN1 transcription factor usingYeast two-hybrid systemA large number of high-quality total RNA was extracted, mRNA was purificated, cDNA library was constructed in the T.halophila. ThWIN1 transcription factor as bait protein, the protein interaction with ThWIN1 transcription factor usingYeast two-hybrid system was studiedThe main innovation points of this study were generalized as follows:1. It was the first time to clone theTHWIN1 gene from halophyte T.halophila.2. For the first time to analysis tissue-specific expression of THWIN1 in the T.halophila. 3. It was the first time to construct the transgenic T.halophila plants overexpressing and silencing THWIN1 were generated.4. Studied the protein interaction with ThWIN1 transcription factor using Yeast two-hybrid system.