Cloning And Functional Analysis Of The C₂H₂ Zinc Finger Protein Gene ThZF1 In Thellungiella Halophila

Agricultural productivity worldwide is subject to increasing environmental constraints, particularly to drought and salinity due to their high magnitude of impact and wide distribution. In the last decade, Arabidopsis thatiana, a genetic model plant, has been extensively used for unravelling the molecular basis of stress tolerance. In their studies, the responses of plants to salt and water stress are described, the regulatory circuits which allow plants to cope with stress are presented, and how the present knowledge can be applied to obtain tolerant plants is discussed. Recently, the salt-tolerant plant Thellungiella halophila was introduced as an attractive model plant to study molecular genetics of salt tolerance, because it is a close relative to Arabidopsis and amenable for transformation unlike that of other tolerant model plants. Thus it may be possible to combine profound genetic knowledge with the expression of extreme tolerance.Zinc finger proteins (ZFPs) play an important role in growth and development in both animals and plants. Recently, some Arabidopsis genes encoding distinct ZFPs have been identified, including AZF2. Cys2/His2-type zinc finger, also called the classical or TFIIIA-type finger, have been found in a number of eucaryotic transcription factors as DNA-binding motifs. It is reported that their involvement in plant development and their important roles under various abiotic stresses. However, the physiological role of their homologues with putative zinc finger motif remains unclear.In the present study, a novel gene, ThZF1, encoding a classical ZFP, was isolated from stress-induced salt cress (Thellungiella halophila, Shan Dong) aided by RT-PCR and RACE (rapid amplification of cDNA ends) strategies and characterized as a transcription factor gene.Its cDNA encodes a protein of 276 amino acid residues with a deduced molecular weight of 30 kD, which PI is 8.27. It is hydrophobic protein without transmembrane helixes. ThZFl contains two conserved C2H2 regions, in which two conserved plant-specific QALGGH sequences was found. ThZFl has region consisting of three acidic residues followed by hydrophobic residues rich in Leu (L box); and have C-terminal short regions containing DLNL sequences (DLN box) as well. The B box is a potential nuclear localization signal, and the L box and DLN box are thought to play roles in protein-protein interactions or in maintaining the folded structure. It shares 79% identity with Arobidopsis ZFP-relate AZF2.The RT-PCR and northern blot analysis were used to investigate ThZFl transcription in response to drought and salt stresses. It is showed that both salinity and drought induced strong expression of ThZFl mRNA in salt cress.Transient expression analysis using fused protein with GFP revealed that ThZFl was localized preferentially in nucleus. ThZFl showed in vitro function to activate HIS marker gene in yeast. The ThZFl was able to bind EP2 repeat element in EMSA analysis. These results suggest that ThZFl functions as an EP2-binding transcription factor in plant.For further understanding of the function of ThZFl under abiotic stress conditions, we generatedtransgenic Arabidopsis azf2 plants over-expressing ThZF1 under the control of the constitutive CaMV 35S promoter. Over-expression of ThZFl seemed to enhance the growth and blooming of seedlings under drought stress. The transgenic plants decrease in transpiration rate and increase in drought tolerance. Moreever, the expression of putative downstream gene EPSPS is induced obviously in transgenic complement plants. Consideration of role of EPSPS, the gene for 5-enolpyruvylshikimate-3-phosphate synthase, in shikimate pathway and is involved in stress, these results suggest that over-expression or ectopic expression of this gene causes somewhere positive effects to plant development or growth and improve their tolerance.This is the first time that a halophytic gene, ThZFl, encoding a classical ZFP, was isolated from model plant salt cress and characterized. Profound research is awaited for understanding the details of...