Functional Characterization Of Ammopiptanthus Mongolicus Zinc Finger Protein Gene Am6f3 And Protease Inhibitor Gene AmPI

Ammopiptanthus mongolicus in the family of leguminosae, is evergreen broad-leafed shrubs exclusively in Inner Mongolia Alashan Desert, with strong resistence to cold, drought and high temperature, hence, they are stress-tolerant gene resource plants of great importance in resistive breeding and researches on stress-tolerance mechnism. Due to abiotic stress tolerance, well-developed root sytem and function of stablizing sands and improving soil via legume, Ammopiptanthus mongolicus could be used in garden greening and environment improvement. Besides, as species survived the tertiary period and the only evergreen broad-leafed endangered tree, they are national conservative plants, and have important scientific value on researches on systematic developmetn of leguminosae, paleoflora, paleogeography, origination and formation of desert plants in the middle of Asia.A zinc-finger protein gene nominated as am6f3 from cold-stressed EST library was gained. And after bioinformatics analysis, we found that it belonged to zf-ANl protein family with conservative domain of AN1 and A20, by which Zn2+ and DNA/RNA could be combined specificly and gene expression could be regulated on transcriptional and translational level.the C terminal a helix could regulate the gene expression as well, as dipolymers or polymers. This protein was speculated to play an important role in transcription, transcriptional regulation and signal transduction as a regulating factor under stress. The plant expression vertor based on pBI121 was constructed with CaMV35S-Am6f3. Tabacco plantlets were transformed by agrobacterium-medicated transformation. It was demonstrated by antibiotics resistance screening that the Am6f3 gene was integrated into the genomes of transformed plantlets.A cold-induced protease inhibitor gene AmPI from Ammopiptanthus mongolicus was gained by substract hybridization, and was introduced into ER2566 by expression vector pTWIN. SDS-PAGE showed that a Mr 35×103 fusion protein was expressed and peaked after 3～4 hour IPTG induction at 37℃, suggesting the fusion protein was expressed in ER2566. The survival rates of E. coli under cold (4℃), heat (48℃), drought (mannitol) and salnity (NaCl) stress showed that E. coli with expressed AmPI were more tolerant to abiotic stresses. And higher survial rates illuminated that AmPI could play a protective role to E. coli.