Study On Functional Characterization Of A Zinc Finger Protein Gene ThZFP3 From Tamarix Hispida

ZFP (Zinc Finger Proteins) transcription factors are one of the most important species of transcription factors in plants, and mainly play a crucial role in the growth and development, resistance to environmental stress and other aspects. In this study, a zinc finger proteins gene that was involved in the resistance to salt stress response was identified from the transcriptome cDNA librarys of Tamarix hispida. The gene named ThZFP3 was 1 335 bp in length, encoded 444 amino acids and belong to the CCCH type zinc finger protein gene family. The estimated molecular weight and isoelectric point of the putative protein were 48 491.6 Da and 8.08, respectively. ThZFP3 was fused to GFP under the control of CaMV 35S promoter, and was introduced into onion epidermal cells by particle bombardment. The results showed that ThZFP3 is a nuclear protein.The transcriptional activation domain of ThZFP3 was identified using yeast two-hybrid system and the ThZFP3 transactivation activity was conferred by its C-terminal domain, located at 225-280 aa.Quantitative real-time PCR assay revealed that the mRNA levels of ThZFP3 was up-regulated in T. hispida under NaCl and PEG treatments, suggesting that ThZFP3 might play an important role in salt and drought tolerance of T. hispida.After constructing the plant overexpression vector of ThZFP3, Arabidopsis thaliana was transformed and the transgenic Arabidopsis plants of T3 generation were stained. The wild type and transgenic lines of Arabidopsis were treated under NaCl and Mannitol. The results showed that compared with WT plants, transgenic lines had higher germination rate, root length, fresh weight and growth. The two lines of the T3 generation of ThZFP3 transformed plants displayed significantly improved tolerance to salt and drought.A suppression vector of pFGC5941-ThZFP5 was constructed. By using transient transformation method, three kinds of transgenic T. hispida plants were generated, including plants overexpressing ThZFP3 (transformed with pROK Ⅱ-ThZFP3), RNAi-silencing ThZFP3 plants (transformed with pFGC5941-ThZFP5), and control plants (transformed with empty pROK Ⅱ). DAB, NBT, and Evans Blue staining showed that compared with control plants and RNAi-silenced ThZFP3 plants, plants overexpressing ThZFP3 has lowest ROS content, and suffers lowest cell membrane damage. These results suggested that overexpression of ThZFP3 significantly improved abiotic stress tolerance of plants. Physiological indexes sush as water loss rate, chlorophyll content, proline content, H2O2 content, MDA content, POD and SOD activity were measured to analysis the stress tolerance in the three kinds of plants. The results showed that, the water loss rate, ROS and MDA content in transgenic plants overexpressing ThZFP3 were lowest,and the overexpression plants showed highly increased chlorophyll content, proline content, SOD and POD activity. These Indicated that overexpression of ThZFP3 in transgenic T. hispida plants increased the reactive oxygen species (ROS) scavenging capability, decreased cell membrane damage, and then improved the plants’ tolerance to stress.