Functional Analysis Of Wheat Transcription Factor TaC2H2ZF Involved In Salt-alkaline Stress Response

Wheat is one of the most important cereal crops in the world,supporting about 40%of the world's population.Soil salt-alkalinization is a major constraint to plant geographical distribution and crop yield.Currently,approximately 7%of the world's land surface is threatened by salinization.It's urgent to analyze the mechanism of plant tolerance to alkalinity,especially for crops.The salt-tolerant traits could be successfully transferred from alien species to common wheat by asymmetric somatic hybridization.In our laboratory,the asymmetric somatic hybrydization between common wheat Jinan 177(Triticum aestivum L.)and intergeneric grass(Thinopyrum ponticum)had been carried out and the hybrid wheat progeny lines,such as Shanrong No.4(SR4)and Shanrong No.3(SR3)were first obtained in the world.In recent years,the application of high-throughput sequencing technology has dramatically progressed our knowledge of salt-alkalinity adaptation mechanisms in wheat.In this study,an alkaline stress up-regulated TaC2H2ZF gene,which containing an approximately 408 bp ORF,was isolated from sequencing-based transcriptome profiling of SR4 in our laboratory.It encodes a C2H2-type zinc finger protein with two typically conserved zinc finger domains and a conserved EAR inhibitory domain,which exhibit different expression patterns under abiotic stress treatment including saline-alkali treatment.Transient expression analysis showed that TaC2H2ZF localizes to the nucleus by using the subcellular localization assay.The transactivation ability of TaC2H2ZF was analyzed using a yeast assay system.It had no in vitro transcriptional activation ability by growing on a medium lacking histidine and leucine but containing 10 mM 3-aminotriazole(3AT)assay,deduced that it is possibly because of the existence of EAR inhibition domain.The pRI101-AN-TaC2H2ZF recombined plasmids were transformed into Agrobacterium tumefciens EHA105.Arabidopsis were transferred by flower-dipping methods,expecting to gain high expression level transgenic plants.After selection by 50 mg/L Kan,the regeneated plants were obtained and PCR analysis of them showed they were all PCR positive lines.In the end,two TaC2H2ZF-heterologous transgenic Arabidopsis overexpression lines(OE1 and OE2)were obtained.To confirm whether the overexpression of TaC2H2ZF could improve salt or alkaline tolerance of transgenic Arabidopsis,The homozygous T3 lines were examined under salt or alkaline stress.As expected,they showed enhanced resistance to salt or alkaline stress,with the concentrations of proline(Pro),malondialdehyde(MDA)and the activities of peroxidase(POD),catalase(CAT)and superoxide dismutase(SOD)under salt or alkaline stress.Contents of Pro was significantly higher in OE lines than in the WT and MU,suggesting that the cytoplasm of OE lines was more stable and effective in preventing abiotic stress damage.Also,under salt or alkaline stress,the MDA levels of OE1 and OE2 lines dropped more rapidly than in the WT and MU,suggesting that less cell membrane damage occurred in OE lines than in the WT and MU.To obtain salt tolerant wheat,the TaC2H2ZF gene was introduced into a wheat variety'Yangmai 20' under Agrobacterium inoculum to shoot apical meristem of wheat seedling.T0 transgenic plants were obtained by the PCR identification.The average transformation efficiency of T0 generation was 5.8%.The salt(200 mM NaCl)or alkaline(80mM NaHCO3:Na2CO3 =9:1)tolerance analysis of the T1 generation transgenic lines shows that the proline content in the transgenic wheat was higher than in control,the MDA content was lower in the transgenic wheat than in control.With the increasing of salinity or alkalinity,the activity of POD and CAT were higher in the transgenic wheat than in negative control.