Functional Analysis Of Saline-Alkaline Stress Responsive Genes TaWRKY46 And TaNRT1.2 From Wheat

Soil salinization has become more serious in recent years,which cause severe effects on plant growth,agricultural production and food supply.Saline and alkaline usually goes hand by hand in soils,and salt stress generally results in osmotic stress and ion damage to plants.In addition to the above stresses,alkaline stress can also affect the pH of soil which impede the normal absorption of ions by plants,disrupt intracellular ion balance and damage plant roots and photosynthetic systems,it also causes great harm on the plant's normal growth.Therefore,it is necessary to exploit saline-alkali stress response mechanisms to address the damage caused by saline-alkali stress on plants.A series of wheat introgression lines were obtained by asymmetric somatic hybridization between common wheat Jinan 177(JN177)and tall wheatgrass,and two saline-alkali tolerant introgression lines designated SR3 and SR4 were selected from them.Experiments in field and the lab both indicated that SR3 is superior for salinity tolerant while SR4 for alkalinity tolerant.The next generation high-throughput sequencing technique was used to analyze the transcriptomes of SR3 and SR4,and two saline-alkali responsive genes were selected for further functional analysis..The first one is a WRKY family transcription factor gene TaWRKY46 whicht was regulated by salt stress in SR3,and the second one is a nitrate transporter family gene TaNRT1.2 which was up-regulated in SR4 under alkali stress.1.Functional Analysis of Wheat Salt Responsive Gene TaWRKY46We cloned the ORF of TaWRKY46 with a length of 669 bp from SR3.TaWRKY46 is a member of WRKY superfamily,which exhibits different expression patterns under various abiotic stress treatments.TaWRK46 is mainly located in the nucleus,and it has in vitro transcriptional activation activity in yeast.TaWRKY46 was transformed into wild-type Arabidopsis thaliana(Col-0)and two TaWRKY46 transgenic lines(OE)with high expression level were screened out.Under normal conditions,the growth of TaWRKY46 OE lines showed no difference with wild-type plants.Under salt stress conditions,the leaf area of the TaWRKY46 OE lines was significantly smaller than the wild type plants,which indicated that TaWRKY46 OE lines were sensitive to salt stress.Under oxidative stress conditions,the leaf area of TaWRKY46 OE lines was significantly larger than the wild type plants,which showed TaWRKY46 OE lines exhibit significant resistance to oxidative stress.The results of DAB and NBT staining showed that TaWRKY46 OE lines accumulated less H2O2 and O2-content than wild plants.The expression level of FeSOD3 and Cu/ZnSOD3 were obviously up-regulated under control conditions and the expression level of ROS synthesis-related genes rbohC,rbohD,and rbohE were down-regulation,therefore we speculated that TaWRKY46 might increase plant oxidative stress tolerance by repressing ROS accumulation.TaWRKY46 OE lines displayed sensitivity under ABA treatment.Real-time quantitative PCR analysis showed that the expression levels of ABA1,ABA2 and ABI5 in TaWRKY46 OE lines were significantly higher than those in the wild type.We speculate that ABA signaling may play an important role in TaWRKY46-mediated salt stress response.2.Functional Analysis of Wheat Alkali Responsive Gene TaNRT1.2We cloned a 471 bp TaNRT1.2 ORF from SR4.It is a nitrate transporter that exhibits different expression patterns under various abiotic stress treatments.TaNRT1.2 was heterologously expressed in wild-type Arabiopsis thaliana(Col-0)and two homozygous transgenic lines(OE)were selected for further analysis.Under normal growth conditions,TaNRT1.2 overexpression does not affect plant growth and development.Under the alkali stress and oxidative stress,the relative leaf area and main root length of TaNRT1.2 OE lines was significantly smaller than that of the wild type plants,which indicated that TaNRT1.2 OE lines were sensitive to alkali stress and oxidative stress.The results of DAB and NBT staining showed that the ROS content of TaNRT1.2 OE line was significantly higher than that of wild type.qRT-PCR results showed that the expression of ROS synthesis-related genes rbohC,rbohD,rbohE,rbohF,and rbohG was significantly up-regulated and the expression of Cu/ZnSOD2,CAT2 and GPX2 genes was down-regulated.In addition,in the presence of ABA,the growth of OE lines showed insensitivity to ABA.The analysis of marker genes in ABA signaling pathway revealed that ABI1,ABI2,and ABI5 were up-regulated in TaNRT1.2 OE lines.We speculate that ROS signaling and ABA signaling may play an important role in the responses of TaNRT1.2 to alkaline stress.