Study On GABA Regulation Of Cold Resistance In Winter Wheat

Wheat（Triticum aestivum）has high nutritional value and is widely cultivated,and plays an important role in wheat production in China to ensure food security supply.There are two types of wheat,winter wheat and spring wheat,and the quality of winter wheat is better than that of spring wheat.In the alpine regions,including the eastern provinces,only spring wheat could be grown for a long time due to the limitation of temperature until 2016 when the first winter wheat variety that could be grown in alpine regions,Dongnongdongmai1（Dn1）,was bred with a return rate of more than 85%,breaking the boundaries of winter wheat cultivation and changing the cultivation system from one year to two years to three years.It has improved the land utilization rate.Therefore,it is of theoretical and practical significance to explore the cold resistance gene resources of Dn1,a winter wheat variety with strong cold resistance,and to explore its cold resistance mechanism to enrich the theory of crop cold resistance and to select and breed new cold resistant crop varieties.γ-Aminobutyric acid（GABA）is a non protein amino acid involved in metabolism,which can enhance plant stress resistance by enhancing antioxidant enzyme activity,altering carbon and nitrogen metabolism,regulating plant hormones,and regulating signal transduction.In the early stage,the research team analyzed the transcriptome database of Dn1 tiller node responding to low temperature,and preliminarily screened that multiple genes in the GABA metabolic pathway were differentially expressed under low temperature,but which genes were related to the strong cold resistance of winter wheat was still uncertain.In this study,the key cold resistance genes in Dn1were further screened by using the weak cold resistance variety Jimai 22（J22）as the control,and functional verification was carried out on Arabidopsis thaliana.Finally,the cold resistance function of GABA was confirmed by applying GABA to 10 different winter wheat varieties with different cold resistance（water treatment as the control）.The main findings were as follows:（1）Eight GABA metabolic enzyme genes（Ta PAO、TaDAO、Ta ODC、Ta ADC、Ta ALDH、Ta Glu DH、Ta GAD and Ta GLYR）responding to the differential expression of low temperature were preliminarily screened according to the previously constructed transcriptome database of Dn1 at low temperature（5℃,-10℃,-25℃）.It is preliminarily believed that GABA metabolism responds to low temperature.（2）q RT-PCR was used to detect the expression patterns of genes related to GABA pathway and polyamine degradation pathway（Ta GAD,Ta Glu DH,Ta GABA-T,Ta GLYR,Ta AAT,TaDAO,Ta ODC,Ta ALDH,Ta PAO,Ta ADC）in wheat tillering node samples treated with natural cooling（5℃,0℃,-25℃）and indoor low temperature（5℃,0℃,-10℃）at various temperature nodes,and compared with weakly cold resistant wheat variety J22,analyzing its cold resistance function,the results showed that the expression level of TaDAO in the polyamine degradation pathway showed a gradually increasing trend with decreasing temperature under both field and indoor treatments,and showed the highest expression level compared to other genes in the metabolic pathway.Therefore,TaDAO was identified as a key gene for cold resistance in the Dn1 GABA metabolic pathway.（3）Cloning of the full CDS length TaDAO sequence,insertion into p CAMBIA230035Su,and a construction of TaDAO vector（p CAMBIA230035Su-TaDAO）,Agrobacterium mediated flower soaking method transfection into Arabidopsis（Col-0）,screening and identification,obtained OE-TaDAO Arabidopsis T₂generation plants.（4）Low temperature treatment was performed on TaDAO overexpressed transgenic Arabidopsis plants to identify their cold resistance.The results showed that there was no significant difference in phenotype among transgenic plants at 4℃,-10℃,WT wilted and the leaf area was significantly smaller than that of overexpressing TaDAO plants;After room temperature recovery culture,the recovery rate of overexpressed TaDAO plants was higher than that of WT,while WT leaves showed significant wilting and withering.The detection of cold resistance physiological indicators showed the relative conductivity,MDA and proline content,and ROS content of transgenic plants overexpressing TaDAO in Arabidopsis were significantly lower than those in WT,while the activities of SOD,POD,and CAT were significantly higher than those in WT.（5）The TaDAO expression levels of 10 different cold resistant winter wheat varieties treated with exogenous GABA under low temperature stress were significantly higher than those of the water control group.In summary,this study analyzed and validated the expression patterns of key enzyme genes in GABA metabolism under Dn1 low temperature stress,and found that TaDAO exhibited the highest expression level compared to other genes in the polyamine metabolism pathway of GABA synthesis.The heterologous expression strategy was applied to obtain TaDAO transgenic Arabidopsis homozygous plants,thereby verifying the cold resistance function of TaDAO.The preliminary revelation of a new mechanism of GABA mediated plant response to low temperature stress provides a new pathway for the study of the regulatory mechanism of Dn1 cold resistance,and has important guiding significance for the application of molecular breeding technology to cultivate new winter wheat varieties with strong cold resistance.