Transcription Factors Expression Pattern And Functional Analysis By Exogenous ABA In Winter Wheat Under Low-temperature Stress

Dongnongdongmai1（Dn1）is the first strong cold-resistant winter wheat（Triticum aestivum L.）variety,which is safe in winter in Heilongjiang Province.A large number of studies have shown that the physiological and biochemical reactions of plants to cold are caused by the gene expression changes caused by low temperature.So far,many genes related to cold resistance have been cloned and their cold resistance has been enhanced.An important role in solving practical problems of production also has a wide range of applications.As an important signaling regulator,ABA plays a key role in plant regulation of abiotic stress.In this study,selecting the six transcription factor genes that are related to stress resistance of Dn1.The transcriptional level was whether by ABA and low temperature induced regulation under natural cooling（5℃,0℃,-10℃,-25℃）.Therefore,this experiment will explore the effects of 6 transcription factors under low temperature stress to determine its relative expression level at low temperature by quantitative method,and make bioinformatics prediction analysis,then subcellular localization of TabZIP1,clone and construct TabZIP1 overexpression vector was transfected into Arabidopsis thaliana and transient expression of TabZIP1 pattern analysis.The study also can complement transcription factor regulation of winter wheat cold-resistant mechanism.The results are as follows:（1）Real-time fluorescence quantitative RT-PCR analysis of gene expression levels of transcription factors in natural fields at 5℃,0℃,-10℃ and-25℃ in tilling nodes of Dn1,which revealed that the gene expression levels of six transcription factor genes（TabZIP1,TaWabi5,TaMYB1,TaMYB80,TaNAC2 and TaWRKY80）in tiller nodes reached the highest at-10℃,the results indicate that the temperature of-10℃ is the key point of cold stress-responsive transcription factor gene response to low temperature regulation in Dn1.After exogenous ABA treatment,except for TaMYB1,the expression levels of the other 5 transcription factors reached the highest at-25℃,which TabZIP1 increased most significantly.（2）To construct fusion GFP green fluorescent protein and pCAMBIA1301 expression vector.Agrobacterium infects the lower epidermis cells of tobacco leaves and subcellular localization of TabZIP1 protein.The subcellular localization of TabZIP1 showed that it was localized in the nucleus,which was consistent with the putative role of transcription factors.（3）TabZIP1 bioinformatics analysis:TabZIP1 full-length 1167 bp,encoding 388 amino acids,TabZIP1 has a conserved BRLZ motif domain,its expression product is hydrophobin,no signal peptide or transmembrane domain;TabZIP1 protein secondary structure is a mixed type;the homologous amino acid protein multi-sequence comparison indicates that the TabZIP1 protein contains typical basic amino acid sequence characteristic domains;phylogenetic analysis shows that:TabZIP1 is closely related to bZIP clusters of monocotyledons,in which wheat and barley are gathered.（4）The TabZIP1 gene of Dn1 was cloned and its length was 1164 bp.It was linked to the plant expression vector and the overexpression vector pCAMBIA1301-35S of TabZIP1 was successfully constructed.The Arabidopsis thaliana was impregnated with pollen tube pathway and overexpressed transfer TabZIP1 plants.Seeds were collected from the transfected Arabidopsis and screened to homozygous seeds in the T₃ generation.The seedlings that had grown to about 4 weeks were treated at low temperature.Physiological indicators showed that the chlorophyll content in the transgenic ox-TabZIP1 strain was higher than In the WT and bzip1 plants,the MDA content and relative conductivity of the transgenic ox-TabZIP1 strain were lower than those of the WT and bzip1 plants.