Physiological And Molecular Mechanism Of Winter Wheat TaGAD And TaGABA-T In Response To Low Temperature Stress

Dongnongdongmai 1(Dn1)is the first wheat variety with strong cold-resistant that can survive safely in high cold area of Heilongjiang Province.At present,many of its related cold resistance genes have been studied.These studies are extremely important for the physiological and molecular studies of winter wheat’s cold resistance in Northeast China.The γ-aminobutyric acid metabolic pathway(GABA)is a branch of the tricarboxylic acid cycle(TCA),which provides the intermediates for TCA.When plants are subjected to abiotic stress,GABA accumulates in large amounts in plants.Glutamate decarboxylase(GAD)and aminobutyrate transaminase(GABA-T)are two key enzymes in GABA pathway,and its function is of great significance to improve the cold resistance of Dn1.In this study,the Dn1 planted under the natural cooling conditions in the field was used as the basic material to determine the gene expression levels of TaGAD and TaGABA-T in the tillers and leaves at four temperature nodes(5 ℃,0 ℃,-10 ℃,-25 ℃),And isolate the TaGAD and TaGABA-T genes from Dn1,perform bioinformatics prediction analysis,further construct an overexpression vector,overexpress in Arabidopsis thaliana,and detect its cold resistance-related physiological indicators,including MDA,electrical conductivity As well as GAD and GABA-T enzyme activity detection,and the expression levels of two genes in Arabidopsis overexpressed were analyzed.The research results are as follows:(1)The TaGAD protein sequence consists of 487 amino acids and TaGABA-T is composed of 508 amino acids.Subcellular localization indicates that this protein is a weak acid unstable protein in the mitochondria.The prediction of the secondary structure of the two proteins is that α-helix(H)and random coil(C)account for most of them,and they have strong hydrophilic ability,and both belong to extramembrane proteins.(2)Under cold stress,TaGAD and TaGABA-T can enhance the cold resistance of winter wheat;after ABA treatment,the expressions of TaGAD and TaGABA-T in Dn1 tiller nodes and leaves increase,Under cold stress,ABA positively regulates the expression of TaGAD and TaGABA-T.(3)The full length of the CDS sequence of TaGAD and TaGABA-T was cloned,respectively 1762 bp and 1590 bp,Overexpression vector was successfully constructed and overexpressed onto Arabidopsis.(4)Sampling wild-type Arabidopsis thaliana and overexpressing Arabidopsis plants at four temperatures(normal temperature,4 ℃,0 ℃,-10 ℃),and tested physiological and molecular indicators.The results showed that TaGAD and TaGABA-T Can improve the cold resistance of Arabidopsis.At the same low temperature,Arabidopsis overexpressed TaGAD and Arabidopsis overexpressed TaGABA-T had lower MDA and relative electrical conductivity than wild type;under cold stress,the expression of TaGAD and TaGABA-T in Arabidopsis overexpressed plants With the decrease of temperature,the activity of GAD and GABA-T enzymes has the same changes as gene expression.This study initially explored the role of TaGAD and TaGABA-T in the cold resistance of Dn1,and also provided support for the ABA signal regulation mechanism at low temperatures,and provided a theoretical basis for further exploration of the cold resistance mechanism of TaGAD and TaGABA-T.