Cloning Of Glutamate Decarboxylase (GAD) And Regulation Function Of ?-aminobutyric Acid (GABA) In Woody Plant

Salt stress,one of the important abiotic stress factors of plants,could have a physiological effect on plant growth and development,or cause serious ecological environmental problems.It would be caused a variety of physiological changes and adaptive responses by salt stress.The processes of these changes and responses were regulated by the perception and transmission of signal molecules to cause the expression of the related genes and would be activated in adjustment and resistence of the damage caused by salt stress.It has been found that the ROS and plant hormones,as signaling molecules were involved in regulation and physiological responses under abiotic stress.Glutamate decarboxylase?GAD?is an enzyme which was encoded by GAD gene widespread in living organisms and exist some important functions in vivo.Glutamic acid could be catalysed by GAD enzyme to generate a small molecule,four-carbon non-protein amino acid named as?-aminobutyric acid?GABA?.It has been proved that GABA plays a significant role in mediating cytoplasmic PH,nitrogen storage,growth and development,metabolism of carbon,molecular signal regulation of stress resistance and so on.The previous studies on the expression patterns of GAD gene family under stress had been already reported,but the roles in stress resistance mechanisms still need a further study between GAD gene and GABA involved in reactive oxygen,hormones and other signal substance at the molecular level.In our study,two GAD genes from Caragana liouana were cloned by RACE technology for the first time.Then ClGADs gene expression pattern and determination of GABA levels were analysed in the seedlings of Caragana liouana treated by salt and exogenous ABA.On the other hand,transcriptomic analysis were performed in the model plant Populus tomentosa separately treated with high?10 mM?and low?0.25 mM?concentrations of GABA under 200 m M NaCl stress.Bioinformatic analysis combined with qRT-PCR experiment were made to determine the gene differential expession of reactive oxygen species,hormone synthesis,signal transduction.The main results were as follows:1.Two full-length cDNA sequences of ClGADs genes were cloned from Caragana liouana by RACE amplification technology according to the transcriptome sequencing information.The cDNA of ClGADs genes were named as ClGAD1 and ClGAD2.Alignment analysis of mRNA sequences showed that the proteins encoded by ClGAD1 and ClGAD2 contained a pyridoxal decarboxylase conserved domain.Alignment analysis of amino acid sequences identified that there were a highly conserved lysine?Lys?site and a tryptophan?TRP?binding site in C-terminal,which play an important role in CaM binding to GAD and catalyzes glutamic acid to GABA.It was showed that,ClGAD1 and ClGAD2 existed in different evolutionary positions by amino acid sequence phylogenetic tree analysis2.The expression patterns of Cl GAD1 and ClGAD2 of Caragana liouana were analyzed by NaCl stress treatment and exogenous abscisic acid?ABA?.It was found that expression patterns of ClGAD1 and ClGAD2 were absolutly different in roots and leaves,but relative expression level of ClGAD2 was much higher than that of Cl GAD1.The result showed that there existed different sensitivity between the two members of GAD genes.Another experiment on Caragana liouana treated by exogenous ABA indicated that the accumulation of endogenous GABA would be significantly increased with low concentration of exogenous ABA in a short period of time.The expression level of Cl GAD1 was significantly lower than that of ClGAD2 in leaves,and expressions of ClGADs in leaves were more sensitive than those in roots.The results suggested GAD gene family members in Caragana liouana play a different role in its response to salt stress.3.After the experiment of Populus tomentosa seedlings with exogenous GABA under salt stress,the physiological indicators were measured,and the gene expression profiling were generated.It was found that the concentration of GABA was rapidly increased in leaves and the expression of gene related to ROS were markedly inhibited with exogenous 0.25 mM GABA within 6 h.0.25 mM GABA also had a great impact on gene expression of synthesis and signal transduction of ABA,ethylene?ETH?,and other hormones.Extrogenous GABA actually played a regulatory role to a certain extent in expression of genes involved in ROS and hormones metabolism.This is closely related to the cytoplasmic pH,Ca²⁺signal pathway and transcription factor signal transduction,whose functional mechanisms need to be further explored.