Spatiotemporal Expression Of GAD Family Genes And Their Multiple Mutant Phenotype Analysis In Arabidopsis Thaliana

?-Aminobutyric acid(GABA)is a four carbon non protein amino acid with signal molecular function.However,in plants,the signaling pathways involved in GABA production,transporting and the response of effector cells are not fully understood.In this paper,Arabidopsis thaliana,the model plant was used as the research materials,the vectors of GUS reporter gene,fluorescent expression of promoter nuclear localization and full-length genes were constructed to investigate the spatiotemporal expression patterns of five members of glutamic acid decarboxylase(GAD)family,which are the key genes to control GABA synthesis.Their expression patterns were comprehensively analyzed from the tissue,gene expression and translation level.The main results are as follows:1.DNA sequence alignment analysis showed that the five members of GAD gene family had different numbers of exons and introns,and the similarity ratio of different full-length gene sequence was 12.69%;cis acting element in promoter analysis showed that all the five member genes were rich in CAAT box,TATA box and MYC binding elements,indicating that all had the same regulatory mode.However,all the coding proteins have high frequency of identical amino acid residuals with highly conserved ratio of 63.36%at the protein level,and all have three identical motif sequences,indicating that the enzyme activity of GADs are regulated by the same functional elements.Furthermore,the predicted crystal structure of protein showed that all five members of GADs have the same calmodulin(CAM)binding sequence.2.At tissue level,GUS staining analysis showed that pGAD1-GUS was expressed in roots,stems and leaf veins in seedlings,and partially expressed in pistils;Expression of pGAD2-GUS was lower in 5-day seedling,but higher in 10-day seedlings.GAD2expressed in root,stem and leaf veins,and it expressed in whole inflorescence including petals,sepals,pistils and stamens of the flowering plants;pGAD3-GUS was higher expressed in 10-day seedling;pGAD4-GUS was expressed both in pistils and stamens of mature plants,and it expressed in seedling tissues,but strongly expressed in pistils;However,pGAD5-GUS was not expressed in seedling tissues,but only expressed in anthers.3.At the transcription level,the fluorescent promoter nuclear expression analysis of showed that pGAD1-H₂B-GFP had strong fluorescence distribution in seeds and shoot meristem of seedlings;pGAD2-H2B-GFP had strong fluorescence expression in pistils and shoot meristem center;pGAD3-H2B-GFP had strong fluorescence in seeds,pollens and pericycles of roots;pGAD4-H2B-GFP had strong fluorescence in pistils of stigma and siliques,and the strong fluorescence mainly distributed in the center of shoot meristem.However,pGAD5-H2B-GFP had strong fluorescence in pollens.4.At the protein translation level,the fluorescent expression of the full-length genes showed that pGAD2-GAD2-GFP had strong fluorescence in the pistils;pGAD3-GAD3-GFP had strong fluorescence in the seeds,and had weak fluorescence signals in the shoot meristem and pollens,and strong fluorescence signals in the pericycles of seedling roots;pGAD4-GAD4-GFP had strong fluorescence in the pistils of stigma,siliques and shoot meristem of seedling,whereas,pGAD5-GAD5-GFP has strong fluorescence in pollens.5.Using the gad1 gad2 gad4 gad5 mutant,the quintuple mutant of gad1 gad2gad3 gad4 gad5 were constructed by using CRISPR/cas9 technology.The phenotype analysis showed that carbon-and nitrogen starvation significantly inhibited the growth of Arabidopsis both in wild-type and mutant seedlings,but the mutants was more sensitive to nutrient stress.In summary,the spatiotemporal expression patterns of five key GABA synthesis genes were comprehensively analyzed using GUS and GFP tracking technology in Arabidopsis thaliana,their specific and local distribution of GAD family members revealed that the distribution of GABA signals had the features of regional distribution and long-distance transportation in different tissues in Arabidopsis thaliana.Phenotype of mutant analysis revealed its sensitivity to nutritional stress.This study provides an important information for further understanding GABA signal pathway in plant development.