| Gamma aminobutyric acid(GABA)exsit in many organisms,and it is important for physiological function of animals,plants and microorganisms.GABA was mainly catalyzed by glutamate decarboxylase(GAD)in plants.In recent years,researcher devote to increase GABA content of tea in the stressed condition through improve proscessing technology to obtain "Gabaron tea".However,there are few research on the GABA metabolism and regulation model of tea plants,including how to improve the GABA content in tea research through variety breeding and improvement.The aim of this paper is to clone GAD1 gene in tea plant,verify GAD1 function and analyze its distribution in different organs of tea by qRT-PCR.Meanwhile,the pormote of GAD1 was cloned,then based on the analysis of the promoter of bioinformatics,the regulatory factor of GAD1 gene was obtained.The results not only contribute to understand the GABA metabolism and regulation mode in tea,but also clarifying the enrichment principle of GABA in tea,which apply theory for improving the varieties breeding and cultivation measures on the high GABA content of tea cultivar.1.Detecte GABA content in different leaves of teaThe results showed that the GABA content in buds is higher than in leaves,among which the highest is in the buds of one bud and one leaf stage.Meanwhile,GABA content reduced gradually with the development of leaves,among which the lowest is in the first leaf of one bud and two leaves stage.2.The clone of CsGADl geneThe complete sequence of CsGAD1 was 2054 bp,the open reading frame(ORF)was 1482bp which encoding 493 amino acids.GAD1 was a cytoplasmic protein,whose molecular weight was 55.49 kD and pI was 5.44.It was a non-secreted protein,and was not transmembrane domain.GAD1 was also a hydrophilic protein including 23 probable phosphorylation sites.Bioinformatics analysis showed that it contains several secondary structure,a-helix structure and ?-pleated sheet structure were accounted for 13.62%,19.25%,respectively,randon coil and other structures were accounted for 67.14%.The analysis result of the genetic and molecular evolution showed that the GAD1 has high homologue with PgGAD,MzGAD and VvGAD.3.Prokaryotic expression of analysis CsGADlThe pMAL-C4X-CsGAD1 expression vector was constructed,and the fusion expression vector was transported to Ecoli.Rosetta good.After IPTG induction,SDS-PAGE result showed that the molecular weight size is close to 55kD,which is consistent with the predicted protein.However in vitro enzyme activity assay,the enzyme activity was not detected.4.Cloning gDNA of CsGAD1 geneThe gDNA sequence of CsGAD1 gene was amplified by the TAIL-PCR technology,And the length was 4619bp.Sequence comparison revealed that it contained 7 exons and 6 introns.5.The expression of GAD1 in different tissues of tea plantThe expression of GAD1 of different tissues of tea plant by qRT-PCR.The results showd that the highest expression level of GAD1 in the root of tea plant.Other tissues have little different expression content.The expression quantity have a bit different in the single bud,the flowers,the epicotyl and fibrous root To compare with other tissues,the expression level is more lower of the leaves in the tea plant.6.The cloning and analysis of CsGADl promoter and functional verification of the related cis-elementsThe sequence of CsGADl promoter was obtained though Genome-walking PCR method,and the length was 1500 bp.The fragment was analyzed by PlantCare software,the result showed that there are several cis-acting elements associated with gene expression,and the transcriptional start site was located at the 193bp upstream of the ORF box.The elements includes light induced LAMP element,temperature response elements LTR and ABA stress components ABRE and so on.CsGAD1 expression in tea seedling was detected after ABA approach,the result showed that ABA induced the expression of CsGADl gene of tea seedling roots and leaves,and there are different expression patterns in different tea tissues. |
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