Cloning The GAD Genes From Tea Plant And Exploring Accumulated Mechanism Of GABA In Camellia Sinensis(L.)O.Kuntze

Gamma aminobutyric acid(GABA)as an important non-protein amino acid exists in many organisms which has multiple physiological functions for aninmal,plants,and microorganisms.Glutamic acid decarboxvlase(GAD)is considered a key enzyme in the process of biosynthesis of GABA.In recent years,researcher more-focus on how to increase GABA content to make“Gabaron” tea through improve processing technology.However,there are a few of study about the GABA metabolism and regulation mechanism inside of tea plants.This research focus on members of GAD gene family in tea tree,basing on early cloned CsGAD1 gene,we keep on cloning 2 GAD genes and named CsGAD2 and CSGAD3.Through the bioinformation method,we compare their function and expression pattern,analyse the relative expression of GAD under adversity stress,and compare their gene expression pattern at transcription level and protein level under anaerobic stress.This study attempt to reveal the metabolism and regulation mechanism about how to enrich GABA in tea plant then provide theoretical basis for improving the cultivaltion model to increase GABA in tea plant.The main contents are as follows:1.The clone of CsGAD2?CsGAD3 geneThe full-length cDNA sequence of CsGAD2 was 1,872 bp,the open reading frame(ORF)was 1,539 bp,which encoding 512 amino acids.The full-length cDNA sequence of CsGAD3 was 1,833 bp,the open reading frame(ORF)was 1,482 bp,which encoding493 amino acids.GAD2?GAD3 was a cytoplasmic protein,whose molecular weight was 58,027.0Da?55,757.20 Da.They were all hydrophilic protein and have not transmembrane domain.Randon coil and other structures were their main secondary structure.CsGAD1 and CsGAD3 had tryptophan in C terminal amino acid in addition to CsGAD2.Therefore,CsGAD2 may not be combined with CaM.CsGADs had high homologue with ginseng,vitis vinifera and solanaceae plant.2.Tissue specific expression of CsGAD2 and CsGAD3The expression of CsGAD2?CsGAD3 in different tissues of tea plant were detected by qRT-PCR.The results showd that the relative expression level of CsGAD2 of leaf were the highest,of flower were the lowest,other tissues had a little different in expression content.The CsGAD3 gene has the highest expression level in flower,the expression level of other tea tissues were more lower than in flower.3.Analysis of CsGADs gene expression in response to ABA,MeJA,and SACsGADs gene were induced expression under the stress of 100?M ABA?100?M SA?100?M MeJA,the results of qRT-PCR showd that CsGADs gene were obvious induced under the sress of ABA,SA,except MeJA treatment.The function were as consistent with some cis-elements of CsGAD1 promoter.4.Prepared the polyclonal antibody against CsGAD1The Recombinant plasmid pET-28a-CsGAD1 was induceded and purified,then the polyclonal antibody against CsGAD1 was final prepared.The Western blotting analysis pointed out that the prepared antibody had favorable sensitivity.5.Analysis of CsGADs change induced by anaerobic stressCsGADs gene were induced expression under the anaerobic stress.The content of GABA reached the highest under the Carbon dioxide treatment in six hours,and CsGAD1 gene had rapid response under the Carbon dioxide treatment in half an hours by qRT-PCR technology.The function were consistent with cis element of CsGAD1 promoter.In the transcription and translation level were not same way,the accumulation of protein was latter.