| ?-GGT(gamma glutamyl transpeptidase)(gamma-glutamyltranspeptidase,?-GGT),also known as gamma glutamine transferase,is widely distributed in living organisms.At present,the existing research results showed that E.coli and B.subtilis can take advantage of GGT gene in theanine(N-ethyl-gamma-L-glutamine)synthesis,and the conversion rate can reach upto 60% with short reaction time and no need of ATP participation.Because theanine has special healthcare function and pharmacological effects,recombinant bacterical GGTs have been used to produce theanine in a large scale.Theanine,as a characteristic component of Camellia sinensis,theacea family and also reported in other species of same family.Other than plant species,theanine reported in prokaryotic organism(mushroom).70% of total was found in fresh tea shoots whereas,1%-2% free amino acid was noticed in dry weight of tea leaves,but the mechanism of theanine syntheisis in tea plant has not been revealed till now.GGTs in microorganism can be used to synthesize of theanine in massive.However,the study about the mechanism of GGTs in tea plants was not yet.In this study,we used "Shuchazao" tea variety as a test material and cloned CsGGTs isolated from the tea plant and analyzed the functions;we also detected the expression of the GGTs in different organs of the tea plant organs.The results are as follows:(1)Based on the GGT homologous sequences obtained from the constructed cDNA library and we cloned the full lengths of two transpeptidase genes from "Shuchazao"(named as CsGGT2 and CsGGT4,respectively).Homologous comparison analysis showed that the CsGGT2 has high homology with GGT1 and GGT2 from Arabidopsis and turnip,and CsGGT4 has high homology with GGT4 of Aabidopsis and GGT3 of radish.(2)qRT-PCR was used to detect the expression of CsGGT2 / CsGGT4 in different organs such as,the bud,the fisrt leaf,the second leaf,the third leaf,the fourth leaf and the fifth leaf,root,stem gene expression were studied and the expression results showed that leaf CsGGT2 expression quantity is the fisrt leaf > bud > stem >the second leaf,the third leaf,the fourth leaf,the fifth leaf and root,CsGGT4 expression quantity is stem > the fisrt leaf > bud > the second leaf,the third leaf,the fourth leaf,the fifth leaf,root.(3)The content of theanine in different organs of tea plant was detected by HPLC.The results showed that root> shoot> first leaf,stem> second leaf,third leaf,fourth leaf and fifth leaf.(4)Prokaryotic and eukaryotic two heterologous expression methods were used to detect the CsGGT2 and CsGGT4 activities.In prokaryotic expression,we tested the crude and pure enzyme activities of CsGGT2 and CsGGT4,respectively.The crude enzyme gamma pancreatic acyl transpeptidase activity was detected and pH optimization study was carried out to find the suitable pH for synthesis of crude enzyme.We tested different pH(5,6,7,8,9,10)of CsGGT2 and CsGGT4 crude enzyme activity,among them pH = 10 optimum for crude enzyme activity of ?-glutamyltranspeptidase in both CsGGT2 and CsGGT4.The HPLC quantification analysis was carried out and the results were showed that the highest theanine content was alkaline condition.LC-MS-MS analysis results were reaveled that the theanine generated content of tea plant CsGGT2/CsGGT4 strains crude enzyme is higher than the control.At the same time,we used the roots of agrobacterium K599 heterologous transformation method to detect the strains of soybean CsGGT2 and CsGGT4 activity,according to the results,after feeding different concentrations of ethylamine hydrochloric and glutamine and take a sample for examination and test in different time periods,the HPLC detection,transgenic strains were not theanine is generated. |
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