The Localization And Functional Characterization Of Theanine Synthetase (TSI) And Glutamine Synthetase (GSII-1.1) In Tieguanyin

Theanine is one of the characteristic components of tea.It is a hallmark of tea quality,and generates important economic value and health benefits.The theanine content,however,decreases during tea fermentation process.As a result,mechanistic studies of theanine biosynthesis in tea leaves would help to elevate theanine quantity in tea drinks,improve tea flavor and quality,enhance health benefits,and promote the applications of tea products.Several key enzymes are involved in theanine synthesis pathway,including theanine synthetase and glutamine synthetase.In 2018,a high-quality full genome sequence of the Camellia sinensis var.sinensis（CSS）was reported,which provides the coding information of these key enzymes in the theanine synthesis pathway.Since transgenic technology has not been fully established for tea plants,the function of these theanine synthesis-related genes can’t be investigated by using knock out or over-expression strategies in vivo.Here I established a transient transfection system in Tieguanyin mesophyll protoplasts and studied the subcellular localization of a theanine synthetase gene TSI and a glutamate synthetase gene GSII-1.1,and studied the function of GSII-1.1 in Arabidopsis thaliana.The method developed here is expected to provide a useful technology for the functional characterization of novel tea genes and for increasing the theanine content in tea.The project has three parts.In Part I,a protocol to extract Tieguanyin mesophyll protoplast is developed.Fresh Tieguanyin seedling were used as experiment material.Different enzyme solutions were tested.The quantities,cell viability,and debris contents of the extracted protoplast were measured.Results showed that the enzymatic hydrolysate formula with 1.6%Cellulase,0.4%Pectolyase,0.1%Macerozyme,0.5 M Mannitol,20 m M KCl,20 m M MES（p H=5.7）,100 m M Ca Cl₂,and0.1%BSA yields the highest number of protoplasts（2.09×10⁶/g fresh weight）among all tested formulas.The enzyme hydrolysate formula with1.4%Cellulase,0.4%Pectolyase,0.2%Macerozyme,0.4 M Mannitol,20m M KCl,20 m M MES（p H=5.7）,100 m M Ca Cl₂,and 0.1%BSA results in the best protoplast activity.In Part II,a Tieguanyin mesophyll protoplast transient transfection protocol was created,and the subcellular localization of the TSI and GSII-1.1 were studied.First of all,a Tieguanyin protoplast transient transfection system is established by using a high-expression HBT-TCP20-GFP carrier.Then,primers of the TSI and GSII-1.1 genes were designed according to the reported gene sequence,followed by extraction of total RNA from tea leaves,reverse transcription into c DNA,PCR amplification of TSI and GSII-1.1 gene fragments,and connection to a HBT-GFP-HA carrier.Then HBT-GFP-TSI and HBT-GFP-GSII-1.1were transferred into Tieguanyin mesophyll protoplasts to observe their subcellular localization under fluorescence microscope.Results showed that the TSI and GSII-1.1 were both located in protoplast cytoplasm.In addition,the GSII-1.1 gene was connected to a PCR^TM8/GW/TOPO gateway carrier.And Arabidopsis thaliana 35S promoter driven GSII-1.1-GFP transgenic plants were generated for analyzing GSII-1.1gene’s function in theanine production.The GSII-1.1 was located in the cytoplasm of leaf,root and stem of the transgenic Arabidopsis thaliana.In Part III,the function of the GSII-1.1 was investigated using the transgenic Arabidopsis seedlings.The transgenic Arabidopsis thaliana seedlings were treated with ethylamine hydrochloride for 0 to 9 days and their theanine contents were analyzed by using UPLC-Qq Q MS.Compared with untreated seedlings,the theanine content of the treated transgenic seedlings did not change significantly from 0 to 3days.However,there were significant differences between the treated transgenic seedlings and wild type on the 9th day.Together,the techniques developed here are expect to provide new tools to clarify the biosynthesis mechanism of theanine in Tieguanyin and potentially improve the economic value and health benefits of Tieguanyin.