| Theanine, a unique non-protein amino acid found almost exclusively in tea plants, isthe main component responsible for the ‘umami’ taste of ‘green’ tea, and has variousphysiological functions toward animals including humans. At present, the synthesis andmetabolism of theanine is still not fully understood and little is known about the genes inthe biosynthetic pathway. Therefore, research on the functional genes and regulationfactors involved in theanine synthesis would make a foundation for further understandingthe pathway of theanine metabolism and producing theanine through transgenic techology.Based on the conclusion that theanine is synthetized in the young roots of a tea plant,Camellia sinensis （L.） O. Kuntze cv. Longjing43, fed with precursors of ZtNH2·HCl andsodium glutamate to activate the pathway of theanine synthesis, was used as the material toconstruct a cDNA library, and partial clones were sequenced to obtain ESTs. Meanwhile,the effects of nitrogen compounds which were added into the culture media on thesynthesis of theanine were systematically studied. The effects of shading and shadingcombination with chemical agents on the content of theanine were also examined, and theexpressions of the unigenes putatively involved in theanine metabolism in tea callus withobvious difference in theanine contents were further analyzed using qRT-PCR （real timequantitative PCR）. This will make a foundation for isolation of functional genes andregulatory factors involved in metabolism of nitrogen and theanine synthesis by usingcDNA microarray or DGE （digital gene expression） in the future. The main results are asfollows.1. We tried many published protocols for RNA isolation from the young roots of teaplants, including the method used for extraction RNA from tender tea leaves, but failed toyield high-quality RNA. Therefore, a modified CTAB method was developed, and highquality RNA was obtained，which was suitable for cDNA library construction.2. Based on SMART technology, a cDNA library was constructed for the tissue ofyoung roots of the tea plant （Camellia sinensis）. The results showed: without amplicationof the library, the cloning efficiency was5×105colonies/μg, the recombinant rate was92%,and the average size of inserted cDNA fragment was over1kb. To generate ESTinformation,5160clones were single-pass sequenced and4860valid sequences from100bp to1850bp in length were generated with average size of617bp after vector trimmingand discarding the sequences less than100bp （some nucleotide sequences reported here are available in the GenBank dbEST database under the accession numbersGE652554–FE861258）, in which648contigs and1161singlets were obtained after initialassembly with Phrap program. The unigenes were subjected to BlastX against thenon-redundant protein sequence database in NCBI and annotated with Blast2go. Based onthe results of BlastX analysis,1041（57.6%）unigenes were found to significantly match theproteins with known and putative function, and282unigenes （14.65%） matchedsignificantly with those having unknown function in the non-redundant protein database.The remaining486unigenes （26.9%） showed no significant homology to any protein in thepublic databases, indicated that they might be new genes. The1041unigenes of known andputative functions were further classified into13categories described by the EUArabidopsis Genome Project. Among them,136known and putative functional unigenes（13.06%） were grouped into the ‘protein synthesis’ category;92unigenes （8.84%） wererelated to the ‘defense’ category and90unigenes （8.65%） were classified into the‘transporter’ category. The secondary metabolism-related category was one of the mostdiverse categories, corresponding to4.52%of the functional genes in the study. In thiscategory, the most abundant sequences were the functional genes encoding cytochromeP450, with a total of seven unigenes. In addition, unigenes encoding flavonol3-O-glucosyltransferase, flavonoid3-glucosyltransferase, p-coumaryl-CoA3’-hydroxylase,anthocyanin5-O-glucosyltransferase, coniferyl alcohol acyltransferase andcaffeoyl-CoA-O-methyltransferase were also found. These are new genes by BlastXanalysis with the dataset of Camellia sinensis in the GenBank. Kegg pathway analysisshowed that many putative functional genes are involved in the metabolic pathway offlavonoid, isoflavone, phenylpropanoid, terpenoid, anthocyanin and flavonol biosynthesis,and the number of unigenes related to flavones is the most. The unigenes involved in primarymetabolism were the most abundant （17.39%）, among them, a large number of unigeneswere found to be related with amino acid metabolism. The homologues of GS, SAMDC（S-adenosylmethionine decarboxylase）, ADC （arginine decarboxylase）, GGT （gamma-glutamyltranspeptidase） and nitrite reductase, cytosol aminopeptidase glutamine amidotransferaseclass-â… were also found. The unigenes related to amino acid metabolism are maininvolved in the pathways of methonine, alanine, glutamate, arginine, proline, phenylalanineand glutathione. The unigenes involved in intracellular transport, signal transduction,protein degradation and store, energy, transcription, cell structure and growth, andunknown function occupied1.16%,6.63%,4.23%,5.57%,9.70%,7.69%and10.56%,respectively. 3. The effects of nitrogen compounds such as theanine precursors, metabolicintermediate in theanine synthesis and NO（nitric oxide） donor SNP（sodium nitroprusside）on the synthesis of theanine in tea callus were investigated by using HPLC （highperformance liquid chromatography） to detect theanine contents; the accumulation oftheanine in the roots of tea plants with different growing periods after addition of（NH4）2SO4into liquid medium was analyzed; the difference of theanine content in roots ofthe tea plant （Longmenkang cultivar） with shading, shading combination with SA （salicylicacid） or ZtNH2·HCl （ethylamine hydrochloride） and that in tender leaves of differentcultivars of tea plants were also preliminary discussed. Our data indicated that25mMZtNH2·HCl could increase the content of theanine in tea callus significantly, especially inthe tea callus grown from the6thday to the12thday. Therefore, tea calli of the control andtreatment groups could be used as materials for studying the genes correlated with theaninemetabolism for qRT-PCR, cDNA microarray or digital gene expression profile analysis.4. In order to obtain a suitable reference genes for analysis of the related geneexpression in calli of the control and treatment groups,7housekeeping genes from theGenBank and ESTs of cDNA library of the young roots（with complete open readingframe）were chosen for primer design. After analysis the amplification efficiency andprimers specificity, the expression levels of the7housekeeping genes in tea calli of thecontrol and treatment groups at different growth stages were determined, and theirstabilities of expressions were analyzed using geNorm and NormFinder softwares. As aresult, β-actin and GAPDH are suitable reference genes under this condition.5. The expression levels of11selected genes with potential role in theaninemetabolism in tea calli of the control and treatment groups （with addition of ZtNH2·HCl incalli medium） at different growth stages were analyzed using qRT-PCR with gene specificprimers designed with primer premier software （version5）, and the results are as follows.The expression levels of GS1-1, gogat, NIR and GDH2were obviously induced in tea callusof the treatment group at the first3days of the callus growth, indicating that theseunigenes might play a role in assimilation of nitrogen compound in the beginning; thetranscript levels of TS2and GDH2were much higher in the treatment group than in thecontrol group from the3rdday to the6thday of callus growth, which is consistent with theincrease of theanine content in the treatment group at the same growth stage. There was noobvious difference in expression abundance of TS1between the control and the treatmentgroups, indicating TS1and TS2might play different roles in theanine synthesis. |
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