摘要
茶氨酸系茶树中的特征性非蛋白质氨基酸，是构成茶叶品质的主要成分之一，影响茶叶的滋味品质，具有重要的保健功能和药理作用。目前与茶氨酸代谢相关的分子机理还知之甚少。因此，研究茶氨酸代谢途径上相关的基因，特别是茶氨酸合成的关键酶基因及调控因子等，将为进一步明晰茶氨酸代谢途径及利用转基因生产茶氨酸提供基础。基于茶氨酸合成于茶树根部以及推定的前体物为盐酸乙胺和谷氨酸钠，作者先在沙培的龙井43茶苗培养介质中添加茶氨酸合成的前体物激活茶氨酸代谢途径，然后选取茶氨酸合成器官——茶苗幼根作为试材，构建cDNA文库并进行ESTs测序分析；另一方面，筛选出茶氨酸含量差异的材料，通过实时荧光定量PCR初步分析茶氨酸代谢相关基因的差异表达。为今后构建基因芯片或数字化的基因表达谱来获取茶氨酸代谢相关的功能基因、调控因子以及深入了解茶氨酸合成及相关氮代谢机理提供研究基础。具体研究结果如下。
1.由于采用现有的茶树RNA提取方法提取茶苗幼根中的RNA难以取得良好的效果，作者在提取茶苗幼根RNA的过程中，对几种RNA提取方法进行了比较，结果显示改良的CTAB法提取茶苗幼根等组织中的RNA完整性好、纯度高，可以满足构建cDNA文库的要求。
2.以茶苗幼根为材料，采用SMART技术构建cDNA文库并对其质量进行了鉴定。结果显示该文库的重组率为92%，库容量为5.0×10~5，平均插入片段长度超过1kb，可以满足后续的大规模测序等要求。从文库中随机挑选克隆并从5’端单向测定5160个克隆，获得4860个有效的ESTs，序列长度从100至1850bp，平均长度为617bp（部分序列已登录GenBank，登录号为GE652554–FE861258）。用Phrap软件进行拼接，共获得1809个unigenes，其中，648个为contigs，1161个为singletons；与NCBI的非冗余核酸数据库进行BlastX比对以及Blast2go注释，将已知或推定的功能基因进行了分类，并通过Kegg分析其中涉及到次生代谢及氨基酸代谢相关的基因。结果显示，1041个unigenes（57.6%）相似于已知或推定功能的蛋白（E-value＜1E-5），282个（15.5%）相似于未知功能的蛋白，486个（26.9%）没有比对上任何蛋白，推定为新的表达基因。将比对上的编码已知或推定功能蛋白的基因进一步分类，蛋白质合成类别占13.06%，与防卫相关的为8.84%，转运蛋白占8.65%。与次生代谢相关的功能基因占4.52%，其中测到序列数量最多的为编码细胞色素P450的功能基因，共有7个；此外，还有编码黄酮3-O-葡糖基转移酶、类黄酮3-葡糖基转移酶、p-香豆酰辅酶A三羟化酶、花青素5-O-葡糖基转移酶、松柏醇酰基转移酶及咖啡酰辅酶A-O-甲基转移酶等基因。Kegg pathway分析结果显示，与次生代谢相关的基因主要参与类黄酮、异黄酮、苯丙素、萜类化合物、二萜、花青素、黄酮和黄酮醇的生物合成代谢途径，其中最主要的是涉及黄酮代谢途径。与初级代谢相关的基因为17.39%，其中氨基酸相关的基因中有涉及氮代谢和茶氨酸合成的谷氨酰胺合成酶、S腺苷甲硫氨酸脱羧酶、精氨酸脱羧酶、γ-谷氨酰转肽酶、亚硝酸还原酶及胞质氨肽谷氨酰胺氨基转移酶等，主要参与蛋氨酸、丙氨酸、谷氨酸、精氨酸、脯氨酸、苯丙氨酸和谷胱甘肽等氨基酸的代谢。其他涉及胞内运输、信号转导、蛋白质降解与储存、能量、转录、细胞结构与细胞生长及未知功能基因分别占1.16%、6.63%、4.23%、5.57%、9.70%、7.69%和10.56%。
3.在茶愈伤组织中添加茶氨酸合成的前体物、代谢中间产物及信号分子等成分，应用高效液相色谱分析愈伤组织生长过程中茶氨酸的变化及其在对照与处理之间的差异。分析了茶苗水培中添加(NH_4)_2SO_4诱导前后的茶氨酸含量变化，同时对遮阴、遮阴与水杨酸或盐酸乙胺组合处理以及不同品种的茶苗嫩叶中茶氨酸含量差异也进行了初步地探讨。试验结果显示，培养基中添加25mM盐酸乙胺后，在茶愈伤组织生长6d和12d时，与对照相比，茶氨酸含量增加最为明显。因此选取对照和盐酸乙胺诱导的茶愈伤组织作为差异表达材料进行后续的候选茶氨酸代谢相关基因的差异表达分析。
4.为了选取合适的内参基因来分析前体物诱导及对照的茶愈伤组织中茶氨酸代谢相关基因的差异表达，作者利用GenBank上登录的茶树基因序列以及通过构建文库测序所得的基因（具有完整的阅读框）共7个持家基因设计引物，在分析这些引物的扩增效率和特异性后，测定了它们在茶愈伤组织生长过程中（对照和愈伤培养基中添加外源物的情况下）的表达水平。利用geNorm和NormFinder软件分析了这些持家基因的稳定性，确定在该条件下合适的内参基因为β-actin和GAPDH。
5.选取11个推定的与茶氨酸代谢相关的基因，利用Primer premier5设计引物，利用qRT-PCR定量分析它们在茶氨酸含量差异明显的茶愈伤组织中表达量的差异。试验结果显示：GS1-1、gogat、NIR和GDH2在愈伤组织培养初始3d的表达量在处理组被明显地诱导，表明它们可能在含氮化合物的起始同化中发挥着作用；TS2及GDH2的表达量在处理组愈伤组织培养的3～6d增加明显，与同期茶愈伤组织培养基中添加前体物茶氨酸含量增加相一致。而TS1的表达量在对照和处理组并没有明显的差异，这可能说明该基因与TS2在茶氨酸的合成中发挥着不同的作用。
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-Ⅰwere 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(NH_4)_2SO_4into 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.
引文
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