Identification of the lysine and histidine transporter family in Camellia sinensis and the characterizations in nitrogen utilization

In plants,the lysine and histidine transporter(LHT)family represent a class of proteins that mediate the uptake,translocation,and utilization of amino acids.The tea plant(Camellia sinensis)is a perennial evergreen with a relatively high level of amino acids.However,systematic identification and molecular characterization of the LHT gene family has rarely been reported in tea plants.In this study,22 CsLHTs were identified from the‘Shuchazao’genome and classified into two groups.The modeled three-dimensional structure and the conserved domains presented a high similarity among the LHTs proteins.Moreover,it was predicted that a few genes were conserved through the analysis of the physiochemical characters,structures and cis-elements in promoters.The expression patterns in tea plants revealed that CsLHT7 was mainly expressed in the roots,and CsLHT4 and CsLHT11 exhibited relatively high expression in both the roots and leaves.Moreover,the expression of all three genes could be induced by organic nitrogen.Additionally,heterogeneous expression of CsLHT4,CsLHT7 and CsLHT11 in Arabidopsis thaliana decreased the aerial parts biomass compared with that in WT plants while significantly increased the rosette biomass only for CsLHT11transgenic plants versus WT plants.Overall,our results provide fundamental information about CsLHTs and potential genes in N utilization for further analysis in tea plants.

Genetic analyses of ancient tea trees provide insights into the breeding history and dissemination of Chinese Assam tea(Camellia sinensis var.assamica)

Chinese Assam tea(Camellia sinensis var.assamica)is an important tea crop with a long history of cultivation in Yunnan,China.Despite its potential value as a genetic resource,its genetic diversity and domestication/breeding history remain unclear.To address this issue,we genotyped 469 ancient tea plant trees representing 26 C.sinensis var.assamica populations,plus two of its wild relatives(six and three populations of C.taliensis and C.crassicolumna,respectively)using 16 nuclear microsatellite loci.Results showed that Chinese Assam tea has a relatively high,but comparatively lower gene diversity(H_(S)=0.638)than the wild relative C.crassicolumna(H_S=0.658).Clustering in STRUCTURE indicated that Chinese Assam tea and its two wild relatives formed distinct genetic groups,with considerable interspecific introgression.The Chinese Assam tea accessions clustered into three gene pools,corresponding well with their geographic distribution.However,New Hybrids analysis indicated that 68.48%of ancient Chinese Assam tea plants from Xishuangbanna were genetic intermediates between the Puer and Lincang gene pools.In addition,10%of the ancient Chinese Assam tea individuals were found to be hybrids between Chinese Assam tea and C.taliensis.Our results suggest that Chinese Assam tea was domesticated separately in three gene pools(Puer,Lincang and Xishuangbanna)in the Mekong River valley and that the hybrids were subsequently selected during the domestication process.Although the domestication history of Chinese Assam tea in southwestern Yunnan remains complex,our results will help to identify valuable genetic resources that may be useful in future tea breeding programs.

Analysis of NPK in Camellia sinensis, Gliricidia sepium, and Musa acuminata Biomasses for Preparation of an Organic Fertilizers Formula for Young Tea Plants (Musa acuminata) and Studying of Their Nutrient Release Capacity in the Biodegradation Process

This study investigates the nitrogen (N), phosphorous (P), and potassium (K) contents in raw biomasses of Camellia sinensis, Gliricidia sepium, and Musa acuminata. Therein, the highest N and P content was seen in Camellia sinensis 116.80 ± 0.08 mg and 66.00 ± 0.14 mg respectively. The highest K content (106.80 ± 0.04 mg) was observed in Musa acuminata. Next, all three types of plant materials were allowed to decompose in water for 3 weeks, and a sample from each was analyzed for NPK after the 1st, 2nd, and 3rd week during decomposition. A significant increase in the release of N, P, and K by the Camellia sinensis to water (P Musa acuminate were not significantly changed (P > 0.05) over time. The ratio for N:P:K was calculated for raw biomass samples and decomposed samples to find the best fitting N:P:K ratio to apply to young tea plants as organic fertilizers. In addition to that, the microbial insight of these organic compounds was analyzed by observing how microbial population increased with decomposition by the enumeration of the total microbial count. A considerable increment in total microbial count was observed up to 3.28 × 106, 1.21 × 1010, 2.18 × 108, and 6.49 × 107 CFU/ml for Camellia sinensis, Gliricidia sepium, Musa accuminata (leaves), and Musa accuminata (trunk) respectively. The presence of phosphate solubilizing bacteria (PSB) and nitrogen solubilizing bacteria (NSB) throughout the decomposition period was confirmed by their growth on NBRIP and a modified nutrient medium that was specifically designed for the identification of ammonifiers respectively. Prepared fertilizer samples were applied to young tea plants that were grown in the Mawanella area in Sri Lanka (7°15'12.42"N 80°26'47.62"E) and according to the results, it is clear that fertilizer mixture 1 (N:P:K, 10:5:10, tea dust + Gliricidia + banana trunk) and fertilizer mixture 2 (N:P:K, 10:5:10, tea dust + Gliricidia + banana leaves) has the potential to increase the growth of young tea plants.

Natural variation of main biochemical components,morphological and yield traits among a panel of 87 tea [Camellia sinensis(L.) O. Kuntze] cultivars

Many attentions have been previously focused to identify the multiple biochemical components related to tea quality and health benefits,however, the natural variation of biochemical components present in tea germplasm has not been adequately evaluated. In this study, the main biochemical components, leaf morphological and yield characteristics were evaluated for four rounds of tea leaves in a panel of 87 elite tea cultivars suitable for black, green, or oolong tea. Significant variations were observed among the tea cultivars, as well as seasonal differences in the levels of the free amino acid(FAA), caffeine(CAF), tea polyphenols(TP), water extract(WE) and TP to FAA ratio(TP/FAA). Results showed that the average levels of FAA showed a seasonal change, with the highest level of 4.0% in the 1st spring tea in the cultivars suitable for green tea and the lowest of 3.2% in summer tea in the cultivars suitable for black tea. The average CAF content was highest 3.2% in the cultivars suitable for oolong tea in the 1st spring and the lowest 2.5% in the cultivars suitable for green tea in summer. Limited seasonal and varietal variations were noticed in the average levels of WE among the three categories of tea. In addition, significant natural variation of the morphological characteristics, bud length varying from 2.5 cm to 8.7 cm, bud density from 190.3 buds · m-2to 1 730.3 buds · m-2, mature leaves biomass from 128.4 kg · hm-2to 2 888.4 kg · hm-2, and yield component traits of 100 buds(one bud with two leaves) dry weight from 3.7 g to37.7 g, tea yield/round from 444.6 kg · hm-2to 905.3 kg · hm-2, were observed. The aim of our evaluation was not only to identify the advantages of seasonal and clonal variations but also to provide a new viewpoint for their further application. Representative accessions were selected from the germplasm to promote the establishment of an inherent biochemical constituent expressing the quality of black, green, and oolong tea. The findings might be utilized to establish early selection criteria to enhance the tea breeding and production program.

Differential metabolites and their transcriptional regulation in seven major tea cultivars (Camellia sinensis) in China

Various genetic and biochemical characteristics exist in tea plant cultivars,and they largely determine production suitability and tea quality.Here,we performed transcriptomic and metabolomic analyses of young shoots of seven tea cultivars and identified major regulatory transcription factors(TFs)for the characteristic metabolites in different cultivars based on weighted gene co-expression network analysis(WGCNA).Phenotypically,we found that‘Tieguanyin’(TGY)and‘Fujian Shuixian’(FJSX),which are suitable for oolong tea,had higher catechin contents.The metabolites of‘Jinxuan’(JX)were more prominent,especially the contents of phenolic acids,flavonoids,terpenes,and tannins,which were higher than those of the other six cultivars.Moreover,‘Fudingdabai’(FDDB),which is suitable for white tea,was rich in amino acids,linolenic acid,and saccharides.At the molecular level,hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase(HCT)(CsTGY12G0001876,and CsTGY06G0003042)led to the accumulation of chlorogenic acid in TGY.The main reason for the higher l-ascorbic acid content in FJSX was the high expression levels of L-galactono-1,4-lactone hydrogenase(GalLDH)(CsTGY13G0000389)and Myo-inositol oxygenase(MIOX)(CsTGY14G0001769,and CsTGY14G0001770),which were regulated by WRKY(CsTGY11G0001197).Furthermore,FDDB,‘Longjing 43’(LJ43),‘Shuchazao’(SCZ)and‘Baihaozao’(BHZ)had higher free fatty acid contents,among which MYB(CsTGY14G0002344)may be a hub gene for the regulation of palmitoleic acid accumulation.More importantly,we found that the shoots of TGY were green with purple,mainly due to the accumulation of anthocyanins and the downregulation of the Mg-protoporphyrin IX nonomethyl ester cyclase(MPEC)(CsTGY10G0001989)gene that affects chlorophyll synthesis.These results will provide a theoretical reference for tea cultivar breeding and suitability.

Cold Stress-induced Glucosyltransferase CsUGT78A15 is Involved in the Formation of Eugenol Glucoside in Camellia sinensis

Eugenol is a natural phenolic compound known for its health-promoting properties and its ability to add a floral scent to tea plants.Plant eugenol glycosides have been identified and shown to make important contributions to fruit floral quality.However,the details of their biosynthesis and metabolism in tea plants are still unknown.Here,eugenol glucoside was unambiguously identified as a native metabolite in the tea plant,and its biosynthesis was shown to be induced by low temperature treatment.Through the analysis of UGTs induced by low temperature,the glycosyltransferase CsUGT78A15 was identified in tea,and its encoded protein was shown to catalyze the glucosylation of eugenol.Vmax/Km ratios showed that eugenol was the most suitable substrate for CsUGT78A15.Sugar donor preference analysis showed that CsUGT78A15 had a higher selectivity for glucose,followed by galactose and glucuronic acid.The expression of CsUGT78A15was correlatedwith the accumulation of eugenol glucoside in different tissues and genotypes of tea.Down-regulation of CsUGT78A15 led to a decreased eugenol glucoside content under cold stress,indicating that CsUGT78A15 plays an important role in the biosynthesis of eugenol glucoside under cold stress.The identification of eugenol glucoside in the tea plant and the discovery of a cold stress-induced eugenol glucosyltransferase in tea provide the foundation for the improvement of tea flavor under cold stress and the biotechnological production of eugenol glucoside.

Anti-inflammatory properties of oolong tea(Camellia sinensis)ethanol extract and epigallocatechin gallate in LPS-induced RAW 264.7 cells

Objective: To evaluate the anti-inflammatory activity of oolong tea ethanol extract(OTEE) and epigallocatechin gallate(EGCG) on lipopolysaccharide-induced murine macrophage cell line(RAW 264.7).Methods: A cytotoxic assay using MTS tetrazolium was conducted to find a nontoxic level of OTEE and EGCG toward RAW 264.7 cells. Interleukins(IL-6, IL-1 b), tumor necrosis factor-a(TNF-a), and cyclooxigenase-2(COX-2) levels were measured by ELISA, and nitric oxide(NO) levels measured by a nitrate/nitrite colorimetric assay to determine the inhibition activity of OTEE and EGCG.Results: Lipopolysaccharide induction increases NO, COX-2, IL-6, IL-1 b, and TNF-a levels compared with the untreated cell(negative control). The positive control,lipopolysaccharide-induced RAW 264.7 without treatments showed the highest level of all pro-inflammatory cytokines and modulators tested in this study. The positive control was used as standard to obtain OTEE and EGCG inhibition activity toward NO, COX-2,IL-6, IL-1 b, and TNF-a. OTEE had a higher inhibition activity toward NO, COX-2, IL-6,and IL-1 b than EGCG; the reverse was seen for TNF-a. However, both OTEE and EGCG suppressed production of NO, COX-2, IL-6, IL-1 b, and TNF-a.Conclusions: OTEE and EGCG have the potential for use as anti-inflammatory drugs,which is shown by their ability to reduce the production of NO, COX-2, IL-6, IL-1 b, and TNF-a in active macrophages.

The leaf extracts of Camellia sinensis (green tea) ameliorate sodium fluoride-induced oxidative stress and testicular dysfunction in rats

Objective:To investigate the effect of Camellia(C.)sinensis in mitigating oxidative damage and reproductive toxicity in testis induced by sodium fluoride in a rat model.Methods:Twenty-four adult male Wister rats were divided into 4 groups,with 6 rats in each group.Group 1 orally received distilled water(1 mL/100 g body weight)daily and served as the control group,while group 2 received drinking water with 100 ppm sodium fluoride per day for 21 consecutive days,group 3 was administered with only C.sinensis extract by gavage at a dose of 100 mg/kg body weight and group 4 received drinking water with 100 ppm sodium fluoride and 100 mg/kg body weight C.sinensis leaf extract per day for 21 consecutive days.At the end of the treatment,the rats were sacrificed under light ether anesthesia.The gonado-somatic index,sperm count and motility,serum level of luteinizing hormone and testosterone were assayed.Lipid peroxidation[malondialdehyde(MDA)level],nitric oxide(NO)production,and activities of antioxidant enzymes-superoxide dismutase(SOD),catalase,and reduced glutathione level(GSH)were also analysed.Results:Sodium fluoride treatment significantly decreased gonado-somatic index,sperm count and motility as well as the serum level of luteinizing hormone and testosterone(P<0.05).The histological examination of testes revealed atrophy and degenerative changes in several seminiferous tubules,along with enhanced interstitial space and a reduced number of Leydig cells.There was a highly significant increase in NO and MDA production(P<0.05),while SOD,catalase activities and GSH level decreased significantly(P<0.05).However,C.sinensis significantly restored testicular weight,sperm parameter,hormonal level(P<0.05),and also reversed MDA and NO generation and antioxidant enzymes activities in the testicular tissue(P<0.05).Conclusions:C.sinensis may have an ameliorative role against sodium fluoride-induced oxidative damage in the testis probably because of its antioxidant property.

Functional mechanism on stem cells by tea(Camellia sinensis)bioactive compounds

Camellia sinensis(tea),one of the most popular commercial crops,is commonly applied in all parts of the world.The main active ingredients of tea include polyphenols,alkaloids,polysaccharides,amino acids,aroma and volatile constitutes,all of which are potentially responsible for the activities of tea.Stem cells(SCs)are the immature and undifferentiated cells by a varying capacity for proliferation,self-renewal and the capability to differentiate into one or more different derivatives with specialized function or maintain their stem cell phenotype.Herein,a thorough review is conducted of the functional mechanism on SCs by tea bioactive compounds.

Identification of miRNAs and target genes regulating catechin biosynthesis in tea (Camellia sinensis)

Micro RNAs（miRNAs） are endogenous non-protein-coding small RNAs that play crucial and versatile regulatory roles in plants. Using a computational identification method, we identified 55 conserved miRNAs in tea（Camellia sinensis） by aligning miRNA sequences of different plant species with the transcriptome library of tea strain 1005. We then used quantitative real-time PCR（qRT-PCR） to analyze the expression of 31 identified miRNAs in tea leaves of different ages, thereby verifying the existence of these miRNAs and confirming the reliability of the computational identification method. We predicted which miRNAs were involved in catechin synthesis using ps RNAtarget Software based on conserved miRNAs and catechin synthesis pathway-related genes. Then, we used RNA ligase-mediated rapid amplification of c DNA ends（RLM-RACE） to obtain seven miRNAs cleaving eight catechin synthesis pathway-related genes including chalcone synthase（CHS）, chalcone isomerase（CHI）, dihydroflavonol 4-reductase（DFR）, anthocyanidin reductase（ANR）, leucoanthocyanidin reductase（LAR）, and flavanone 3-hydroxylase（F3H）. An expression analysis of miRNAs and target genes revealed that miR529d and miR156 g-3 p were negatively correlated with their targets CHI and F3H, respectively. The expression of other miRNAs was not significantly related to their target genes in the catechin synthesis pathway. The RLM-RACE results suggest that catechin synthesis is regulated by miRNAs that can cleave genes involved in catechin synthesis.

Induced Polyploidy as a Tool for Increasing Tea(Camellia sinensis L.) Production

Tea （Camellia sinensis L.） represents different ploidy levels. In the present paper, we reviewed the recent data on the diploid, aneuploid and polyploid formed their origin and chemically induced polyploidy and predicted it role in teagenetic improvement for better yield. Different polidy levels had different effects on tea physiology. Tetraploid and triploid had more vigour and hardness due to increased size of cells, while triploid could have even more vigours due to increased size of cells and sterilities. Chemically induced polyploidy had been found an important tool for improving plant physiology and production, therefore, induced polyploids should be produced to overcome the problem of low yield and limited rainfall in tea growing areas.

Structural,morphological,and optical properties of tin(Ⅳ) oxide nanoparticles synthesized using Camellia sinensis extract: a green approach

Tin oxide(SnO) nanoparticles were cost-effectively synthesized using nontoxic chemicals and green tea(Camellia sinensis) extract via a green synthesis method. The structural properties of the obtained nanoparticles were studied using X-ray diffraction, which indicated that the crystallite size was less than 20 nm. The particle size and morphology of the nanoparticles were analyzed using scanning electron microscopy and transmission electron microscopy. The morphological analysis revealed agglomerated spherical nanoparticles with sizes varying from 5 to 30 nm. The optical properties of the nanoparticles’ band gap were characterized using diffuse reflectance spectroscopy. The band gap was found to decrease with increasing annealing temperature. The O vacancy defects were analyzed using photoluminescence spectroscopy. The increase in the crystallite size, decreasing band gap, and the increasing intensities of the UV and visible emission peaks indicated that the green-synthesized SnOmay play future important roles in catalysis and optoelectronic devices.

THE INHIBITORY EFFECT OF EXTRACT OF CAMELLIA SINENSIS AND EXTRACT OF CAMELLIA PTILOPHYLLA CHANG ON DNA POLYMERASE OF EHRLICH ASCITES CARCINOMA CELLS

Objective: To detect the effect of extract of Camellia Sinensis (ECS) and extract of Camellia Ptilophylla Chang (ECPC) on DNA polymerase (Pol) of Ehrlich ascites tumor cells Methods: Referring to the method of K Ono, Pol was extracted from Ehrlich ascites tumor cells in mice Pol α, β, and γ were separated by phosphocellulose column chromatography and were identified The effect of ECPC and ECS on Pol was studied Results: ECPC and ECS were shown to inhibit the activity of Pol α, β, and γ IC 50 values of ECS on Pol α , β, and γ were 10 2μg/ml, 9 9μg/ml and 28 9μg/ml respectively IC 50 values of ECPC on Pol α, Pol β and Pol γ were 5 6μg/ml, 15μg/ml and 14 7μg/ml respectively The modes of inhibition of ECPC on Pol α, Pol β and Pol γ were noncompetitive with respect to template DNA The Ki values of ECPC on Pol α , β, and γ were 2 68±0 12μg/ml, 2 24±0 12μg/ml , 2 56±0 18μg/ml Conclusion: ECPC and ECS were shown to have inhibitory effect on DNA polymerase of tumor cells The mode of inhibition of ECPC on Pol α, Pol β and Pol γ were noncompetitive with respect to template DNA

Genome-Wide Characterization of the Cellulose Synthase Gene Superfamily in Tea Plants(Camellia sinensis)

The cellulose synthase gene superfamily,including Cellulose synthase A(CesA)and cellulose synthase-like(Csl)gene families,is responsible for the synthesis of cellulose and hemicellulose,respectively.The CesA/Csl genes are vital for abiotic stress resistance and shoot tenderness regulation of tea plants(Camellia sinensis).However,the CesA/Csl gene family has not been extensively studied in tea plants.Here,we identified 53 CsCesA/Csl genes in tea plants.These genes were grouped into five subfamilies(CsCesA,CsCslB,CsCslD,CsCslE,CsCslG)based on the phylogenetic relationships with Arabidopsis and rice.The analysis of chromosome distribution,gene structure,protein domain and motif revealed that most genes in CsCesA,CsCslD and CsCslE subfamilies were conserved,whereas CsCslB and CsCslG subfamily members are highly diverged.The transcriptome analysis showed that most CsCesA/Csl genes displayed tissue-specific expression pattern.In addition,members of CsCslB4,CsCesA1/3/6,CsCslB3/4,CsCslD3,CsCslE1 and CsCslG2/3 subfamilies were up-regulated under drought and cold stresses,indicating their potential roles in regulating stress tolerance in tea plants.Furthermore,the expression levels of CsCslG2_6 and CsCslD3_5 in different tissues and cultivars,respectively,were positively correlated with the cellulose content that is negatively related with shoot tenderness.Thus,these two genes were speculated to be involved in the regulation of shoot tenderness in tea plants.Our findings may help elucidate the evolutionary relationships and expression patterns of the CsCesA/Csl genes in tea plants,and provide more candidate genes responsible for stress tolerance and tenderness regulation in tea plants for future functional research.

Screening of Adoptive Elite Tea (Camellia sinensis) Clones

The research screening of adoptive elite tea clones was conducted at NTRI, Mansehra during 2011-2012. Nine clones 101Aa, 105aa, 108aa, 561aa, ll7aa, 219ab, 470bb and 180bd were evaluated for seedling performance. Randomized complete block design was used with three replications. Data was recorded on various morphological characters after 8 months. The results showed that high survival percentage, shoot length, number of roots plant-1, number of leaves plant1 and root length were observed in clone 105aa. While the highest fresh weight and dry weight of leaves were observed in clones 117aa and 105aa. The clone 105aa was drought resistant, high survival percentage and root growth. On the basis of the results, clone 105aa was recommended for cultivation through cuttings in the hilly areas of Pakistan where unequal rainfall distribution was a major hitch.

In Vitro Antioxidant and Free Radical Scavenging Activity of Curcuma longa, Acorus calamus and Camellia sinensis

The purpose of the current study was to determine the total phenolic and flavonoid content and total antioxidant activity of the Curcuma longa, Acorus calamus, and Camellia sinensis ethanolic extracts and their free radical scavenging activity. The study concluded that the Curcuma longa, Acorus calamus, and Camellia sinensis ethanolic extracts have a good source of phenolics, flavonoids, and antioxidant sources in turn which opens the high possibility of the extracts being used as food preservatives. The DPPH assay for scavenging free radicals showed that the IC50 value was above 123% of Curcuma longa ethanolic extract, 129.9% μg/ml of Acorus calamus ethanolic extract and 25% of Camellia sinensis ethanolic extracts shows very strong inhibition of the free radicals. Thus, comparing the DPPH assay for scavenging free radicals of Curcuma longa, Acorus calamus and Camellia sinensis ethanolic extracts with the positive control ascorbic acid, Curcuma longa and Camellia sinensis ethanolic extracts showed strong inhibition of the free radicals.

Influence of Geographical Regions on Catechin and Caffeine Levels in Tea (Camellia sinensis)

This study aimed at investigating the influence of different growing conditions in two regions, Timbilil and Kangaita, on the levels of catechins and caffeine. Fresh leaf samples of 60 clones cultivated in both sites were obtained, processed and assayed for levels of caffeine, total and individual catechins using Reverse Phase High Performance Liquid Chromatography (RP-HPLC). The obtained data was subjected to analysis of variance using GENSTAT-C statistical software. Safe for a few clones in the Kangaita site had significantly higher total and individual catechins than those grown in the Timbilil site. A mean epigallocatechin gallate (EGCG) content of 7.9% was observed for Kangaita clones compared to 6.7% in Timbilil. A similar trend was observed for the rest of the individual catechin fractions with a mean epicatechin gallate (ECG) of 3.3% in Kangaita and 2.5% in Timbilil, a mean epigallocatechin (EGC) of 5.2% in Kangaita compared to 5.0% in Timbilil and a mean epicatechin (EC) content of 1.5% in Kangaita compared to 1.4% in Timbilil. Similarly in the total catechins analyses, clones in Kangaita had a mean value of 18.7% compared to 16.2% observed for the Timbilil site clones. However, the Timbilil site clones had significantly high caffeine contents (mean, 4.2%) compared to the Kangaita site clones (mean, 3.9%) although with a few exceptions. Significant interactions (P < 0.05) between clone and region are also observed, suggesting that levels of these biomolecules are bound to differ with the growing region but the extent of variation will vary depending on the clonal genotype. The observed differences in levels of these biologically important biomolecules among the two regions shows the importance of the underlying conditions on the production of tea.

基于SSR标记的古茶树(Camellia sinensis)遗传关系分析

本研究利用30个SSR标记,结合已有的128个茶树品种指纹,对福建省园岭林场的4株古茶树和1株铁观音进行了遗传关系分析。结果表明,30个SSR标记共产生了124个等位片段,每个标记的等位片段数1~9个;多态性信息量较高,平均为0.507。遗传聚类分析表明,铁观音品种‘Tieguanyin2’与Fujian组的‘Tieguanyin’、‘Jinmudan’、‘Mingke1’和‘Zimudan’等遗传距离较近,属于铁观音品种。4株古茶树(YL01,YL02,YL03和YL04)间遗传距离较近,属于Fujian组,推测为八仙茶古茶树品种。本研究的5株茶树与已知的128个茶树品种间没有直接的亲子关系。YL02、YL03和YL04聚类较近,不匹配位点数较少,可能来源于共同的亲本。DNA分子指纹检测表明,8对核心SSR标记可对所有参试茶树进行有效的鉴别,这为古茶树的品种鉴定和产权保护提供了有效的分子证据。

Optimization of Agrobacterium tumefaciens-Mediated Transformation Systems in Tea Plant(Camellia sinensis)

In this study, an efficient plant regeneration protocol in vitro and transformation by Agrobacterium-mediated method of Camellia sinensis was achieved, which would lay the foundation for genetic improvement of tea plant by genetic engineering technology. The cotyledon callus of C.sinensis were used as the receptors for transformation by Agrobacterium tumefaciens EHA105 containing PS1aG-3. Some factors which affected the result of Agrobacterium-mediated transformation of C. sinensis were studied on the basis of GUS transient expression system. The optimum system of Agrobacterium-mediated transformation was that the cotyledon callus were pre-cultured for 3 d, and then infected by EHA105 for 15 min followed by 3 d co-culture in the dark on the YEB medium containing 150 μmol·L^(-1) acetosyringone(AS). The transient expression rate of GUS gene was 62.6%. After being delayed selective culture for 3 d, infected callus were transferred into the differentiation medium and the root induction medium both of which were supplemented with 100 mg·L^(-1) spectinomycin, and then resistant seedlings of C. sinensis were obtained. The conversion rate was 3.6%.

Computational Identification of miRNAs and Their Target Genes from Expressed Sequence Tags of Tea(Camellia sinensis)

MicroRNAs （miRNAs） are a newly identified class of small non-protein-coding post-transcriptional regulatory RNA in both plants and animals. The use of computational homology based search for expressed sequence tags （ESTs） with the Ambros empirical formula and other structural feature criteria filter is a suitable combination towards the discovery and isolation of conserved miRNAs from tea and other plant species whose genomes are not yet sequenced. In the present study, we blasted the database of tea （Camellia sinensis） ESTs to search for potential miRNAs, using previously known plant miRNAs. For the first time, four candidate miRNAs from four families were identified in tea. Using the newly identified miRNA sequences, a total of 30 potential target genes were identified for 11 miRNA families; 6 of these predicted target genes encode transcription factors （20%）, 16 target genes appear to play roles in diverse physiological processes （53%） and 8 target genes have hypothetical or unknown functions （27%）. These findings considerably broaden the scope of understanding the functions of miRNA in tea.