Journal of Tea Science

Progress in Functional Gene Cloning of Camellia sinensis

LU Jian-liang, LIN Chen, LUO Ying-ying, ZHANG Guang-hui, LIANG Yue-rong

Journal of Tea Science. 2007, 27(2): 95-103. doi:10.13305/j.cnki.jts.2007.02.001

Abstract ( 411 )

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Progress of separated genes involved in catechins metabolism, caffeine synthesis and aromatic compounds formation were reviewed in this paper, according to published papers and searching results from National Center for Biotechnology information (NCBI). Genes related to stress reaction and basic metabolism were also included.

Study on the Gallic Acid in Pu-erh Tea

LU Hai-peng, LIN Zhi, GU Ji-ping, GUO Li, TAN Jun-feng

Journal of Tea Science. 2007, 27(2): 104-110. doi:10.13305/j.cnki.jts.2007.02.002

Abstract ( 794 )

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Gallic acid is a characteristic phenolic compound in Pu-erh tea with notable bioactivity. The content of gallic acid in Pu-erh tea, the change of gallic acid during the pile-fermentation, and the relation between extract fraction bioactivity and its gallic acid content were studied. Results showed that the average content of gallic acid in Pu-erh tea was 9.01βmg/g, but striking dissimilarity existed between the different samples; The gallic acid content first increased to some extent then decreased during the pile-fermentation of the solar green tea of [Camellia sinensis (Linn.) var. assamica (Masters) Kitamura]. It was found that the extracted fractions which rich in gallic acid showed higher antioxidative activities by DPPH radical scavenging assay.

Research on Conditions of Theanine Biosynthesis by Genetically Engineered Escherichia coli

WANG Li-yuan, WANG Xian-bo, CHENG Hao, ZHOU Jian

Journal of Tea Science. 2007, 27(2): 111-116. doi:10.13305/j.cnki.jts.2007.02.003

Abstract ( 367 )

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Expression conditions of the genetic engineering E.coli which expressed E.coli γ-GGT were investigated. The optimum IPTG concentration and inductive temperature were determined. Factorial experiment design was employed to determine the effects of the reactive conditions (such as microbe concentration, pH, substitute concentration, reactive temperature) on theanine biosynthesis. Results showed that theanine biosynthesis was inhibited by the high microbe concentration. By the higher concentration of L-Gln, the more theanine was obtained and the convertion rate against Gln was declined. It also was manifested that pH9.5 and 32℃~37℃ were optimum conditions for theanine biosynthsis.

Preparation, Analysis and Application of Black Tea Flavor Used in Cigarette

JIANG Mei-hong, BAO Chong-yan, NIAN Xiao-kui, LIU Li-fen, ZHU Dong-lai, ZHE Wei

Journal of Tea Science. 2007, 27(2): 117-119. doi:10.13305/j.cnki.jts.2007.02.004

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The black tea flavor used in cigarette was made from Yunnan black tea leaf by using special technique. The product possessed the characteristic aroma of Yunnan black tea and as well as the flavor roasting aroma. The volatile compositions in the flavor were collected with SDE, and forty-eight compositions were identified and determined by GC-MS, the flavor substances contained in tobacco, such as (E)-3,7-dimethyl-2,6-octadien-1-ol,1,6- octadien-3-ol,3,7-dimethyl-, phenylethyl alcohol, benzyl alcohol were been defined there, those low aliphatic acids 2-methyl-butanoic acid, hexanoic acid and benzaldehyde, benzeneacetaldehyde, 3-methyl-butanal, etc are important ingredients which devoted to the tobacco. The black tea cigarette flavor was added into cigarettes as tobacco flavoring and evaluated by panelists, the results indicated that the extract could enrich the tobacco flavor, reduce the irritancy of smoke and improve taste obviously.

Molecular Identification, Bioinformatic Analysis and Prokaryotic Expression of the Cyclophilin Gene Full-length cDNA from Tea Plant (Camellia sinensis)

ZHANG Ya-li, ZHAO Li-ping, MA Chun-lei, CHEN Liang

Journal of Tea Science. 2007, 27(2): 120-126. doi:10.13305/j.cnki.jts.2007.02.005

Abstract ( 442 )

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A cDNA clone, encoding cyclophilin, obtained by random sequencing of young shoot cDNA library from tea plant (Camellia sinensis). The full-length cDNA of the cyclophilin gene was 949 bp(GenBank accession No. DQ904327), containing a putative ORF of 495 bp, encoding 164 amino acids and the predicted MW were 17.47 kD and pI was 8.54, respectively. A “CAAT” signal in 5′ untranslated region and a polyadenylated signal of “AATAA”poly-A in 3′untranslated regions of cyclophilin mRNAs were found. The presumed amino acid sequences of tea plant were aligned with those of other 26 organisms through CLUSTAL W. The phylogenetic analysis based on the Neighbor-Joining method showed the similarity was greater than 85% between cyclophilin genes of tea plant and Oryza sativa (japonica cultivar-group), Solanum tuberosum, Triticum aestivum etc. Primers were designed on the open reading frame of the cyclephilin gene of tea plant to construct the expressive vector pET/Csin-Cyp. A recombinant protein about 23 kD in the Escherichia coli BL21 (DE3) was induced.

Molecular Cloning and Sequence Analysis of Chalcone Isomerase Gene of Tea Plant (Camellia sinensis)

MA Chun-lei, ZHAO Li-ping, ZHANG Ya-li, CHEN Liang

Journal of Tea Science. 2007, 27(2): 127-132. doi:10.13305/j.cnki.jts.2007.02.006

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Tea is one of the most popular non-alcoholic healthy beverages in the world, which possesses great value as a source of secondary metabolic products, such as catechins. Isolation and cloning of important functional genes of tea plant (Camellia sinensis) is of crucial significance for using biotechnology method to regulate the metabolism of tea plant. In this paper, the chalcone isomerase gene, which was an important functional gene of catechins biosynthesis pathway, was cloned from tea plant by using EST sequencing and RACE (rapid amplification of cDNA ends) approaches. The full-length cDNA of chalcone isomerase gene is 1 163 bp (GenBank Accession No. DQ904329), containing a 723bp open reading frame (ORF) encoding a 240 amino acid protein, and its 3′ untranslated region has an obvious polyadenylation signal. The deduced protein molecular weight was 26.4 kD and its theoretical isoelectric point was 5.19. Sequence analysis result showed that it is closely related with that of Lycopersicon esculentum.

Differential Gene Expression Profiles Analysis of Tea Plant Induced by Tea Looper (Ectropic oblique) Attack Using DDRT-PCR

WEI Chao-ling, GAO Xiang-feng, YE Ai-hua, YANG Yun-qiu, JIANG Chang-jun

Journal of Tea Science. 2007, 27(2): 133-140. doi:10.13305/j.cnki.jts.2007.02.007

Abstract ( 409 )

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DDRT-PCR technique was used to explore the differential gene expression profiles analysis of tea plant induced by tea looper (Ectropic oblique) attack. Results showed that 222 differential expression fragments were obtained, which was 32.8% of total fragments, by amplified in anchor primers A3, A4, A6, A7, A8 and random primers R1-R8,R12, respectively. Twenty fragments of which were re-amplified by RT-PCR. Based on the BLASTx search in GenBank, it were found that eight of the fragments shared no homology, five of the fragments shared homology with the unknown proteins, and seven of the fragments shared higher score homology with the known proteins, which can be divided into six groups related to photosynthetic pigment protein in photosystem II(C15), abiotic resistance induced protein(D22), glucoside metabolism enzyme(A45), nucleic acid and protein transcription and regulation factors(A12, D63), auxin-regulated protein(E94) and VIP2 protein(D73). The fragments of C15, A45, D22, D73 and E94 were firstly found in the studies of molecular mechanism of plant-insect interaction, up to day.

Extracting Rate of Three Kinds of Pesticide in MadeTea During Brewing Process and Risk Assessment on Human Health

WU Xue-yuan, SHENG Xuan, FAN Wei, TANG Feng, YUE Yong-de

Journal of Tea Science. 2007, 27(2): 141-146. doi:10.13305/j.cnki.jts.2007.02.008

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Three kinds of pesticide (Triazophos, Endosulfan and Lambda-cyhalothrin )were sprayed separately in tea garden. The green shoots were harvested from treated plots at 2, 5, 9, 14 and 21 days after the treatment, and manufactured into green tea. The concentration of the above mentioned pesticides in made tea, tea infusion and infused leaves was determined by gas chromatography. Dissipation behavior of pesticide residue in made tea and tea infusion were investigated. Results indicated that the residue level of pesticide in made tea and tea infusion decreased gradually while the plucking interval increased. The concentration of pesticide residue in tea infusion was positively related with that in made tea. The average extracting percentage of residue from made tea to infusion was 29.06％（triazophos）、5.11％（endosulfan）and 1.73％（lambda-cyhalothrin）respectively,and the insoluble residue remained in infused leaves. In comparing the intake amounts of pesticide residue possibly to human body via the tea infusion drinking with ADI of the pesticides, the results showed that the risk to human health from three kinds of pesticide residue by infusion drinking is very small,which is only ranged in 10^-3~10^-5 level.

Toxicity of Five Insecticides on Predatory Mite(Anystis baccarum L.) and Their Effects on Predation to Tea Leafhopper (Empoasca vitis Göthe)

ZENG Zhao-hua, ZHOU Zhe, WEI Zhi-juan, CHEN Shao-bo, YOU Min-sheng

Journal of Tea Science. 2007, 27(2): 147-152. doi:10.13305/j.cnki.jts.2007.02.009

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The toxicity of five insecticides, azadirachtin, imidacloprid, endosulfan, biphenthrin and DDVP, on predatory mite (Anystis baccarum L), and effects on the functional response of A. baccarum mite in preying the tea leafhopper (Empoasca vitis (Göthe)) were investigated. Results revealed that, in contrast to other kinds of insecticide, azadirachtin, imidacloprid showed a rather weak toxicity and less influence on the predation function of A. baccarum mite; the functional response model of A. baccarum mite preying on tea leafhopper were all conformed to Holling’s type II curve in spite of pretreated with sublethal doses of these insecticides. However, there were some changes in the parameters of the model, e.g., the maximal predatory capacities of A. baccarum mite that pretreated with sublethal dose of insecticides were reduced, the handling time was prolonged, the predatory rates were declined, and the searching capability were weakened than that of control. These findings suggested that insecticides at sublethal level had harmful influence on the predation of A. baccarum mite.

Preliminary Studies on Pathogenic Activity and Physical and Chemical Characteristics of Toxins from Gloeosporium theae-sinensis

LIU Shou-an, HAN Bao-yu, FU Jian-yu, CUI Lin, LI Gang

Journal of Tea Science. 2007, 27(2): 153-158. doi:10.13305/j.cnki.jts.2007.02.010

Abstract ( 350 )

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Tea anthracnose fungi, Gloeosporium theae-sinensis, can produce pathogenic exo-toxin, which induced the lesions of tea leaves and the symptoms being similar to those resulted from G. theae-sinensis in the fields. The cultural filtrate of G. theae-sinensis was used to toxin bioassay, so as to make a study of the optimum conditions for toxin-producing and characteristics of toxins. The optimum toxin-producing conditions were as follows: pH 5.0, and 25℃ cultivating for 16 days in the manner of vibration with Czapek-Dox liquid medium. The method of immerging the tea shoots into toxin solution was appropriate for the bioassay of toxins.

The Detection Method of Nitrite in Tea

GU Xiao-ling, LU Cheng-yin*, LIU xin, YU Liang-zi, CHEN-Li-yan, WANG-Qing-hua

Journal of Tea Science. 2007, 27(2): 159-162. doi:10.13305/j.cnki.jts.2007.02.011

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A spectophotometric method on the determination of nitrite in tea was investigated, which is based on the diazotization of nitrite with p-aminobenzene sulfonic acid under acidic conditions then coupled with N-(1-Naphthyl)- ethylenediamine dihydrochloride in forming the colour-producing response. Because of the interference of polyphenols, pigment and ascorbic acid in tea, the optimum operating conditions of measurement was established. The method showed a good analytical characteristics with correlation coefficients of 0.9993, variation coefficient of 4.6%, and recovery was ranging from 72.90% to 86.53%. It is a rapid, accurate, simple method and applicable for the determination of nitrite in tea.

Study on the Acute Toxicity and Genetic Toxicity of Cold-Brewing Tea

YANG Jian, SHI Da-liang

Journal of Tea Science. 2007, 27(2): 163-166. doi:10.13305/j.cnki.jts.2007.02.012

Abstract ( 499 )

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“Cold-making tea” is a new type of tea. To ensure the safety of cold-brewing tea, it is necessary to conduct the research on its property and safety. Results showed that the mice were ingested the tea sample by means of maximal tolerant dosage(40βml/kg·BW)method, The median acute oral lethal dose(LD₅₀)>40βml/kg·BW and it equals to LD₅₀>20βg/kg·BW of the dried tea sample. It can be classified as the class of nontoxic . Based on the above results, three-treatment levels（the maximum dosage 20βml/kg·BW）of the tea infusion were ingested into the mice stomach. It was showed that the result of mice micronucleual test of bone marrow cell was negitive. which means the tea sample was no teratogenicity on sperm and had no micronucleus effect on Kunming mice under the test levels. Results showed that the tea sample was nontoxic and safe.

Effects of Scenting Technique on the Transferring of Pesticide Residue in Jasmine Tea

SI Hui-qing, PANG Xiao-li*, JI Dang-ling

Journal of Tea Science. 2007, 27(2): 167-171. doi:10.13305/j.cnki.jts.2007.02.013

Abstract ( 698 )

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：In this paper,methamidophos and fenvalerate were selected,which represent water-soluble pesticide and fat-soluble pesticide respectively, and sprayed in the proportion of 1β000 and 800 in jasmine garden. The fresh jasmine flower was pluched and processed for jasmine tea. The pesticide residue in jasmine flower and jasmine tea were determined respectively. Results indicated that the pesticide residues of the fresh jasmine flower can transfer into jasmine tea through manufacturing process. The water content of refined tea is the critical factor in the transferring of water-soluble pesticide (methamidophos), while the jasmine flower amount is the critical factor in the transferring of fat-soluble pesticide (fenvalerate).

Evaluation of Chinese Tea Science Literature Based on the Statistical Analysis of The Cited Rate

ZHU Yong-xing

Journal of Tea Science. 2007, 27(2): 172-178. doi:10.13305/j.cnki.jts.2007.02.014

Abstract ( 435 )

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The cited rate of Chinese tea science literature in the recent 20 years or more were searched. With these data the statistical analysis on several citation indexes of tea science literature were carried out, including the cited frequency, the cited rate on piece base, and their distribution characters on journals, institutions and authors. Based on these statistical analysis, the author discussed several aspects about the valuation of tea science literature, tea institutions and the relatied authors in China.

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