Journal of Tea Science

Full-length cDNA Cloning and Bioinformatic Analysis of ACC Synthase Gene from the Tea Plant (Camellia sinensis)

ZHANG Ya-li, QIAO Xiao-yan, CHEN Liang

Journal of Tea Science. 2008, 28(4): 235-241. doi:10.13305/j.cnki.jts.2008.04.013

Abstract ( 335 )

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Ethylene is one of the important plant hormones. It has significant physiological role in the life of plant. ACC (1-aminocyclopropane-1-carboxylic acid) synthase (ACS) is a key enzyme in the ethylene biosynthesis in plants. It plays important roles in the regulating ethylene biosynthesis. Degenerate primers were designed based on the conservative ACS gene sequence of other plants in the GenBank to clone the full-length cDNA of ACS genes of the tea plant by using the RACE and RT-PCR methods. It is deposited to the GenBank (Accession No. EF205149). The tea plant ACS gene cDNA is 1 579 bp in length, encoding 478 amino acids, the putative molecular weight is 53.7 kD and the pI is 8.039. The phylogenetic trees constructed by the software MEGA 3 showed that ACS gene of tea plant was closely related to that of Diospyros kaki. The cloning of ACS gene full-length cDNA from the tea plant provides a possibility for understanding the molecular mechanism of physiological role on molecular level in the tea plant.

Construction of E. coli Recombinant Engineered Strain for Theanine Biosynthesis with GS Gene Embedded

ZHU Wen-xian, LI Xing-hui, WANG Li-yuan, FANG Wan-ping, CHENG Hao

Journal of Tea Science. 2008, 28(4): 242-248. doi:10.13305/j.cnki.jts.2008.04.012

Abstract ( 309 )

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An experiment on the construction of E. coli recombinant strain for theanine biosynthesis with GS gene embedded was reported. In this experiment, Glutamine Synthetase gene in E. coli BL21 was ligated with the pET32a vector in order to produce theanine. The ligation product was transformed to E. coli BL21 and the engineered E. coli strain was constructed successfully. The glutamine Synthetase was expressed with induction of 0.1 mmol/L IPTG in 28℃. The activity of γ-glutamyl transpeptidase of the engineered strain of fresh E. coil engineered strain reached 41.79 U /mg prot, and was 126.64 times higher than that of the original strain one and the yield of theanine from L-Gln and ethylamine was 6.3 mg/ml. The ability of theanine biosynthesis of recombinant was enhanced obviously compared with that of original E. coli BL21.

Analysis of Differential Expression Genes in Cold-induced Tea Plant

ZOU Zhong-wei, FANG Wan-ping, ZHANG Ding, DUAN Yun-shang, LI Xing-hui

Journal of Tea Science. 2008, 28(4): 249-254. doi:10.13305/j.cnki.jts.2008.04.011

Abstract ( 302 )

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The differential expression was investigated between cold-induced and normal environment in tea plant (Camellia sinensis) with cDNA-AFLP (cDNA Amplified Fragments Length Polymorphism). Expression profiles were generated around 86 special cDNA fragments (16 pairs primer, 256 primer combinations). Ten differentially expressed transcript-derived fragments were isolated. Cha1 revealed similarities (77%) to one kind of low temperature and salt responsive protein in Arabidopsis thaliana. Cha5 revealed similarities (89%) to Arabidopsis thaliana 60 S ribosomal protein L7 (RPL7B), ChaH8 had high similarities (79%) to Nicotiana tabacum A10 mRNA for stress-inducible H1 histone-like protein. Cha9 had high similarities to Populus EST from severe drought-stressed opposite wood. Other 6 fragments showed no homology to the known sequences in GenBank, perhaps they were new genes.

Rapid Molecular Identification of Euproctis pseudoconspersa Infected with EpNPV by PCR Method

FU Jian-yu, HAN Bao-yu

Journal of Tea Science. 2008, 28(4): 255-259. doi:10.13305/j.cnki.jts.2008.04.006

Abstract ( 290 )

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According to nucleotide sequences of the two key genes, A13-1 lef-8 gene and A13-1 polyhedrin gene of EpNPV, two pairs of special primers were designed. Two typical DNA fragments with the size of 538 bp and 281 bp were amplified by PCR method. The two PCR products were cloned into T vector and sequenced, Blasted with the DNA sequence of EpNPV, both of them were matched absolutely. That meant that these two DNA fragments can be used as molecule markers to identify the Euproctis pseudoconspersa Strand infected with EpNPV. The present investigation constructed a molecular biological method to detect EpNPV DNA, to evaluate the biological control effect of EpNPV and to study the infecting route of EpNPV into E. pseudoconspera.

Preparation of the Antibody Against EupsNPV Polyhedrin and Its Utilization in Detection of the Viral Pesticide

WANG Li-zhong, XIAO Qiang, ZHANG Chuan-xi

Journal of Tea Science. 2008, 28(4): 260-266. doi:10.13305/j.cnki.jts.2008.04.005

Abstract ( 359 )

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The 741 bp coding region of polyhedrin gene (ph) was amplified from Euproctis pseudoconspersa nucleopolyhedrovirus（EupsNPV）genome and its prokaryotic expression vector pET-28-a-Ep-ph was constructed. Then the prokaryotic expression plasmid was transformed into Escherichia.coli. BL21（DE3）for expression. The expressed fusion protein was separated by SDS-PAGE and retrieved from the gel. New Zealand white rabbit was immunized with the purified fusion protein and the antiserum against ph with a titre of 6.4×10⁴ was prepared. Western blotting indicated that the prepared polyclonal antibody was specific for polyhedrin. The EupsNPV was detected by indirect ELISA and its regression equation was obtained: y=0.4152x-0.8299 with correlation coeffient r=0.9897(P<0.01). Indirect ELISA detection showed that the antibody could be used for quantitative detection of the NPV in a commercial bio-pesticide and provided an accurate and effective method for detection.

Effect of Ultra high Pressure on the Aromatic Character of Pu’er Tea

ZHANG Jun-song, ZHANG Chang-ji, ZHENG Hong-yang, MAO Duo-bin, YANG Gong-min

Journal of Tea Science. 2008, 28(4): 267-272. doi:10.13305/j.cnki.jts.2008.04.004

Abstract ( 464 )

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The volatile compounds of ultra high pressure (UHP) (400 MPa, 25℃, 20 min) treated Pu’er tea were isolated by solid-phase microextraction and analyzed by capillary gas chromatography-mass spectrometry (GC-MS). The relative contents of constitutes were determined by area normalizing method. The results showed that the mass fraction of main aromatic compounds was increased obviously after UHP treatment. The mass fraction in the contents of β-Ionone, guaiacol, cedrol, geranyl acetone, 2,6-dimethoxyphenol, linalool oxide, 2,3-dimethoxytoluene and dihydroactinidiolide was increased 92.31%、80.77%、75.00%、73.08%、63.64%、54.92%、54.54% and 34.92% respectively, the geraniol (0.22%), cadinol (0.15%) and damascenone (0.15%) were only found in UHP treated Pu’er tea in compared with the sample before UHP treatment. The organoleptic results demonstrated that UHP process can increase the stale taste and enhance the pure stale characteristic aroma of Pu’er tea obviously. The authors regarded that UHP treatment is a potential technique in Pu’er tea processing.

Study on Adsorption Behaviors of Theanine on HD-8 Macroporous Anion Resin

GONG Yu-shun, HUANG Jian-an, LIU Zhong-hua, YANG Zhi-hui, LI Juan

Journal of Tea Science. 2008, 28(4): 273-276. doi:10.13305/j.cnki.jts.2008.04.010

Abstract ( 366 )

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The investigation on the adsorption behaviors of theanine on HD-8 macroporous anion resin were conducted. Effect of PH on adsorption of theanine was not obvious within range of 2~7. The adsorption data fit well into the Freundlich isotherm equation within temperature range of 303~323 K and investigative concentration range. The rate of adsorption was under control by some factors simultaneously such as film diffusion and chemical reaction.

Separation, Purification and Structure Identification of Flavonol Glycosides in Black Tea

GAO Qing-qing, JIANG He-yuan, ZHANG Jian-yong, YUAN Xin-yue, KOU Xiao-hong, GAO Qi, LIU Xiao-hui, KE Chang-qiang

Journal of Tea Science. 2008, 28(4): 277-281. doi:10.13305/j.cnki.jts.2008.04.003

Abstract ( 439 )

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Column chromatography, HPLC and prepared HPLC were used together to separate and purify the ethyl acetate extract of Dajielee black tea, and the mixture of two flavonol glycosides was obtained. The molecule NMR methods weights and structure of these two flavonol glycosides were identified by using the HPLC/MS and NMR methods.

Analysis on Aromatic Constituents of Fragrant Flower [Chimonathus praecox (L.)Link] in Aroma Releasing

XIE Chao, WANG Jian-hui, GONG Zheng-li

Journal of Tea Science. 2008, 28(4): 282-288. doi:10.13305/j.cnki.jts.2008.04.002

Abstract ( 382 )

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The change of essential oil which extracted by SDE method was studied by the gas chromatography (GC), GC/MS and evaluation methods during Lamei [Chimonathus praecox (L.) Link] blooming. The number and content of fragrant components of essential oil were changed during aroma releasing. Results showed 26, 37, 43, 49 aromatic components in Lamei were identified by GC/MS data during the following four stages (bud, early spread, spread, blooming). In compared with the unbloomed bud, the content of alkyl compounds raised 0.590, 7.152 and 13.820 times and the alkenes compounds raised 0.424, 1.078 and 4.286 times, the alcohol compounds raised 0.895, 3.324 and 5.091 times and the ester compounds raised 1.071, 2.182 and 15.101 times, respectively with the increase of opening of Lamei flower. The ketone compounds were appeared only during the later period of blooming. The content of ketone were increased steadily during the full blooming period and was 12.3 times higher than that in spreading flowerperiod. Organoleptic evaluation showed that the Lamei buds expressed a low aroma and with green grass taste, and the specific aroma was only appeared during the flower-spreading period. It is characteized that the intensity of aroma was increased from low to high with the increasing of the flower opening. On the basis of above experiments results, the trend of fragrant components’ content by GC and GC/MS method and the results from organoleptic evaluation method were similar.

Determination of Polyphenolic Content and Antioxidant Activity of Kudingcha Made from Ilex kudincha C. J. Tseng

LIU Li-xiang, LIANG Xing-fei, SUN Yi, Tanguy Laura, YE Hong, ZENG Xiao-xiong

Journal of Tea Science. 2008, 28(4): 289-293. doi:10.13305/j.cnki.jts.2008.04.009

Abstract ( 384 )

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The hot water extract (crude extract) of kudingcha made from Ilex kudincha C. J. Tseng and its four fractions of chloroform, ethyl acetate, n-butanol, and water were prepared. The total polyphenol contents and the antioxidant activity of the extracts were determined by Folin-Ciocalteu method and DPPH assay, FRAP assay, and TEAC assay, respectively. The results showed that kudingcha extracts contained high polyphenol content and showed potent antioxidant activity. The antioxidant activities of crude extract and its fractions decreased in the order of ethyl acetate fraction>butanol fraction>crude extract>water fraction>chloroform fraction according to the DPPH assay and FRAP assay, which were the same with the exception of the rank order of water fraction and chloroform fraction obtained from TEAC assay. Satisfactory correlations between the total polyphenol content and antioxidant activity and between measuring methods of antioxidant activity were observed. It suggests that the polyphenols in the extracts are largely responsible for the antioxidant activities.

Recent Advance on the Application of Near-infrared Spectroscopy in Tea

ZHOU Jian, CHENG HaoP, WANG Li-yuan

Journal of Tea Science. 2008, 28(4): 294-300. doi:10.13305/j.cnki.jts.2008.04.008

Abstract ( 592 )

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The recent advance on the application of Near-infrared spectroscopy in tea was introduced in this paper. Its advantage and disadvantage in comparing with the other analytical methods were also analyzed. The main reasons which limited the development of near-infrared spectroscopic technique was analyzed and the range of the application in tea was predicted in the paper. This article also gives some advices about the development of near-infrared spectroscopy in tea.

Evaluation of Nitrogen Status in Tea Plants by SPAD

YANG Yi-yang, MA Li-feng, SHI Yuan-zhi, RUAN Jian-yun, LI Xing-hui

Journal of Tea Science. 2008, 28(4): 301-308. doi:10.13305/j.cnki.jts.2008.04.007

Abstract ( 442 )

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Determination of nitrogen concentrations in tissues is an important diagnostic means for N status of plants. A pot experiment with tea plants applied with different amounts of N fertilizer was carried out to evaluate the usefulness of SPAD-502 as a diagnostic tool. The results showed that the yield of young shoots and N concentration of mature leaves responded to N application rates with a pattern of an initial linear increase before a platform was reached. There was a significantly positive linear relationship between the SPAD reading and total N concentration of mature leaves. The similar pattern of an initial linear increase followed by a platform was observed for the relationship between SPAD reading and the yield. These results suggested that the N status of tea plants may be characterized by the measurement of SPAD reading of mature leaves and the SPAD is a potentially useful rapid diagnostic tool in N fertilization recommendation. However, this needs further validation from field experiments. Those factors which may influence the precision of SPAD measurement, such as the point of test on a leaf, selection of leaves from twig (position), weather condition, in situ or in vitro analysis, surface cleaning and the number of leaves for a composite sample were also investigated.

Studies on the Extraction of Tea-leaf Protein

CAI Zhi-ning, WANG Chun-yan, LIANG Cui-jin, LI Qing-yu

Journal of Tea Science. 2008, 28(4): 309-312. doi:10.13305/j.cnki.jts.2008.04.001

Abstract ( 416 )

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：The investigation was designed by using these reference factor including the solid to liquid ratio, extraction time, pH value and extraction temperature, as to determine the optimum technological parameters of tea-leaf protein extraction process and the optimum combination. Results showed that the optimum condition is as follows: solid to liquid ratio of 1:8, pH11, 60℃, 60 min. The protein extraction rate was 21.89% under these conditions. The extraction process of tea protein and the equipment used are simple. The optimal pH of sedimentation is pH 4.0.

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