Journal of Tea Science

Clone, Expression and Functional Analysis of Dihydroflavonol 4-Reductase Gene of Tea Plant (Camellia sinensis)

WANG Yun-sheng, XU Yu-jiao, HU Xiao-jing, JIANG Xiao-lan, YANG Qing, LI Wei-wei, LIU Ya-jun, GAO Li-ping, XIA Tao

Journal of Tea Science. 2013, 33(3): 193-201. doi:10.13305/j.cnki.jts.2013.03.010

Abstract ( 508 )

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Dihydroflavonol 4-reductase (DFR) is a key enzyme in the biosynthesis of catechins in tea plant. However, the functions and the zymologic properties of DFR were not deeply identified in recent researches. The open reading frame of DFR gene, which encoding a 347 amino acids protein, was cloned from tea plant (Camellia sinensis) by RT-PCR. The deduced protein molecular weight was 38.69βkD and its theoretical isoelectric point was 6.02. The gene was cloned into the expression vector SUMO for expression in prokaryotic cells. The SDS-PAGE results showed that the dihydroflavonol 4-reductase peoteins was expressed in Escherichia coli BL21. The optimal inducing conditions including time, temperature and IPTG concentration were studied. The deduced protein was purified and its activity was detected by HPLC-MS method. The results indicated that purified protein showed the DFR activity, catalyzed the reduction reaction of DHQ and DHM. The research provides a valuable foundation for better understanding the substrate specificity and enzymatic properties of CsDFR.

The Gene Cloning and Expression Analysis of SCPL in Tea Plant (Camellia sinensis)

QIU Chuan-hui, LI Wei-wei, WANG Yun-sheng, LI Ming-zhuo, LUO Yang, LIU Ya-jun, GAO Li-ping, XIA Tao

Journal of Tea Science. 2013, 33(3): 202-211. doi:10.13305/j.cnki.jts.2013.03.011

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In recent years, serine carboxypeptidase-like proteins (SCPL) have been found that they are involved in plant secondary metabolites with the function of transferring acyl. The full-length cDNA of three CsSCPL were cloned from Camellia sinensis by RT-PCR technology. Bioinformatics analysis showed that the three CsSCPL proteins contained SCPL family's characteristic structures, such as one substrate binding and three catalysis conserved regions, a number of N-glycosylation sites and a conserved catalytic triad Ser-Asp-His amino acid active catalytic site and so on. The phylogeny analysis showed that CsSCPL probably possessed acyltransferase function. Quantitative RT-PCR analysis showed that the CsSCPL genes expressed in bud, leaf, stem and root. The relative expression of CsSCPL1 and CsSCPL3 in the leaves was significantly higher than that in stems and roots, while CsSCPL2 was highly expressed in the root. The CsSCPL genes were constructed into expression vector pET-32a(+) for over expression in prokaryotic cells and optimal inducing conditions including time, temperature were studied. The SDS-PAGE showed that recombinant proteins with formula weight 70βkD were induced successfully by IPTG, which coincided with the prediction.

Progress in Plant Cold-stress-responsive miRNAs and the Application in Cold Resistance Research of Camellia sinensis

ZHU Quan-wu, FAN Kai, XIE Yan-lan, DONG Ji-fen, Zhan Yu-wen, LUO Yao-ping

Journal of Tea Science. 2013, 33(3): 212-220. doi:10.13305/j.cnki.jts.2013.03.007

Abstract ( 549 )

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MiRNAs(MicroRNAs) negatively regulate the expression of gene by targeting mRNA for cleavage or translational repression in a sequence-complementary dependent manner. Plant miRNAs not only involved in the regulation of growth and development but also played an important role in response to cold as well as other abiotic stresses. This article described plant miRNAs biogenesis and mechanism, and analyzed the effect of miRNAs in gene regulatory networks under cold stress. The knowledge for understanding the role of cold-stress-responsive miRNAs and the new ideas for improving cold tolerance of Camellia sinensis were provided in the paper.

Research on Enzymatic Characteristics of Anthocyanin Reductase of Tea Plant [Camellia sinensis (L.) O. Kuntze]

YANG Qin, ZHAO Lei, LIU Ya-jun, LIU Li, WANG Yun-sheng, GAO Li-ping, XIA Tao

Journal of Tea Science. 2013, 33(3): 221-228. doi:10.13305/j.cnki.jts.2013.03.008

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Anthocyanidin reductase (ANR) is a key enzyme in the biosynthetic pathway of proanthocyanidins(PAs), which catalyzes anthocyanidins into the corresponding 2, 3-cis-flavan-3-ols. For researching enzymatic characteristics of the enzyme, this study was carried out to express and purify the protein by prokaryotic expression and Cobalt ion affinity column purification. The optimal conditions of CsANR1 were observed at 40℃ and pH 6.5. The more substrate preference of CsANR1 was showed on cyanidin over delphinidin. Moreover, Cu²⁺, Co²⁺, Fe²⁺, Mn²⁺, Zn²⁺ and Hg²⁺inhibited the enzyme activity and the enzyme activity decreased 50% after storing 15 days.

Optimization and Application of Real-time Fluorescence Quantitative PCR for Detection of Ectropis obliqua Nucleopolyhedrovirus

YUAN Zhi-jun, ZHANG Chuan-xi, XIAO Qiang, YIN Kun-shan

Journal of Tea Science. 2013, 33(3): 229-236. doi:10.13305/j.cnki.jts.2013.03.006

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Primers were designed based on p16 gene from Ectropis obliqua nucleopolyhedrovirus in order to optimize the detection method of SYBR Green I real-time fluorescence quantitation PCR for EoNPV, and the standard curve of SYBR Green I real-time fluorescence quantitative PCR for p16 gene was established using the recombinant plasmid DNA as template. Statistic analysis showed that there was a good linear relationship between Ct value and the logarithmic value of plasmid concentrations (R²=0.9981). The sensitivity of the method was 10² copies/μl, and wide detection range of 6 orders of magnitude was obtained. Larave infected at different time were sampled and detected by the method, and the results showed that there was a good linear relation between the logarithmic value of the multiples of gene copies and the infection time (R²=0.9935). Experimental results indicated that the method can identify biological pesticides from qualitative and quantitative aspects, and distinguish the high homology nuclearpolyhedrosisvirus (Euproctis pseudoconspersa nuclearpolyhedrosisvirus) from EoNPV accurately.

Determination of Aflatoxins B1 in Tea by High Performance Liquid Chromatography-Fluorescence Detector with Post-column Photochemical Reaction

ZHAO Hao-jun, WANG Kun, YANG Wei-hua, YANG Chao-yi

Journal of Tea Science. 2013, 33(3): 237-241. doi:10.13305/j.cnki.jts.2013.03.001

Abstract ( 626 )

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A new method for the sensitive determination of Aflatoxins B1 in tea by high performance liquid chromatography with photoelectric reactor and fluorescence detector was established. A solution of V(acetonitrile)∶V(H₂O)=86∶14 was used to extract Aflatoxins B1 from tea. The extracted solution was then purified by a multifunctional and immuneaffinity column, respectively. The peak area and the concentration of Aflatoxins B1 showed a good linear relationship within the range from 0.591βμg/L to 5.91βμg/L with a linear correlation coefficient (r) of 0.9994. The recoveries at the concentrations studied [low level (7.090βμg/kg), high level (14.180βμg/kg)] were between 85.6% and 98.9% with a relative standard deviations ranging from 1.7% to 1.8%. The limit of detection (LOD) is 0.1βμg/kg (S/N=3). The limit of quantitation (LOQ) is (0.591βμg/kg). The new method was used to analyze eight tea samples collected from the local markets and negative results were obtained. The method is suitable for detection of Aflatoxins B1 in tea with high selectivity and sensitivity.

Research Advance on Mechanism of Regulating Lipid Metabolism by Active Ingredients of Dark Tea

CHEN Zhi-xiong, QI Gui-nian, ZOU Yao, LI Jian-hua

Journal of Tea Science. 2013, 33(3): 242-252. doi:10.13305/j.cnki.jts.2013.03.002

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The effect of regulating lipid metabolism by dark tea has been proved through various studies, while the active ingredients and mechanism of the effect are explored gradually. In this paper, the chemical and quantitative characteristics of the active and special ingredients in dark tea have been summarized, and the safety assessment of dark tea has also been made. Based on these, the way of regulating lipid metabolism by these ingredients has been discussed according to the researches on their mechanisms of function. The lipid-lowering mechanism of dark tea, and its multiple effect of various active ingredients were discussed and concluded in this paper.

Study on the Variation of Important Functional Compositon in the Tea Seeds on its Maturation Period

ZHENG De-yong, CHANG Yu-xi, YE Nai-xing, YANG Jiang-fan

Journal of Tea Science. 2013, 33(3): 253-260. doi:10.13305/j.cnki.jts.2013.03.012

Abstract ( 408 )

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The variation of important functional composition in the tea seeds, including the oil content, fatty acid composition of tea seeds oil, the content of vitamin E and squalene in tea seeds oil were studied by GC and HPLC on its maturation period. The tea seed samples come from 4 tea germplasms: Fuding dabaicha, Fuyun No.6, Huangdan and Rougui. The results showed that, the nutrients accumulated, the moisture content decreased and the oil content increased on the tea seed maturation process. The oil content increased more rapid at the earlier maturation stage, then slowed down at the later stage and reached a maximum value, the average seed oil content of these 4 germplasm was 24.24%. The vitamin E content in the tea seeds oil increased by the ‘dual wave peak’ pattern, and then increased fast at later stage to a maximum value, the vitamin E content of Fuyun 6 reached 1β060.29βμg/g. The squalene content of tea seeds oil increased at earlier maturation stage, decreased at later stage, the Rougui’s squalene content was 161.42 μg/g. The palmitic acid relative content in tea seeds oil decreased slowly, and the relative content of stearic acid in tea seeds oil increased slowly on the tea seeds maturation. The oleinic acid relative content in tea seeds oil increased firstly, decreased at medium stage, and then increased continually to the end. The oleinic acid relative content in matured tea seeds oil of Fuding dabaicha, Fuyun No. 6, Huangdan and Rougui was 50.43%、45.98%、52.62% and 52.98% respectively. The linoleic acid relative content in tea seeds oil decreased firstly, increased at medium stage, and then decreased continuing to the end. The linoleic acid relative content in matured tea seeds oil of Fuding dabaicha, Fuyun No.6, Huangdan and Rougui was 31.65%、27.36%、24.66% and 24.07% respectively.

Effects of Different Potassium Level on Leaf Photosynthesis of Tea Seedling

LIN Zheng-he, ZHONG Qiu-sheng, CHEN Chang-song, CHEN Zhi-hui, YOU Xiao-mei

Journal of Tea Science. 2013, 33(3): 261-267. doi:10.13305/j.cnki.jts.2013.03.013

Abstract ( 497 )

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Vegetative propagated 10-month-old tea [Camellia sinensis (L.) O. Kuntze cv. Ruixiang] seedlings grown in pots were fertilized three times weekly for 24 weeks with nutrient solution containing 0, 100, 200, 600 or 2β000βμmol/L potassium. The portable photosynthesis system (LI-6400) was used to determine leaf photosynthesis of tea. The result showed that K-deficient leaves showed decreased CO₂ assimilation and stomatal conductance, water use efficiency but increased intercellular CO₂ concentration. Result also showed that biomass, content of K and chlorophyll were decreased in K-deficient leaves. The tea seeding appeared potassium deficiency symptoms when fertilized with nutrient solution containing 0, 100βμmol/L potassium(K content of leave 6.63βmg/g, 6.85βmg/g).

Application of Tea Embedding Particle in Cigarette Holder

YAO Er-min, SONG Hao, LI Xiao, LI Jin-cheng, JING Tian, JI Xiao-nan, XU Xiu-feng

Journal of Tea Science. 2013, 33(3): 268-272. doi:10.13305/j.cnki.jts.2013.03.003

Abstract ( 427 )

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By using the PVA-H₃BO₃ cross-linking method to make tea embedding particles by imbedding the tea powder, using the cigarette holder which uses these particles as filter material to assess the smoking quality and investigating the conventional ingredients and chemical components of mainstream smoke. Compared with the blank control sample, the results indicated that the tea embedding particles could decrease irritancy, enhance cigarette flavor and improve aftertaste obviously. Total particulate matter (TPM) and tar were decreased 5.80%, 6.04% respectively, nicotine did not change significantly. The flavor, enhancing smoke concentration and mellowing smoke components were increased in different degree, such as benzyl alcohol, dihydro-actinidiolide,1-(2-furanyl)-ethanone,5-methyl- 2-furancarboxaldehyde, megastigmatrienone were increased 188.10%, 151.65%, 80.11%, 63.97% and 57.73% respectively, while the harmful components, such as 2-methyl-phenol, 2,3-dimethyl-Phenol, benzylnitrile and 2-methyl-9H-Fluorene decreased significantly, were reduced 38.47%、24.46%、60.09%、65.40% respectively.

Application Research on the Application of Heatpipe Technology to Tea Processing Energy Conservation

ZHENG Peng-cheng, TENG Jing, GONG Zi-ming, WU Yun-hui, MA Chi

Journal of Tea Science. 2013, 33(3): 273-278. doi:10.13305/j.cnki.jts.2013.03.004

Abstract ( 424 )

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In order to reduce the energy consumption during tea processing, the technology principle of heat pipe transferring heat and the characteristics of heat pipe heat exchangers were briefly introduced in this paper. The heat pipe technology was applied to the electron hot air ball-breaking dryer for waste heat recovery, and the results indicated that the exhausted temperature of electron hot air dryer was decreased from 103℃ to 50℃, the thermal efficiency was increased from 40.71% to 78.57% and the electricity was saved 25βkW·h in 1βh. The effect of energy conservation is remarkable.

Effects of Different Biomass Materials on the Amelioration of Acidic Tea Garden Soil

XIE Shao-hua, ZONG Liang-gang, CHU Hui, WANG Zhang-yi, QIU Xiao-lei, MA Ai-jun, HE Ren-hong

Journal of Tea Science. 2013, 33(3): 279-288. doi:10.13305/j.cnki.jts.2013.03.009

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The amelioration effects of different biomass materials applied to three acidic tea garden soils were examined with the incubation experiments. The results indicated that the pH of three soils (LC, LG and LX) increased by 0.44, 0.31 and 0.26 units averagely while the contents of soil exchangeable acidity, exchangeable aluminum, total soluble aluminum, and total monomeric aluminum decreased significantly after biomass materials treatment for 45 days. Among different biomass materials, the amelioration effects of cow dung, straw compost and commercial organic fertilizer were more prominent than the others. The straw carbon was more effective than other materials in increasing the exchangeable cation contents and the degree of soil base saturation, as well as the soil cation exchange capacity and soil organic matter contents. These results help to better understand the mechanisms of the amelioration.

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