Journal of Tea Science

Research Advance on Tea Biochemistry

WAN Xiaochun, LI Daxiang, ZHANG Zhengzhu, XIA Tao, LING Tiejun, CHEN Qi

Journal of Tea Science. 2015, 35(1): 1-10. doi:10.13305/j.cnki.jts.2015.01.002

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Tea biochemistry is a subject of life science on Camellia Sinensis. It mainly focus on characteristic secondary metabolites of tea plants, especially those in the fresh tea shoots using biochemical and molecular techniques as research tools. Those metabolites include catechins, caffeine, theanine and volatile terpenes, which contribute greatly to tea quality and healthy effects. The major research fields of tea biochemistry involve the biosynthesis of secondary metabolites in tea plants, their transformation during tea processing and their effects on final tea quality and healthy function. Recently, there have several breakthroughs in the metabolites biosynthesis pathway, tea genome, specific tea germplasm metabolome, the processing metabolome, as well as the chemistry of tea quality. Since tea biochemistry is the fundamental in tea science, its achievements provide the theoretical basis and method approaches to tea cultivation and breeding, tea primary and further processing, tea trading and tea culture. With the development of tea industry and life science & technology, it is believed that the advancement of tea biochemistry will push forward tea industry to a high level at the sustainable development.

Research Progress on Germplasms of Tea Plant (Camellia sinensis)

MA Jianqiang, YAO Mingzhe, CHEN Liang

Journal of Tea Science. 2015, 35(1): 11-16. doi:10.13305/j.cnki.jts.2015.01.003

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Tea germplasms are the necessary material bases for tea breeding, genetic research and production, as well as the drivers of sustainable development for tea industry. In this paper, the research progress on tea plant germplasm resources, including collection, conservation, evaluation and utilization, was reviewed, and the problems existed in the research on tea plant germplasm resources in China were proposed.

The Cloning and Prokaryotic Expression of Polyphenol Oxidase Gene in Pear (Pyrus Pyrifolia Nakai)

CHEN Dongsheng, WANG Kunbo, LI Qin, LI Juan, HUANG Jian′an, LIU Zhonghua

Journal of Tea Science. 2015, 35(1): 17-23. doi:10.13305/j.cnki.jts.2015.01.004

Abstract ( 488 )

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：The polyphenol oxidase (PPO, GenBank accession No. JQ861265) genes were cloned by PCR from Pyrus Pyrifolia Nakai. The full length of PPO gene was 1β782βbp without introns, coding a precursor peptide. PPO precursor consists of 593 amino acids with a molecular weight of about 65.8βkDa. It has a theoretical PI of 8.4 and has a transit peptide consisting of 47 amino acids. The mature PPO without transit peptide consists of 547 amino acids with a molecular weight of about 60.8βkDa and a theoretical PI of 6.69. There are 2 Cu-binding domain in the α-helix of PPOs. The protein accumulation got a peak in 3-6 hours. The induced proteins (precursor PPO and mature PPO) can oxidize catechins into theaflavins in vitro.

The Cloning of Transcription Factor Gene CsDREB-A4 and The Response to Temperature Stress in Camellia sinensis

LIU Zhiwei, XIONG Yangyang, LI Tong, YAN Yajun, HAN Hongrun, WU Zhijun, ZHUANG Jing

Journal of Tea Science. 2015, 35(1): 24-34. doi:10.13305/j.cnki.jts.2015.01.006

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The CsDREB-A4 gene, which encoding to the DREB transcription factor, was cloned by PCR using DNA as template from tea plant (Camellia sinensis) cultivar ‘Yingshuang’, based on transcriptome data of ‘Yingshuang’. The open reading frame lengths of CsDREB-A4 gene was 708βbp, encoding 235 amino acids. The transcription factor of CsDREB-A4 contained the AP2 DNA binding domain, and had high homology with DERB transcription factors of Glycine max, Solanum lycopersicum, Vitis vinifera, Arabidopsis thaliana, and so on. Phylogenetic tree, hydrophilicity, disordered residues, two- and three-dimension structure of CsDREB-A4 transcription factor were also predicted and analyzed. Results showed that the disordered residues of CsDREB-A4 factor were obvious. The three-dimension structure of CsDREB-A4 factor was similar to AtERF1. The CsDREB-A4 factor, the majority of amino acid were hydrophilic, belonged to A4 group of DREB subfamily of AP2/ERF family transcription factors. The three-dimension structure was similar to AtERF1, the CsDREB-A4 gene was induced by high temperature and low temperature treatment in ‘Anjibaicha’, ‘Yingshuang’ and ‘Yunnanshilixiang’, respectively. The expression levels of CsDREB-A4 gene reached a maximum at 24 h in the three tea plant cultivars, which increased 23, 4, 43 times when exposed at 4℃. In ‘Yingshuang’ and ‘Yunnanshilixiang’, the expression levels of CsDREB-A4 gene were up-regulated more longer time and higher than that in ‘Anjibaicha’. The expression level of CsDREB-A4 gene was inhibited in ‘Yingshuang’ and ‘Yunnanshilixiang’ when exposed at 38℃ temperature, except at 8 h. In ‘Anjibaicha’, the expression level of CsDREB-A4 gene was increased obviously, and reached 2 720 times in 12 h than that in wild type tea plant.

The Gene Cloning and Expression Analysis of C4H in Tea Plant (Camellia sinensis)

YAO Shengbo, WANG Wenzhao, LI Mingzhuo, XU Yujiao, WANG Yunsheng, LIU Yajun, GAO Liping, XIA Tao

Journal of Tea Science. 2015, 35(1): 35-44. doi:10.13305/j.cnki.jts.2015.01.007

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Cinnamate 4-hydroxylase (C4H) is a key enzyme in the phenylpropanoid pathway in tea plant. The gene can influence the biosynthesis of secondary metabolites such as lignin and flavonoids. The cDNA full-length of C4H gene was cloned from tea plant by rapid amplification of cDNA ends with a 1β518βbp open reading frame encoding a protein of 505 amino acids. The deduced protein molecular weight was 58.15βkD and its theoretical isoelectric point was 9.29. A 1β840βbp promoter sequence was isolated by genome walking method. The promoter region not only has the basic transcriptional elements of TATA-box and CAAT-box, but also has many potential inducible and tissue-specific cis-acting elements. Quantitative RT-PCR analysis showed that the CsC4H gene expressed in bud, leaf, stem and root. The gene was cloned into the expression vector pYES-DEST52 for eukaryotic expression in Saccharomyces cerevisiae WAT11. The enzyme reaction products were detected by LC-MS method. The results indicated that cinnamic acid was para-hydroxylated by target proteins to generate p-coumaric acid.

Cloning and Expression Analysis of Auxin Receptor Gene CsTIR1 in Tea Plant (Camellia sinensis)

CAO Hongli, YUE Chuan, ZHOU Yanhua, WANG Lu, HAO Xinyuan, ZENG Jianming, YANG Yajun, WANG Xinchao

Journal of Tea Science. 2015, 35(1): 45-54. doi:10.13305/j.cnki.jts.2015.01.009

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Indole-3-acetic acid (IAA or auxin), functioning via its signal transduction, plays a pivotal role in plant growth and development regulation. Transport inhibitor response 1 (TIR1) protein, an auxin receptor, is one of the most critical components in IAA signaling pathway. The full-length cDNA sequence of CsTIR1 gene was obtained by using RACE technique, and submitted to GenBank with accession number JX050147. The CsTIR1 cDNA length was 2β315βbp, and contained a 1β746βbp open reading frame (ORF), encoding 581 amino acid residues. The molecular weight and theoretic isoelectric point of CsTIR1 protein are 65.18 kD and 5.64, respectively. In addition, CsTIR1 protein had the highest sequence similarity about 82% and the closest genetic relationship to Nicotiana tabacum. The CsTIR1 was predicted to contain one F-box and six leucine-rich-repeat (LRR) domains, which forming the ‘stem’ and ‘cap’, respectively. And its tertiary structure is shaped as a mushroom. Semi-quantitative RT-PCR results suggested that CsTIR1 expression showed a tissue-specificity among root, stems, leaves and flowers. The further investigation indicated that the transcript of CsTIR1 was regulated by phytohormones. In a time-course assay, it was found that CsTIR1 was significantly up-regulated when tea plant treated with three different IAA concentration and various plant hormones (ABA, GA₃, MeJA and BR), and showed the highest expression level under 50 μmol·L^-1 IAA concentration. Finally, the expression of CsTIR1 was detected in bud dormancy-active cycle during winter and CsTIR1 showed a low transcription level in dormant buds but expressed abundantly in active buds.

Cloning and Expression Analysis of GDP-D-mannose Pyrophosphorylase cDNA in Camellia sinensis

XIAO Yao, ZHOU Tianshan, LI Jiao, ZHANG Jiaxin, YU Youben

Journal of Tea Science. 2015, 35(1): 55-63. doi:10.13305/j.cnki.jts.2015.01.010

Abstract ( 427 )

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Ascorbic acid (Vitamin C, Vc) is an important antioxidant in tea plant, playing important roles in metabolism and responses to abiotic stress, and tea quality is positive correlation with the content of Vc. GDP-D-mannose pyrophosphorylase (GMP) is an important enzyme in the synthesis of ascorbic acid. The full-length cDNA sequence of GMP gene was isolated from the shoots of Camellia sinensis by RT-PCR and RACE. The entire GMP cDNA was 1β510βbp, containing a 1β086βbp complete open reading frame which encoding a protein with 361 amino acids and a calculated molecular weight of 39.599βkDa. Blast analysis showed that GMP gene in Camellia sinensis was most closely to Actinidia with 96% amino acids similarity. Quantitative real-time PCR analysis showed that the expression levels of GMP gene in the third leaves were highest while the stem is the lowest, and the different varieties also existed an obvious differences. High temperature stress stimulate the expression of GMP gene and the accumulation of Vc in incipient stage, then reduced rapidly.

Gene Cloning and Expression Analysis of GAGP in Tea Plant

LI Yuanhua, LU Jianliang, FAN Fangyuan, SHI Yutao

Journal of Tea Science. 2015, 35(1): 64-72. doi:10.13305/j.cnki.jts.2015.01.012

Abstract ( 515 )

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By using SSH, the differences in gene expression of root from Fudingdabai tea plant infected by VA mycorrhiza were analyzed and the diversity sequences was obtained. The sequence alighment showed that the down-regulated expression sequence possibly contained 10 unknown genes and the up-regulated expression sequence possibly contained 5 known genes. The GAGP (gap-pol) genic full-length sequence was obtained by using RACE. The length of GAGP gene was 3146bp (GenBank, Accession no., KJ946251), with 2β769βbp ORF (1^st-2β769^th), the sequence encoded 923 amino acid. Bioinformatics indicated that the GAGP protein’s molecular weight was about 106.9βkD, IEP was 8.42, located in mitochondria. The study also showed expression degree of GAGP was distinct in different cultivars, while it responded obviously to biological and non-biological stress.

Effect of Withering Temperature on Dynamic Changes of Main Biochemical Components and Enzymatic Activity of Tea Fresh Leaves

HUA Jinjie, YUAN Haibo, WANG Weiwei, JIANG Yongwen, LIU Qianlu, CHEN Gensheng, WANG Fang

Journal of Tea Science. 2015, 35(1): 73-81. doi:10.13305/j.cnki.jts.2015.01.014

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By using of two leaves and a bud shoot as fresh tea leave material, setting three different withering temperature (20, 28, 36℃), sampled leaves with different moisture content, then the contents of tea polyphenols, total amino acids, flavonoids, total soluble protein, soluble sugar, PPO activity, and POD activity were determined to study the effect of withering temperature on dynamic changes of main biochemical components and enzymatic activity of tea fresh leaves, and the effects of different withering temperature on main biochemical components of rolled leaves, fermented leaves and fired tea were also analysed. The results show that with the increasing of withering time, moisture content of tea fresh leaves decrease gradually, the contents of tea polyphenols, total soluble protein and soluble sugar decrease gradually accordingly, and the contents of tea polyphenols were no difference between 28℃ and 36℃, but are obviously higher than that in 20℃; the contents of soluble protein and soluble sugar are negatively correlated with temperature; the contents of total amino acids and flavonoids increase gradually, and are positively correlated with temperature; the released amount of CO2 decreased at first, then increase and decrease finally, and the amount is the largest when the withering light temperature is 36℃; polyphenol oxidase activity shows a slow downward trend, peroxidase activity shows an upward trend, and they are the highest at 28℃. The contents of theaflavins, thearubigins, and polyphenols are also the highest at 28℃; the results of sensory evaluation also shows that the liquor color and taste of black tea at 28℃ had the highest score, with the best quality.

Effects of Different Nitrogen Application Rates on Nitrification and pH of Two Tea Garden Soil

WANG Feng, CHEN Yuzhen, YOU Zhiming, WU Zhidan, JIANG Fuying, ZHANG Wenjin, WENG Boqi

Journal of Tea Science. 2015, 35(1): 82-90. doi:10.13305/j.cnki.jts.2015.01.016

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Two tea garden soils (yellow soil and red soil) derived from Wuyishan City were incubated at 25℃with a soil moisture content of 60% water holding capacity (WHC) for 46 days. The dynamics of nitrification and pH of soil after urea application at different rates were measured. The results show that urea is hydrolyzed fast (2-6βd) with a high nitrification activity in yellow soil. However, the urea is hydrolyzed relatively slow (16βd) in red soil. The nitrification rate in two tea garden soils are 81.32% and 73.48% respectively, and the nitrification activity in yellow soil is significantly higher than that in red soil (P<0.05). With or without nitrogen application, changing tendency of the contents of NO₃^-N in two tea garden soils show as ‘J’ type with an obvious delay step for 11-16 days. The nitrification in these tea garden soils follows the exponential model N=N₀e^kt(P<0.01). Compared with the control treatment, the net amount of nitrified nitrogen, net nitrification rate and k value increase with the urea N application rates, however, the nitrification rates decreased with the urea N application rates. With or without Nitrogen Application, the N₂O fluxes from yellow tea garden soil were higher than those from red tea garden soil(P<0.05). Compared with the control treatment, soil pH decreased by 0.16-0.52 and 0.11-0.25 pH units with the urea N application rates. These results indicated that two highly acidic tea garden soils showed high nitrification activity(delay step were 11-16 days), and be conducive to absorb NH₄⁺-N. Urea application could decrease soil pH, and the nitrogen application rate is one of the main factors accelerating acidification of tea garden soil.

Preliminary Research on Induced Biodegradation of PCBs of Tea Saponin

LI Xuelian, FANG Zhiguo, XIA Huilong

Journal of Tea Science. 2015, 35(1): 91-96. doi:10.13305/j.cnki.jts.2015.01.017

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Two bacterial strains named temporally as WTS and YTS were separated from the soil, and they could utilize tea saponin for growing and showed significant promotion effect on the biodegradation of PCBs. Under the condition of coexistence with tea saponin, the degradation rate constants of PCB 77, PCB 118 and PCB 138 were increased by 4.3 times, 4.8 times and 2.8 times, respectively, by WTS, and 7.1 times, 9.1 times and 8.9 times. respectively, by YTS compared to those treatments without tea saponin. The results indicated that tea saponin could promote the biodegradation of PCBs, implying that tea saponin might be a potential inducer for the biodegration of PCBs.

Screeining and Identification of the Endophytic Bacterial Strain Against Ice Nucleation Active Bacteria of Tea Plant

HUANG Xiaoqin, ZHANG Lixia, LIU Huixiang, CHEN Zongmao, LI Duochuan

Journal of Tea Science. 2015, 35(1): 97-102. doi:10.13305/j.cnki.jts.2015.01.018

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In order to protect the tea plant against freezing injury, the screening of antagonistic bacteria against INA bacteria No. 8 (Pantoea ananatis) and No. 14 (P. agglomerans) isolated from tea plant was carried out in Shandong Province. The strain Y1,an antagonistic bacteria against INA bacteria was isolated from the endophytic bacteria strains of tea plant. Based on the morphological characteristics and 16βS rDNA sequence analysis, the strain Y1 was identified as the Bacillus amyloliquefaciens. Through this study, the endophytic bacteria strain against the ice nucleation bacteria was obtained and identefied, it will be beneficial to the development of the biological control of ice nucleation bacterial.

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