Journal of Tea Science

Identification and Expression Analysis of Tea Plant (Camellia sinesis) miR164a and Its Target Gene

KONG Lei, ZHU Xiangxiang, WANG Yiwei, XIE Xiaofang, JIANG Changjun, LI Yeyun

Journal of Tea Science. 2018, 38(6): 547-558. doi:10.13305/j.cnki.jts.2018.06.001

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Plant miR164 is involved in complex physiological processes such as plant growth and development regulation and stress responses by negatively regulating the target gene NAC transcription factors. The identification, cloning and sequence analysis of miR164a and its target gene NAC1 in tea plant were carried out by high-throughput sequencing of microRNA and degradome analysis. The expression patterns of Csn-miR164a and its target gene CsNAC1 during leaf development and under high or low temperature stresses were also studied. The length of Csn-miR164a precursor is 126βbp, and the mature sequence is 21βbp. The CsNAC1 ORF is 912βbp, encoding 303 amino acids. There are a typical NAM domain at the N terminus of CsNAC1 protein and a Csn-miR164a recognition site at the C-terminus. Quantitative real-time PCR analysis showed that the expression of Csn-miR164a was the highest in the bud and the lowest in the seventh leaves. Under high and low temperature treatments, the expression of Csn-miR164a was down regulated, while the expression level of CsNAC1 increased. The expression of Csn-miR164a was negatively correlatied with the expression of the target gene CsNAC1. The results revealed that Csn-miR164a and its target gene CsNAC1 might be closely related to the growth and stress responses in tea plant.

Cloning and Expression Analysis of Na⁺/H⁺ Antiporter Gene CsNHX1 and CsNHX2 in Tea Plant (Camellia sinensis)

CHEN Jiangfei, YU Jinming, YANG Jiankun, YU Youben, XIAO Bin, YANG Yajun, WANG Weidong

Journal of Tea Science. 2018, 38(6): 559-568. doi:10.13305/j.cnki.jts.2018.06.002

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The Na⁺/H⁺ antiporter (NHX) plays an important role in plant growth, development and stress response. In this study, the full-length cDNA sequences of CsNHX1 and CsNHX2 (GenBank: MG722977 and MG515211) were cloned from tea cultivar ‘Longjingchangye’. Bioinformatics analysis showed that the full-length cDNA of CsNHX1 and CsNHX2 were 1β691βbp and 1β757βbp, all containing a 1β626βbp open reading frame and encoding 541 amino acids. The molecular weights of CsNHX1 and CsNHX2 were 59.5βkD and 59.7βkD and pI were 7.07 and 8.79, respectively.The results of protein sequence analysis showed that CsNHX1 and CsNHX2 contained the conserved Na⁺/H⁺ exchange domain, and belong to the typical transmembrane proteins. Phylogenetic analysis of plant NHX revealed that CsNHX1 and CsNHX2 are the member of Class I that localized to the vacuolar membrane in IC subfamily. In addition, qRT-PCR results showed that the expressions of CsNHX1 and CsNHX2 were induced by drought, low-temperature and salt stress. In contrast, exogenous ABA could not induce the expressions of CsNHX1 and CsNHX2. In addition, the expression level of CsNHX1 in tea plant decreased significantly, but that of CsNHX2 increased gradually under heat stress, indicating that CsNHX1 and CsNHX2 were differently involved in tea plant responding to environmental stress, and possibly through different responding modes.

Isolation and Functional Analysis of Promoter for N-methyltransferase Gene Associated with Caffeine Biosynthesis in Tea Plants (Camellia sinensis)

LIU Ping, REN Qiujing, KANG Xin, ZHANG Yuanyuan, LIN Xiaorong, LI Bin, GAO Xiong, CHEN Zhongzheng

Journal of Tea Science. 2018, 38(6): 569-579. doi:10.13305/j.cnki.jts.2018.06.003

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Caffeine is an important bioactive compound in tea plant, and N-methyltransferase (NMT) is the key enzyme for its biosynthesis. In this research, the promoter of NMT1 was isolated from Camellia sinesis Yinghong 9 by hiTAIL-PCR, and its cis-elements were analyzed by Plant CARE and other online software. According to cis-elements composition, serial 5' shorten promoter were re-amplified from NMT1 promoter and cloned into pBI121 vector fusion with GUS gene respectively. The constructed vectors then transformed into tobaccos. The function of cloned promoter and its response to environmental factors were analyzed by GUS staining and quantitative PCR. The results showed, the isolated NMT1 gene promoter was 767βbp, containing basic cis-acting elements such as TATA-box, CAAT-box and some other response elements involved in the plant abiotic stress. Using recombinant technique, the CaMV35S promoter in pBI121 was replaced by the 5' shorten NMT1 promoters, resulted in 4 new vectors fusion with GUS and marked as pA, pB, pC and pD. The transient expression with the four constructed vectors in tobacco leaves revealed that NMT1 promoters with different lengths all could regulate the expression of GUS, and the activity of GUS enhanced along with promoter length increased. The pA vector which owned full cloned NMT1 promoter was selected for transgenosis and successfully gained transgenic tobacco. Expression of GUS could be detected in different tissues of transgenic tobacco, The expression level followed leaf > stem > root and the expression level in leaves reached 3 times higher than that in roots. When transgenic tobacco treated with light, temperature, simulated drought and abscisic acid, there were significant changes in the expression of GUS in leaves, except at the temperature of 40℃. The results showed that the function of NMT1 promoter was influenced by the environmental stresses.

Cloning and Functional Analysis of the CsMYB Promoter In Tea Plant (Camellia sinensis L.)

ZHENG Shizhong, JIANG Shengtao, LIU Wei, CHEN Meixia, LIN Yuling, LAI Zhongxiong, LIN Jinke

Journal of Tea Science. 2018, 38(6): 580-588. doi:10.13305/j.cnki.jts.2018.06.004

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The 1β828βbp full-length gDNA of CsMYB and a 1β038βbp 5′-flanking sequence of CsMYB (named proMYB) were cloned from the shoots of tea cultivar ‘1005’by gDNA cloning and genome-walking method. Bioinformatics analysis showed that the gDNA of CsMYB contained 2 introns and 3 extrons. The cis-acting element analysis showed that the proMYB promoter contained 5′-UTR Py-rich stretch motifs, TATA-box, CAAT-box core elements putative elements responding to hormone, light, stresses and some cis-elements with unknown or specific function, suggesting the inducible character of proMYB. It was also implied that CsMYB might be involved in abiotic stress responses and hormonal signal transduction in tea plant. The transient expression in transformed tobacco showed that the proMYB could drive the expressions of downstream genes.

Determination of 2,4-Epibrassinolide in Different Teas by Ultra Performance Liquid Chromatography Coupled to Tandem Mass Spectrometry with Dispersive Solid Phase Extraction

ZHU Li, CHEN Hongping, CHAI Yunfeng, MA Guicen, HAO Zhenxia, WANG Chen, LIU Xin, LU Chengyin

Journal of Tea Science. 2018, 38(6): 589-594. doi:10.13305/j.cnki.jts.2018.06.005

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An efficient method for analyzing 2,4-Epibrassinolide in different teas was developed based on ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). Homogenized samples were extracted with acetonitrile solution, cleaned up with three sorbents including C₁₈, strong anion exchanger (SAX) and graphitized carbon black (GCB). The compound was separated on a HSS T3 column using electrospray Ionization (ESI) mode, detected by scheduled multiple reaction monitoring (SMRM) and quantified by matrix-matched external standard curves. The compound showed good linearity between the concentration of 0.8-800βμg·L^-1, with high significant correlation (R²>0.999). The limit of quantitation (LOQ, S/N=10) ranged from 0.55 to 1.46βμg·kg^-1. In addition, the spiked (20, 40, 200βμg·kg^-1) recoveries of tea samples were 75.5%-93.6%, and RSDs were 0.4%-7.0%. The method was accurately and sensitively applied to commercial tea samples, which fulfilled all requirement for the determination of 2,4-Epibrassinolide in tea samples.

Determination of 21 Pesticide Residues in Tea by Ultra Performance Liquid Chromatography Orbitrap High Resolution Mass Spectrometry

JIN Lili, HAO Zhenxia, GAO Guanwei, CHAI Yunfeng, WANG Chen, CHEN Hongping, LU Chengyin

Journal of Tea Science. 2018, 38(6): 595-605. doi:10.13305/j.cnki.jts.2018.06.006

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A ultra performance liquid chromatography orbitrap high resolution mass spectrometry method for the determination of 21 pesticides in tea was developed. The tea samples were extracted by methanol-water (V_methanol∶V_water=1∶1), and cleaned up by a mixture of adsorbents containing primary secondary amine and strong cation exchanger. The chromatography analysis was performed on a C₁₈column. The target analytes were analyzed by high resolution mass spectrometry under Full Scan mode. The results showed that the linearities of 21 pesticides ranged from 0.5βμg·L^-1 to 200βμg·L^-1 with correlation coefficients higher than 0.99. At the spiked levels of 10, 50 and 100βμg·kg^-1, the average recoveries were from 70% to 125%, with the relative standard deviations (RSDs) below 15%. The limits of quantitation were 10βμg·kg^-1. The method is simple, rapid, sensitive and appropriate for the determination of 21 pesticide residues in tea.

Research Advance on Biosynthesis, Regulation and Function of Anthocyanins in Tea Plant

PANG Dandan, ZHANG Fen, ZHANG Yazhen, WEI Kang, WANG Liyuan, CHENG Hao

Journal of Tea Science. 2018, 38(6): 606-614. doi:10.13305/j.cnki.jts.2018.06.007

Abstract ( 524 )

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Anthocyanins, a type of polyphenols and natural antioxidants, can benefit human health such as lowering blood lipids, pressure and against cancer. Purple tea cultivars enriched in anthocyanins, attract increasing attention in the market. Based on recent studies of anthocyanins, the types, biosynthesis pathway and physiological functions of anthocyanins in tea plants, as well as the internal and external factors affecting the biosynthesis of anthocyanins in tea plant were reviewed, which would provide theoretical foundation for the development and utilization of tea anthocyanins.

Antioxidant and Anti-aging Activities of White Tea

ZENG Qi, REN Fazheng, LEI Xingen, HOU Caiyun

Journal of Tea Science. 2018, 38(6): 615-624. doi:10.13305/j.cnki.jts.2018.06.008

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To in vitro and in vivo evaluate and compare the antioxidant and anti-aging activities of white tea with different sources of raw materials, Baihaoyinzhen (YZ), Baimudan (MD), Shoumei (SM) were prepared and extracted to measure the main efficacy components. In vitro antioxidant assay, the antioxidantβactivitiesβwereβ also tested. For in vivo anti-aging test, the mice were subcutaneously injected with D-galactose and separately orally administered white tea extracts for aging model analysis. The contents of main efficacy components in YZ extract were higher than those in MD and SM extracts. The contents of tea polyphenols and flavonoids in SM extract were higher than those in MD extract. In vitro, YZ extract had better antioxidant capacity, and there was no obvious difference between SM and MD extract. In vivo, the degree of liver lipid peroxidation in YZH and YZL mice were significantly (P<0.05) decreased. The antioxidant enzyme activities were significantly (P<0.05) increased in serum of SMH and SML mice. But it did not show a dose-dependent manner. The 3 kinds of white tea extracts could up-regulate the expression levels of antioxidant enzymes and anti-aging genes to improve the oxidative stress state and it showed a dose-dependent manner. And there was no hepatotoxicity in these group mice.

Studies on the Identification and Isolation of Lactobacillus plantarum ssp. plantarum JJZ21 and Its Fermentation Properties

JIANG Jinzhong, YANG Pengming, WANG Zibu, MU Ande, WANG Xingchun

Journal of Tea Science. 2018, 38(6): 625-633. doi:10.13305/j.cnki.jts.2018.06.009

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To clarify the fermentation properties of microorganisms in fermentation layering of tea-seed-water-milk (TSWM), identification and isolation of microorganism in 5βh fermented TSWM were conducted. Based on morphological observation, 16βS rDNA, pheS gene sequencing and comparison, and phylogenetic tree analysis, a bacterial strain isolated from the fermented TSWM was identified as a strain of Lactobacillus plantarum ssp. Plantarum and named as L. plantarum ssp. Plantarum JJZ21 (LPJJZ21). Its culture preservation number was CCTCC M 2016471. In the fermentation process of TSWM, the total number of LPJJZ21 was increased rapidly after 5βh, peaked at 12βh to 15βh, then gradually reduced until 22βh, finally stayed stable after 22βh. Accompanied by the increasing of LPJJZ21, the dry matter, soluble sugar, soluble protein contents and pH decreased significantly, with highly significant (P<0.01) and negative correlation. Through consuming the soluble protein, soluble sugar and other substances in fermented TSWM, JJZ21 secreted acidic organic matter (such as lactate acid and so on), leading to the gradual decrease of pH in fermented TSWM, which were the prerequisite for the floating up of fat bodies in fermented TSWM and fermentation layering of TSWM.

A Preliminary Research on Identification and Simulated Fermentation of the Mucor Fungi from Hubei Green Brick Tea

WANG Chunyan, XIAO Changyi, LI Shigang, TU Xuan, HE Jiangang

Journal of Tea Science. 2018, 38(6): 634-642. doi:10.13305/j.cnki.jts.2018.06.010

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This research had been carried out to explore the main fungi in the green brick tea fermentation heap. The fungi from Hubei green brick tea were separated and purified through gradient dilution method. One fungus was identified based on morphological characteristics and the sequence alignment of ITS gene and LSUrDNA. The mice intragastric administration was operated with the fungi as dry powder to analyze the toxicological action. In addition, the strain LC-2 was used to simulate fermentation experiment. The results indicated that, the morphological characteristics of strain LC-2 was highly similar to Mucor racemosus Fres. Phylogenetic analyses showed the similar results. No toxicological action was observed in the mice after oral administration. The simulate fermentation tea tasted sweet and soft, and the liquor color seemed reddish yellow. There was no significant difference between the quality of normal green brick tea and the simulate fermentation tea. And the strain LC-2 was proved to be the main fungus participated in the fermentation of green brick tea, which could be used in the automated production of green brick tea.

Study on the Moderate Scale of Tea Family Farm—Based on the Survey Data of Anhui Province

FU Lei, SUN Tong, XIA Tao, WEN Xiaomeng

Journal of Tea Science. 2018, 38(6): 643-651. doi:10.13305/j.cnki.jts.2018.06.011

Abstract ( 509 )

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Based on the scientific definition of tea family farm, the DEA model was used to analyze the operational efficiency of 100 tea family farms in Lu'an, Anhui Province. Further, the evaluating system of the moderate scale of tea family farm was constructed by AHP, and the scale questions of the tea family farm were discussed. The results showed that 16 out of 100 tea family farms were in the effective stage of scale, and the comprehensive effectiveness of 2-2.667βhm² were much higher than 0-0.667βhm²and 6.667βhm² or above. Therefore, the moderate scale of the tea family farm in the main tea producing areas of Anhui Province was 2-2.667βhm². Based on this, a number of suggestions to improve the land circulation system, train new tea family farmers, carry out a variety of forms of training, improve the service system of agricultural socialization and improve the support policy were proposed.

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