茶树CsPLK基因的鉴定与表达分析

茶树中富含茶氨酸、儿茶素和咖啡碱等重要功能成分,具有较高的价值功效,茶树在生命周期中经常遭受逆境胁迫,维生素B6(VB6)在植物体内参与逆境应答,吡哆醛激酶(pyridoxal kinase,PLK)是VB6补救途径中的关键酶。为进一步了解PLK在茶树生物合成中的功能和作用机理,该研究基于茶树基因组数据库,以龙井43为材料,采用逆转录PCR(RT-PCR)的方法从茶树中克隆出CsPLK的基因。结果表明:该基因序列长为1 179 bp,编码393个氨基酸; CsPLK蛋白和已知物种中PLK蛋白具有较高的同源性,都是核糖激酶超家族成员;通过构建pET-CsPLK载体进行原核表达,并鉴定出重组蛋白有很强的催化活性;组织表达特异性分析表明,叶中的表达量比茎、根的高,在根中最低;荧光定量PCR表示,低温诱导CsPLK上调表达,干旱诱导CsPLK下调表达,发现该基因在茶树中有明显的逆境应答,推测CsPLK在茶树的生长发育、逆境胁迫发挥重要作用。     

The Relationship between Branch Canker and Twig Dieback of Tea Caused by Macrophoma theicola

During a recent survey of the inoculum source of twig dieback of tea caused by Macrophoma theicola, symptoms of canker were observed on branches of a number of tea cultivars. Most cankered areas contained minute black pycnidia of M. theicola, which ooze white conidial masses in strands when incubated under moist conditions. Macrophoma theicola was also consistently isolated from cankered branches. When healthy branches of tea were inoculated with conidia of M. theicola obtained from diseased branches, cankers similar to those occurring in nature developed in the inoculated areas, and M. theicola was re‐isolated from all experimentally induced cankers. Isolates derived from conidia of M. theicola also caused dieback on healthy tea twigs, indicating that fruiting bodies on the cankered branches are the main inoculum source of twig dieback of tea. There was a positive correlation between the severity of twig dieback and the prevalence of branch canker among the tea cultivars surveyed. Six of the cultivars surveyed were highly resistant to M. theicola.     

Nonaqueous fractionation and overexpression of fluorescent‐tagged enzymes reveals the subcellular sites of L‐theanine biosynthesis in tea

l ‐Theanine is a specialized metabolite in the tea (Camellia sinensis) plant which can constitute over 50% of the total amino acids. This makes an important contribution to tea functionality and quality, but the subcellular location and mechanism of biosynthesis of l ‐theanine are unclear. Here, we identified five distinct genes potentially capable of synthesizing l ‐theanine in tea. Using a nonaqueous fractionation method, we determined the subcellular distribution of l ‐theanine in tea shoots and roots and used transient expression in Nicotiana or Arabidopsis to investigate in vivo functions of l ‐theanine synthetase and also to determine the subcellular localization of fluorescent‐tagged proteins by confocal laser scanning microscopy. In tea root tissue, the cytosol was the main site of l ‐theanine biosynthesis, and cytosol‐located CsTSI was the key l ‐theanine synthase. In tea shoot tissue, l ‐theanine biosynthesis occurred mainly in the cytosol and chloroplasts and CsGS1.1 and CsGS2 were most likely the key l ‐theanine synthases. In addition, l ‐theanine content and distribution were affected by light in leaf tissue. These results enhance our knowledge of biochemistry and molecular biology of the biosynthesis of functional tea compounds.     

施肥对油茶园土壤呼吸和异养呼吸及其温度敏感性的影响

油茶是中国南方重要的木本食用油料树种,研究施肥对油茶园土壤呼吸及其温度敏感性的影响,对于估算中国南方典型种植园林温室气体排放及其对气候变化的响应具有重要意义。设置对照(CK)、施肥(OF)、断根(CK-T)和断根施肥(OF-T)4个处理,采用静态箱-气相色谱法,通过多年观测,分析探讨施肥对油茶园土壤呼吸和异养呼吸及其温度敏感性的影响。结果表明:(1)施肥对油茶园土壤呼吸和异养呼吸无显著影响。研究期间,各处理(OF、CK、OF-T、CK-T)土壤CO_2通量依次为(77.91±2.59)、(73.71±0.97)、(66.82±1.02)mg C m~(-2)h~(-1)和(66.84±3.94)mg C m~(-2)h~(-1);(2)各处理土壤呼吸温度敏感性(Q_(10))表现为OF-T(1.96±0.01)CK-T(1.79±0.03)OF(1.77±0.01)CK(1.75±0.03),其中,OF-T处理下Q_(10)显著高于其他3个处理,即施肥显著增加了断根处理土壤呼吸Q_(10);(3)施肥显著增加了土壤表层NH_4~+-N和NO_3~--N含量,Q_(10)与土壤表层NH_4~+-N和NO_3~--N含量表现出显著的正相关关系。     

On some biochemical physiognomies of two common Darjeeling tea cultivars in relation to blister blight disease

Abstract

Tea, Camellia sinensis (L.) O. Kuntze, an agro-based industry, is considered as one of the prime sector of exporting resources and thus considered as “cash-crop”. Earlier report shows that tea is native to eastern and northern India, which was cultivated and consumed there since long back. Presently, more scientific reports confirmed the health-benefit traits of tea and awareness increased to a greater extent and in this regard, tea has gained its best worldwide popularity. Darjeeling Tea attained its highest acceptance globally for its pre-eminence in flavour, colour and taste and thus crop improvement is the prime interest to the Indian Scientific Community. Blister blight disease, a common disorder of tea bushes (Exobasidium vexans, a Basidiomycetes fungus) causes drastic damage of tea plantation. Depending on quality production, two common cultivars were released by TRA, Jorhat, Assam viz. Bannockburn – 157 (B-157) and Ambei Valai - 2 (AV-2), of which B-157 is susceptible to the Blister Blight and AV-2 is fairly resistant cultivar. Some biochemical considerations between the two cultivars have been made here for understanding the biochemical reasons behind the resistant characteristics. Plant secondary metabolites, like total phenol, proanthocynadin, total soluble protein provide defending feature against disease onset. AV-2 cultivar shows advantage over B-157 in these regard. Depending on band intensity analysis of native gels, acid phosphatase, catechol oxidase, peroxidase and superoxide dismutase occur in superior amount in AV-2 cultivar than that of B-157. The specific role of these enzymes in blister blight disease compatibility of two cultivars studied has been discussed.     

Cancer therapy and prevention by green tea: role of ornithine decarboxylase

Summary. Green tea which is widely consumed in China, Japan and India, contains polyphenolic compounds, which account for 30% of the dry weight of the leaves. Most of the polyphenols are flavanols, of which (−)-epigallocatechin-3-gallate (EGCG) is most abundant. Epidemiological studies revealed that the incidences of stomach and prostate cancers are the lowest in the world among a population that consumes green tea on a regular basis. It has also been reported that the quantity of green tea consumed, plays an important role in reducing cancer risk and in delaying cancer outbreak and recurrence. Various systems were used to confirm anti-cancer activities of green tea and/or EGCG. These included experimental animals in which cancer was induced chemically. Cultured cells transformed chemically or by oncogenes were also used. These studies clearly demonstrated that green tea or EGCG have anticancer and cancer preventive properties. The mechanisms of these activities have also been studied in details. It has been shown that green tea and its active components interfere with signal transduction pathways. Thus the activities of various protein kinases are inhibited, the expression of nuclear proto-oncogenes declines and the activity of ornithine decarboxylase (ODC) is reduced. ODC, which catalyzes the rate-limiting step in the biosynthesis of polyamines is closely linked with cellular proliferation and carcinogenesis. Inhibitors of ODC, like α-difluoromethylornithine (DFMO) have long been used for cancer prevention and therapy. It has been suggested that polyamine depletion by green tea could offer one explanation for its anti-cancer activities. Received July 27, 2001 Accepted September 8, 2001     

Leaf Wettability Difference Among Tea Leaf Ages and Analysis Based on Microscopic Surface Features

The wettability of leaf surface, commonly represented by contact angle (CA), affects various physiological and physical processes. The present study aims to better understand the wettability of tea leaves and elucidate its influence on the energy barrier of the droplet condensation process. The CA values of different leaf ages (young, mature and old) of five famous tea cultivars (Maolu, longjing 43, Huangjinya, Zhongcha 108 and Anji Baicha) were measured via the sessile drop method, and the micro-morphology of two cultivars leaves (Maolu, Zhongcha 108) was investigated by a 3D super depth-of-field digital microscope. Specifically, two radically distinctive types of CA trends were observed, one was the decreased firstly and then increased slightly with the increase of leaf age, while the other stayed constant. The valley depth or maximum height (R_Z) of Maolu leaf surface increased with the leaf age while the R_Z of Zhongcha 108 leaf remained unchanged by comparing the microscopic features. The Maolu mature leaf CA decline attributed to the young leaf was hydrophilic (θ < 90°), and it was considered that surface structures like folds and pits on old leaf played a crucial role in making CA increased. Small deviation in CA can lead to significant error in calculation of the contact angle function of energy barrier in phase change. It will have great significant for simulating and better understanding the formation of frost on tea leaves.     

普洱地区茶叶内生细菌与根际土壤细菌群落结构分析

[目的]茶叶内生细菌、根际土壤细菌在普洱茶的发酵中起着重要的作用,还可以促进茶树生长,诱导茶树抗病性.研究其群落结构组成及相互关系可为微生物资源开发利用提供理论依据.[方法]本研究以普洱地区茶树叶片和根际土壤为材料,采用高通量测序技术,对茶叶及根际土壤细菌的16S核糖体RNA基因(16S rRNA)进行测序,比较分析茶...     

RNA-seq analysis of green tea polyphenols modulation of differently expressed genes in Enterococcus faecalis under low pH

Enterococcus faecasslis (E. faecalis) is a resident bacterium in the host. The increase in internal stress like low pH may affect the biological effects of E. faecalis. The prebiotic-like function of tea polyphenols can enhance the beneficial effects of its tolerance to environmental stress. In this study, RNA-sequence analysis was used to explore the protective effect of green tea polyphenols (GTP) on E. faecalis under low pH stress. A total of 28 genes were found to be responsive to GTP under low pH stress, including 16 up-regulated and 12 down-regulated. GTP intervention can partly relieve some undesired negative influences, such as the down-regulation of the base excision repair gene and amino acid transport and metabolism gene. The significantly changes were associated with selenocompound metabolism and aminoacyl-tRNA biosynthesis after the intervention of GTP. The present study provided new insights into the growth and continuous adaptation of E. faecalis under stress.     

Hypotriacylglycerolemic and Antiobesity Properties of a New Fermented Tea Product Obtained by Tea-Rolling Processing of Third-Crop Green Tea (Camellia sinensis) Leaves and Loquat (Eriobotrya japonica) Leaves

We manufactured a new fermented tea by tea-rolling processing of third-crop green tea (Camellia sinensis) leaves and loquat (Eriobotrya japonica) leaves. The mixed fermented tea extract inhibited pancreatic lipase activity in vitro, and effectively suppressed postprandial hypertriacylglycerolemia in rats. Rats fed a diet containing 1% freeze-dried fermented tea extract for 4 weeks had a significantly lower liver triacylglycerol concentration and white adipose tissue weight than those fed the control diet lacking fermented tea extract. The activity of fatty acid synthase in hepatic cytosol markedly decreased in the fermented tea extract group as compared to the control group. The serum and liver triacylglycerol- and body fat-lowering effects of the mixed fermented tea extract were strong relative to the level of dietary supplementation. These results suggest that the new fermented tea product exhibited hypotriacylglycerolemic and antiobesity properties through suppression of both liver fatty acid synthesis and postprandial hypertriacylglycerolemia by inhibition of pancreatic lipase.     

Detection of nucleopolyhedroviruses in the eggs and caterpillars of tea looper caterpillar Hyposidra infixaria (Walk.) (Lepidoptera: Geometridae) as evidence of transovarial transmission

The transovarial transmission of Hyposidra infixaria nucleopolyhedroviruses (HyinNPV) in the tea looper caterpillar H. infixaria was investigated. H. infixaria larvae reared from eggs (surface sterilised) collected from a tea garden showed typical signs of nucleopolyhedrosis virus infection. The polyhedrin gene amplified from larvae confirmed HyinNPV infection and provided clear evidence of vertical virus transmission from parent to offspring (transovarial transmission). This study reveals important aspects of HyinNPV that could be exploited in biological control programmes; HyinNPV can persist in larval populations and be passed on to the next generation, and thus it may be useful for the long-term control of H. infixaria.     

Theanine transporters identified in tea plants (Camellia sinensis L.)

Theanine, a unique non‐proteinogenic amino acid, is an important component of tea, as it confers the umami taste and relaxation effect of tea as a beverage. Theanine is primarily synthesized in tea roots and is subsequently transported to young shoots, which are harvested for tea production. Currently, the mechanism for theanine transport in the tea plant remains unknown. Here, by screening a yeast mutant library, followed by functional analyses, we identified the glutamine permease, GNP1 as a specific transporter for theanine in yeast. Although there is no GNP1 homolog in the tea plant, we assessed the theanine transport ability of nine tea plant amino acid permease (AAP) family members, with six exhibiting transport activity. We further determined that CsAAP1, CsAAP2, CsAAP4, CsAAP5, CsAAP6, and CsAAP8 exhibited moderate theanine affinities and transport was H⁺‐dependent. The tissue‐specific expression of these six CsAAPs in leaves, vascular tissues, and the root suggested their broad roles in theanine loading and unloading from the vascular system, and in targeting to sink tissues. Furthermore, expression of these CsAAPs was shown to be seasonally regulated, coincident with theanine transport within the tea plant. Finally, CsAAP1 expression in the root was highly correlated with root‐to‐bud transport of theanine, in seven tea plant cultivars. Taken together, these findings support the hypothesis that members of the CsAAP family transport theanine and participate in its root‐to‐shoot delivery in the tea plant.     

基于整合方法分析茶树响应病原真菌胁迫的共有模式

为探讨茶树(Camellia sinensis)对病菌胁迫的共有响应模式和抗病机制,运用生物信息学方法对多组RNA-seq数据进行提取、整合及功能富集,结合多种工具和数据库资源对主要调控分子及蛋白互作模块加以分析。结果表明,病原真菌胁迫下,茶树有较多细胞色素P450家族成员表达显著上调;类固醇和激素的代谢过程、苯丙烷合成途径被激活,有丝分裂细胞周期调控、DNA甲基化等生物过程及光合作用途径受到抑制;主要调控分子如转录因子WRKY和NAC、激酶RLK-Pelle和CAMK等以上调为主。差异表达的蛋白互作模块分析表明,有丝分裂周期调控、基于微管运动、淀粉和蔗糖代谢、细胞壁多糖合成、光合作用、类黄酮代谢模块明显下调,木质素合成和萜类生物合成模块上调;且模块之间可能存在互作。病菌胁迫激活的木质素和萜类合成途径的关键基因包括阿魏酸-5-羟基化酶基因F5H、过氧化物酶基因POD和萜类合成酶基因HMGR等。细胞色素P450基因可能在病菌胁迫中起关键作用,增强木质素和萜类物质的合成、削弱光合作用可能是茶树响应真菌胁迫的核心模式。     

Bioinformatics analysis to identify possible mechanisms of action of curcumin against tea geometrid

Curcumin, a bright yellow chemical produced from curcuma longa root, is an effective insecticide, but how it works against pests such as tea geometrid (E. obliqua) is unclear. Here possible protein targets of curcumin were identified in the PubChem database, and possible targets in tea geometrid were identified using RNA sequencing; then, both sets of candidate targets were subjected to Ingenuity Pathway Analysis. The results predict that curcumin may affect several signalling proteins in tea geometrid, including TOP2B, TP53, MDM4, YWHAB, TOP2A, MDM2 and EP300; these proteins are involved in key biological processes including the cell cycle; DNA replication, recombination and repair; cellular assembly and organization; and cell death and survival. These results illustrate the power of integrative informatics and chemical fragment analysis for focusing mechanistic studies of botanical insecticides.     

4种茶菊对干旱胁迫的形态和生理响应

以4种茶菊幼苗为材料,采用盆栽控水法,研究了不同强度干旱胁迫下茶菊形态、生理生化及光合生长的响应特性,并对其进行耐旱性评价。结果显示,随着干旱胁迫程度的增强,各茶菊幼苗叶片数增量、叶面积、生物量、叶片相对含水量、净光合速率(Pn)、蒸腾速率(Tr)和气孔导度(Gs)降低;根冠比、丙二醛(MDA)含量、脯氨酸(Pro)含量、可溶性蛋白(SPC)含量和叶绿素(Chl)含量增加;胞间CO2(Ci)浓度先降后升;乳荷、黄滁龙株高增量持续降低,而玉人面、繁白露株高增量先升后降;玉人面叶超氧化物歧化酶活性(SOD)呈先升后降趋势,其它3个品种则持续升高。运用隶属函数法对抗旱能力进行综合评定,不同茶菊品种耐旱性由高到低为乳荷黄滁龙繁白露玉人面。     

Probing behavior of the tea green leafhopper on different tea plant cultivars

The probing behaviors of the tea green leafhopper, Empoasca vitis (Gothe), on 9 tea cultivars were studied using video-text Direct Current-Electrical Penetration Graph, i.e., DC-EPG. The following 7 types of waveforms produced by the leafhopper stylet probing were determined: A, stylet pathway formation; S, salivation when stylets pierce into and stay in phloems; C, active ingestion before stylets reach phloems; E and F, passive ingestion in phloems; R, the insect resting with its stylet inserted into the leaf tissue and NP without probing. The 9 tested tea cultivars were categorized into 3 groups by the cluster analysis according to the number of probes per insect, waveform durations, or duration per probe of various waveforms on different tea cultivars. Waveforms S, E and F correlated to the main feeding activity of the leafhopper and may provide valuable information on predicting the resis-tance level of the tea plants to the leafhopper. The resistance level of the 9 tea cultivars to the leafhopper was ranked based on the durations of waveforms S, E and F, as well as the duration per probe including various waveforms. The ranking order of the resis-tance was: Longjingchangye > Hangdan > Zhenghedabaicha > Qianmei 601 > Hongyafoshuo > Zhongcha 102 > Zhongcha 302 > Longjing 43 > Anjibaicha, which corresponded to the resistance level determined by the population density (infestation) of the leaf-hopper on the 9 tea cultivars in the tea fields. Our study suggests that this simple and convenient DC-EPG technique might have great potential as a reliable tool to predict the resistance of tea cultivars to the tea leafhopper.