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。     

Pathogen resistance of transgenic tobacco plants producing caffeine

Caffeine (1,3,7-trimethylxanthine) is a typical purine alkaloid, and produced by a variety of plants such as coffee and tea. Its physiological function, however, is not completely understood, but chemical defense against pathogens and herbivores, and allelopathic effects against competing plant species have been proposed. Previously, we constructed transgenic tobacco plants, which produced caffeine up to 5 microg per gram fresh weight of leaves, and showed them to repel caterpillars of tobacco cutworms (Spodoptera litura). In the present study, we found that these transgenic plants constitutively expressed defense-related genes encoding pathogenesis-related (PR)-1a and proteinase inhibitor II under non-stressed conditions. We also found that they were highly resistant against pathogens, tobacco mosaic virus and Pseudomonas syringae. Expression of PR-1a and PR-2 was higher in transgenic plants than in wild-type plants during infection. Exogenously applied caffeine to wild-type tobacco leaves exhibited the similar resistant activity. These results suggested that caffeine stimulated endogenous defense system of host plants through directly or indirectly activating gene expression. This assumption is essentially consistent with the idea of chemical defense, in which caffeine may act as one of signaling molecules to activate defense response. It is thus conceivable that the effect of caffeine is bifunctional; direct interference with pest metabolic pathways, and activation of host defense systems.     

茶叶主要化学品质指标和茶树体部分微量元素的钙铝调控效应

以一年生茶树扦插苗为材料,采用水培法研究了添加钙铝对茶叶主要化学品质及茶树钙、铝、锌、铁吸收积累的调控效应.结果表明:(1)适量铝(10或20mg·L-1)有利于提高茶叶茶多酚、咖啡碱、黄酮、可溶性总糖和氨基酸的含量;添加钙可提高上述化学成分的含量,且在高铝浓度(30mg·L-1)下提高的幅度最大.(2)适量的铝可促进茶树对铝和铁的吸收和积累,而高浓度的铝(30mg·L-1)抑制茶树对铝和铁的吸收与积累;添加铝可降低茶树根对钙和锌的吸收,但适量添加铝不影响茎和叶对钙和锌的积累.(3)添加钙可提高茶树体钙的含量,降低铝和锌的吸收与积累,但对铁的吸收与积累没有明显影响.研究表明,铝和钙可调控茶叶化学品质含量和茶树体微量元素的吸收;合理控制茶园土壤铝积累,并适量补充钙可能有利于提高茶叶品质,创建生态高值茶园.     

土壤微量营养元素对武夷肉桂茶品质的影响

对丹岩区和名岩区各5个取样点的武夷肉桂茶树鲜叶生化成分进行分析。结果表明,名岩区肉桂茶树鲜叶中茶多酚、氨基酸、咖啡碱含量总体均高于丹岩区,名岩区的茶树鲜叶具有比丹岩区更优的物质基础。名岩区产的肉桂成品茶的主要生化成分茶多酚、氨基酸和咖啡碱含量亦略高于丹岩区,其他成分差异不显著。测定武夷肉桂茶树鲜叶和成品茶微量营养元素表明,二者在全铜含量上呈显著性相关,且丹岩区肉桂茶树鲜叶的Cu含量显著高于名岩区。对土壤微量营养元素分析表明,丹岩区的Cu、Zn含量显著高于名岩区,而Fe、Mn含量差异不显著。     

茶树CsCCD基因家族全基因组鉴定及乌龙茶LED补光晾青下表达分析

类胡萝卜素裂解双加氧酶(carotenoid cleavage dioxygenase, CCD)家族成员能催化类胡萝卜素裂解生成挥发性芳香物质并参与植物激素的合成。为探究茶树CsCCD基因家族成员生物学功能及基因表达模式,采用生物信息学手段进行了茶树全基因组CsCCD基因家族成员的鉴定,预测分析了其基因结构、保守基序、染色体定位、蛋白的理化性质、进化特性、互作网络、启动子顺式作用元件,并通过RT-qPCR测定了茶树不同叶位、乌龙茶加工过程中光照处理下CsCCD的相对表达量。共鉴定出11个茶树CsCCD基因家族成员,含有1–11个外显子、0–10个内含子不等;平均氨基酸个数为519 aa,平均分子质量为57 643.35 Da;聚类分析显示,CCD1、CCD4、CCD7、CCD8和NCED5个亚族各自聚成一类。茶树CsCCD基因家族主要含有胁迫响应元件、激素响应元件、光响应元件与多因素响应元件,且以光响应元件最多(142个)。进一步对茶树CsCCD基因在茶树不同叶位及乌龙茶加工过程中LED补光晾青过程的表达模式分析发现,CsCCD1、CsCCD4在成熟叶中表达量高于嫩叶及嫩茎,且随做青...     

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...     

茶叶中咖啡因和茶多酚提取技术研究

本文研究茶叶品种和质量、提取剂种类和萃取溶剂量对咖啡因和茶多酚提取得率、提取率和纯度的影响,结果表明:茶叶质量高,咖啡因和茶多酚的含量、得率和纯度就高。95％乙醇提取咖啡因和茶多酚的得率、提取率和纯度最高。1％氧化钙水溶液单纯提取咖啡因的得率、提取率和纯度较高。增加萃取次数和萃取溶剂量可提高咖啡因和茶多酚的得率,对产品纯度没有影响。     

丛枝菌根真菌和施氮量对茶树生长、矿质元素吸收与茶叶品质的影响

采用盆栽法研究了不同施氮水平下接种丛枝菌根（arbuscular mycorrhiza,AM）真菌Glomus mosseae对茶树生长、矿质元素吸收及茶叶品质的影响。结果表明,适量的施氮利于AM真菌的侵染和菌根发育,当施氮过量时则会抑制菌根发育。在不同施氮水平下接种AM真菌均提高了茶树地上部、地下部和总干物质量,其中又以接种AM真菌同时施氮量为0.53g kg-1的茶树总干物质量最大,为对照的1.63倍。不同矿质元素受AM真菌和氮肥的影响不一致,在一定施氮水平下接种AM真菌可提高茶树叶片中N、P、K、Ca、Zn和Fe含量,降低Mn和Cu含量;显著增加根中N、P、K、Mg和Zn含量,降低Mn含量,施高浓度的氮（1.06 g kg-1）显著降低了根系Ca和Fe含量。不同施氮水平下AM真菌处理可增加茶叶中可溶性糖和可溶性蛋白含量,提高了茶叶中茶多酚、咖啡碱、氨基酸和水浸出物含量,降低酚氨比,显著改善茶叶品质。本实验条件下,茶树施氮量为0.53 g kg-1时,接种AM真菌改善茶叶品质的效果最佳。     

Purine Alkaloid Biosynthesis in Young Leaves of Camellia sinensis in Light and Darkness

14 C] adenine to young leaves of tea (Camellia sinensis L.). Light did not have any significant influence on the levels of radioactivity associated with the purine alkaloids. The long-term effects of light on caffeine production were studied using young shoots from plants that were maintained in almost complete darkness (1% full sunlight) by being covered with black lawn cloth. In the control shoots of the naturally-grown plants there were net increases in the total purine alkaloid contents of 2,430 nmoles shoot⁻¹, while in shoots that had been in darkness for 7 days much lower increases, 564 nmoles shoot⁻¹, were observed. Caffeine synthase (CS) activity was 332±55 pkat shoot⁻¹ in light which is ca. 40% higher than the 237±37 pkat shoot⁻¹ in plants kept darkness for 7-days. However, a similar pattern of the metabolism of [8-¹⁴C] adenine was observed in naturally-grown and dark-grown shoots. These findings indicate light is not essential for the biosynthesis of caffeine in young tea shoots. The lower net formation of caffeine in shoots maintained in darkness is an indirect consequence of the reduced growth rate of the young shoots in the absence of light. Received 17 January 2000/ Accepted in revised form 13 March 2000     

Theacrine (1,3,7,9-tetramethyluric acid) synthesis in leaves of a Chinese tea,kucha (Camellia assamica var. kucha)

Theacrine (1,3,7,9-tetramethyluric acid) and caffeine were the major purine alkaloids in the leaves of an unusual Chinese tea known as kucha (Camellia assamica var. kucha). Endogenous levels of theacrine and caffeine in expanding buds and young leaves were ca. 2.8 and 0.6-2.7% of the dry wt, respectively, but the concentrations were lower in the mature leaves. Radioactivity from S-adenosyl-L-[methyl-14C]methionine was incorporated into theacrine as well as theobromine and caffeine by leaf disks of kucha, indicating that S-adenosyl-L-methionine acts as the methyl donor not only for caffeine biosynthesis but also for theacrine production. [8-14C]Caffeine was converted to theacrine by kucha leaves with highest incorporation occurring in expanding buds. When [8-14C]adenosine, the most effective purine precursor for caffeine biosynthesis in tea (Camellia sinensis), was incubated with young kucha leaves for 24 h, up to 1% of total radioactivity was recovered in theacrine. However, pulse-chase experiments with [8-14C]adenosine demonstrated much more extensive incorporation of label into caffeine than theacrine, possibly because of dilution of [14C]caffeine produced by the large endogenous caffeine pool. These results indicate that in kucha leaves theacrine is synthesized from caffeine in what is probably a three-step pathway with 1,3,7-methyluric acid acting an intermediate. This is a first demonstration that theacrine is synthesized from adenosine via caffeine.     

Purine base pattern of camellia irrawadiensis

Theobromine and caffeine in the flush shoot leaves of hybrids of tea and other camellia plants were assayed by HPLC. In C. sinensis and C. taliensis of section Thea the caffeine content exceeded 2% and the theobromine content was below 0.2%. However, in C. irrawadiensis of the same section, the theobromine content was more than 0.5% while the caffeine content was below 0.02%. The theobromine content of tea hybrids was also below 0.2%. In sections other than Thea, C. sasanqua, C. japonica and C. vernalis did not contain detectable amounts of theobromine or caffeine.     

Antimicrobial Activities of Tocklai Vegetative Tea Clones

Thirty-one Tocklai vegetative (TV) tea clones contained caffeine and total catechin 44.39 and 227.55 mg/g dry weight of leaves, respectively. The (−)-epigallocatechin gallate (EGCG) was the most abundant (109.60 mg/g) followed by -(−)-epigallocatechin (EGC, 44.54 mg/g), (−)-epicatechin gallate (ECG, 41.74 mg/g), (−)-epicatechin (EC, 27.42 mg/g) and +catechin (4.25 mg/g). Total catechins were highest in TV 20 (509.7 mg/g) and lowest in TV 6 (71.7 mg/g). The tea clones that contain high level of total catechin exhibited the strongest antimicrobial activity. Among caffeine and flavanol compounds, theaflavins (TF) present in black tea possess a similar antimicrobial potency as EC present in fresh leaves, and that the conversion of catechins to TF during fermentation in making black tea tends to alter their antimicrobial activities. The bioactive molecules other than catechins present in tea leaves may also contribute towards antimicrobial activity.     

一种水稻酰基辅酶A结合蛋白cDNA的鉴定(英文)

对一水稻ｃＤＮＡ 克隆（Ｒ１９０８） 的分析表明, 其可能编码水稻酰基辅酶Ａ 结合蛋白（ａｃｙｌＣｏＡｂｉｎｄｉｎｇ ｐｒｏｔｅｉｎ,ＡＣＢＰ）。Ｓｏｕｔｈｅｒｎ 杂交显示水稻（ Ｏｒｙｚａ ｓａｔｉｖａ Ｌ．） 基因组中仅有一个该基因的拷贝。Ｎｏｒｔｈｅｒｎ 分析表明水稻的ＡＣＢＰ基因在水稻的根、茎、叶、叶鞘、黄化苗和幼穗中皆表达,而以黄化苗的绿苗叶鞘中的表达强度高于绿苗叶片。     

Evaluation in tobacco of the organ specificity and strength of therolD promoter,domain A of the 35S promoter and the 35S2 promoter

In order to study the expression in plants of therolD promoter ofAgrobacterium rhizogenes, we have constructed chimaeric genes placing the coding region of thegusA (uidA) marker gene under control of tworolD promoter fragments of different length. Similar results were obtained with both genes. Expression studies were carried out in transformed R₁ progeny plants. In mature transformed tobacco plants, therolD-gus genes were expressed strongly in roots, and to much lower levels in stems and leaves. This pattern of expression was transmitted to progeny, though the ratio of the level of expression in roots relative to that in leaves was much lower in young seedlings. The degree of root specificity inrolD-gus transformants was less than that of a gene constructed with domain A of the CaMV 35S promoter,domA-gus, but the level of root expression was much higher than with the latter gene. However, the level of expression of therolD-gus genes was less than that of agus gene with a 35S promoter with doubled domain B, 35S²-gus. TherolD-gus genes had a distinctive pattern of expression in roots, compared to that of the two other genes, with the strongest GUS activity observed in the root elongation zone and in vascular tissue, and much less in the root apex.     

白化和黄化茶树品种绿茶游离氨基酸、儿茶素类及咖啡碱差异分析

为探究以白化和黄化茶树品种鲜叶为原料制成的绿茶滋味品质和代谢物差异,对广德市6个白化品种绿茶(奶白茶)和14个黄化品种绿茶(黄金芽茶)进行感官审评和代谢物分析。结果表明,奶白茶滋味鲜爽而收敛性略弱;黄金芽茶滋味收敛性强而鲜度低于奶白茶。游离氨基酸总量以及呈现鲜味、甜味的游离氨基酸在奶白茶中的含量显著高于黄金芽茶,而贡献收敛性的儿茶素类化合物和没食子酸含量以及呈现苦味的咖啡碱含量在奶白茶中显著低于黄金芽茶。偏最小二乘法判别分析(PLS-DA)表明导致两种绿茶滋味差异的标志性化合物有7种,分别是茶氨酸、精氨酸、谷氨酸、天冬氨酸、表没食子儿茶素没食子酸酯(EGCG)、表儿茶素没食子酸酯和咖啡碱。味觉活性值(Dot)最高的EGCG在黄金芽茶中的呈味贡献显著高于奶白茶。因此,游离氨基酸、儿茶素类化合物、没食子酸和咖啡碱含量差异导致白化和黄化茶树品种绿茶滋味不同。     

Further investigation of the biosynthesis of caffeine in tea plants (Camellia sinensis L.). Methylation of transfer ribonucleic acid by tea leaf extracts.

1. The tRNA methyltransferase activity in vitro of leaves, cotyledons and roots of 85-day-old tea seedlings was studied. 2. The activity of extracts prepared from tea leaves with Polycar AT (insoluble polyvinylpyrrolidine) had optimum pH7.7 and was greatly influenced by thiol compounds, but only slightly by metal ions and ammonium acetate. 3. The activities of extracts, expressed per mg of protein, were as follows: roots greater than leaves greater than cotyledons. The only methylated base isolated after incubation with these preparations was 1-methyladenine. 4. The results did not support the view of involvement of methylation of nucleic acids in caffeine biosynthesis in tea plants. In contrast, it is suggested that theophylline is synthesized from the specific methylated precursor in nucleic acids, namely 1-methyladenylic acid, via 1-methylxanthine.     

茶树专用特效叶面肥在春茶上的应用效果

通过在茶园喷施茶叶专用特效叶面肥的应用研究,结果表明：越冬芽早发多发,春茶提早开采,春茶增值。绿茶感官品质及与品质密切相关的有效成分茶多酚、氨基酸、咖啡碱等含量均有提高且效果显著。茶叶专用特效叶面肥值得在茶叶生产上推广应用。     

茶树叶片类脱水素蛋白的Western-blot分析

为了解茶树脱水素种类与功能,采用Western-blot技术,研究了不同季节及越冬过程中茶树叶片脱水素蛋白家族的表达模式。结果显示:(1)茶树叶片总蛋白提取采用酚-甲醇/醋酸铵沉淀法,用时短、蛋白浓度高、SDSPAGE电泳条带清晰,背景干净,满足茶树Western-blot技术要求。(2)在不同季节及越冬期中发现14~95kD共9种不同分子量的茶树类脱水素蛋白,其中95、65、48、37、34和14kD等6种蛋白表达量较为稳定,季节与越冬期变化不明显;58kD脱水素仅在冬季表达,越冬期不断上升,2月份增加到最高,表达丰度高;28kD脱水素蛋白在冬季表达量高,越冬期与茶树抗寒力变化规律一致;21kD脱水素在夏季和越冬期后期有较高的表达。研究表明,这3种脱水素可能在茶树抗逆中起着重要作用。