茶树肌动蛋白基因（CsActin1）全长cDNA克隆与生物信息学分析

以茶树（Camellia sinensis）萌动芽为材料,根据茶树萌动芽芽抑制消减杂交文库中分离得到的肌动蛋白（actin）基因的5′-片段设计引物,利用3′-RACE技术克隆了其cDNA全长序列,该基因cDNA全长1 470 bp,命名为CsActin1（GenBank登录号HQ235647）。序列分析表明,CsActin1开放阅读框长1 134 bp,编码377个氨基酸,5′非编码区100 bp,3′非编码区236 bp。推测的蛋白质分子量为41.70 kD,等电点约为5.31,具有肌动蛋白家族的特征信号序列（YVGDEAQs.KRG和WIAKaEYDE）和肌动蛋白相关蛋白的特征信号序列（LLTEApLNPkaNR）。CsActin1与GenBank中注册的其它植物肌动蛋白核苷酸序列的相似性在80%以上,氨基酸序列相似性在95%以上。与其它植物肌动蛋白的进化树分析结果表明,茶树肌动蛋白与杨树的两个肌动蛋白间的亲缘关系最为密切。并对推导的蛋白结构进行了分析。     

分子标记在茶树遗传育种上的应用

综述了近年来分子标记在茶树种质资源和品种鉴别、遗传多样性、遗传演化、遗传稳定性及分子遗传图谱构建等方面的应用 ,并展望了其应用前景。     

Diversity and genetic differentiation among populations of Indian and Kenyan tea (Camellia sinensis (L.) O. Kuntze) revealed by AFLP markers

AFLP markers were successfully employed to detect diversity and genetic differentiation among Indian and Kenyan populations of tea (Camellia sinensis (L.) O. Kuntze). Shannon's index of diversity was used to partition the total phenotypic variation into between and within population components. On average, most of the diversity was detected within populations, with 79% of the variation being within and 21% being between populations of Indian and Kenyan tea. A dendrogram constructed on the basis of band sharing distinctly separated the three populations of tea into China type (sinensis), Assam type (assamica) and Cambod type (assamica ssp. lasiocalyx) in a manner consistent with the present taxonomy of tea, the known pedigree of some of the genotypes and their geographical origin. Principal coordinate (PCO) analysis grouped Assam genotypes both from India and Kenya supporting the suggestion that the Kenyan clones have been derived from collections made in this region. The China types were more dispersed on the PCO plot which is a reflection of wider genetic variation. As would be expected, clones collected from the same region exhibited less overall genetic variation. AFLP analysis discriminated all of the tested genotypes from India and Kenya, even those which cannot be distinguished on the basis of morphological and phenotypic traits. Received: 2 May 1996 / Accepted: 14 June 1996     

Chemical Diversity in Indian Oregano (Origanum vulgare L.)

The terpenoid composition of the essential oils of 17 different populations of Origanum vulgare L., collected from wild populations and subsequently grown under similar conditions in the sub‐temperate region of the Western Himalaya, was studied. Analysis by GC (RI) and GC/MS allowed the identification of 51 components, representing 90.15 to 99.94% of the total oil. The two classes of the phenolic compounds and the monoterpenoids were predominant in all the essential oils. On the basis of the major constituents, i.e., marker compounds, and by comparison of the results with previous reports, new chemotypes could be identified. Principal component analysis was performed to determine the chemical variability within the different populations of O. vulgare collected and grown under similar conditions. Based on the marker compounds, six chemotypes with significant variations in their terpenoid profile were noticed within the 17 populations.     

Micropropagation of tea (Camellia sinensis (L.) O. Kuntze) using Thidiazuron

The effect of thidiazuron (TDZ) on the micropropagation of Camellia sinensis (China hybrid) was compared with that of benzylaminopurine (BAP) using nodal segments from in vitro raised seedlings. Extremely low concentrations of TDZ (1pM–100nM) alone were effective in inducing shoot bud proliferation and maintaining high rates of shoot multiplication on hormone-free media. On the other hand, higher concentrations of BAP (1–10M) and its continued presence were required to initiate and sustain shoot proliferation. While wider ranges of BAP combined favourably with auxins like NAA or IBA, only specific combinations of TDZ and NAA were effective for shoot proliferation. TDZ treated explants yielded healthy shoots, with sturdy leaves, even during the initial stages of growth, whereas, the effect of BAP was cumulative over subcultures in attaining a high proliferative rate.     

Polyphenols as Potential Indicators for Drought Tolerance in Tea (Camellia sinensis L.)

Plant polyphenols have gained prominence in quality of plant products and in human health. An experiment was conducted to determine the association of tea polyphenols with water stress and their suitability as indicators for drought tolerance. The experiment was conducted in a ‘rain-out’ shelter, and consisted of six tea clones (BBK 35, TRFK 6/8, TRFK 76/1, TRFK 395/2, TRFK 31/30, and TRFK 311/287) and four levels of soil water contents (38, 30, 22, and 14% v/v), which were maintained for a period of 12 weeks. The treatments were arranged in a completely randomized design and replicated three times. Plant growth was monitored over 6 weeks, and a water stress index was calculated to determine water-stress tolerant clones. Total polyphenols in tea shoots was analyzed and a regression analysis done. The results indicate that declining soil water content (SWC) reduced both growth and content of polyphenols in tea. Tolerant clones maintained a high polyphenol content at low SWC, and also showed less fluctuation in phenolics when subjected to changes in SWC. There was significant (P<0.001) correlation of total polyphenol content with shoot growth and WSI of tea, and a linear relationship (r²=0.97) between SWC for tea and both, water stress index and shoot polyphenol content. We report that there is a potential to use polyphenols as indicators for selection of drought-tolerant tea cultivars.     

茶树不同器官组织总RNA提取方法的研究

从茶树组织中提取高质量的总RNA,是开展茶树基因组学、功能基因组学研究的重要前提,而RNase、多酚类物质严重干扰茶树总RNA的分离提取。鉴于茶树组织总RNA提取过程难易不一、总RNA提取质量良莠不齐的现状,现对材料用量、提取液、DNA和蛋白质抽提液、RNA沉淀试剂、多酚氧化抑制剂等进行了比较研究,建立了一种适合茶树各器官组织总RNA提取的简单高效的方法(简易CTAB-LiCl法),并与实验室常用的改良Tri-Reagent法、改良CTAB法进行了比较。核酸定量和琼脂糖凝胶电泳检测结果显示,简易CTAB-LiCl法从茶树各器官组织中提取到的总RNA质量高、得率高。总RNA的得率是改良CTAB法的1.6-5倍。因此,简易CTAB-LiCl法具有效率高、适用范围广,且操作简单、实验成本低的特点。RT-PCR和cDNA-AFLP实验表明,提取的总RNA能够用于后续的分子生物学研究。     

Metabolomic and Pathway Changes in Large-Leaf,Middle-Leaf and Small-Leaf Cultivars of Camellia sinensis (L.) Kuntze var. niaowangensis

As an economically important crop, tea is widely cultivated in more than 50 countries and has numerous health benefits. Metabolomics has considerable advantages in the analysis of small molecules and has been widely used in tea science. We applied a metabolomic method to evaluate the dynamic changes in metabolites and pathways in the large-, middle- and small-leaf cultivars of Camellia sinensis (L.) Kuntze var. niaowangensis grown in the same area from Yunwu Mountain. The results indicate that flavonoid biosynthesis, stilbenoid, diarylheptanoid and gingerol biosynthesis, citrate cycle (TCA cycle), and propanoate metabolism may play important roles in the differences among cultivars. The levels of tea polyphenols, flavonoids and amino acids may impact the sensory properties of teas of different cultivars. Our results may help to elucidate the mechanism underlying the difference in tea quality and offer references for the breeding of high-quality tea cultivars.     

Differential display-mediated identification of three drought-responsive expressed sequence tags in tea [Camellia sinensis (L.) O. Kuntze]

There is no information on drought-modulated gene(s) in tea [Camellia sinensis (L.) O. Kuntze], a woody and perennial plant of commercial importance. Using differential display of mRNA, three drought-modulated expressed sequence tags (ESTs) were identified. Northern and BLAST analysis revealed that clonedr1 (droughtresponsive), induced only by drought but not by ABA, showed significant scores with PR-5 (pathogenesis related) family of PR-protein gene. Another clonedr2, repressed by drought but not by ABA, had nucleotide repeats for polyasparate that are also present in chicken calsequestrin-like mRNA. Clonedr3, responded similarly to clonedr2 but did not show significant homology with the reported genes, hence appears to be novel. Identification of these ESTs is an initial step to clone the full length genes and their promoters     

Purification and Partial Characterization of β-Glucosidase from Fresh Leaves of Tea Plants （Camellia sinensis （L.） O. Kuntze）

β-Glucosidases are important in the formation of floral tea aroma and the development of resistance to pathogens and herbivores in tea plants. A novel β-glucosidase was purified 117-fold to homogeneity,with a yield of 1.26%, from tea leaves by chilled acetone and ammonium sulfate precipitation, ion exchange chromatography (CM-Sephadex C-50) and fast protein liquid chromatography (FPLC; Superdex 75, Resource S). The enzyme was a monomeric protein with specific activity of 2.57 U/mg. The molecular mass of the enzyme was estimated to be about 41 kDa and 34 kDa by SDS-PAGE and FPLC gel filtration on Superdex 200, respectively. The enzyme showed optimum activity at 50℃ and was stable at temperatures lower than 40℃. It was active between pH 4.0 and pH 7.0, with an optimum activity at pH 5.5, and was fairly stable from pH 4.5 to pH 8.0. The enzyme showed maximum activity towards pNPG, low activity towards pNP-Galacto, and no activity towards pNP-Xylo.     

Changes in endogenous gibberellin activity during winter dormancy in tea (Camellia sinensis (L.) O. Kuntze)

Changes in gibberellin (GAs) activities were determined in tea shoots during winter dormancy and subsequent to dormancy release. Free GA-like activity was extremely low at the initiation of dormancy and remained so during the dormancy period. Conjugated GA-like activity (ficin hydrolyzable and β-glucosidase hydrolyzable compounds) remained high during the dormancy period. With an increase in free GA activity, conjugated GA activity decreased in tea shoots prior to dormancy release. The possible role of free and conjugated GAs in dormancy and following its release is discussed in relation to winter dormancy in tea shoots. IHBT Communication number 9846     

Agrobacterium tumefaciens-mediated transformation of embryogenic tissues of tea (Camellia sinensis (L.) O. Kuntze)

Embryogenic tissues of tea were cocultivated withAgrobacterium tumefaciens LBA4404. The plasmid pBi121, which contains the neomycin phosphotransferase II (nptII) gene providing kanamycin resistance as a selectable marker and the β-glucuronidase (uidA) reporter gene, was used as binary vector. The highest transformation frequency (12 transformants/g fresh weight [FW] of treated embryogenic tissue) was obtained with 5-day-old tissues grown in liquid medium and cocultivated withAgrobacterium for 2 d in the same medium but containing 50 μM acetosyringone. There was improvement in the recovery of kanamycin-resistant tissues when tissues were first grown for 10 d on a medium containing 350 mg/L Timentin to prevent bacterial overgrowth, before application of the selection pressure. Resistant tissues obtained after 6 wk on kanamycin-selection medium showed stableuidA expression. Polymerase chain reaction demonstrated the presence of the transgenes, while Southern hybridization confirmed their integration into the genome. Transgenic plants were regenerated from transformed tissues within 4 mo after coculture.     

Clonal variation of tea [Camellia sinensis (L.) O. Kuntze] in countering water deficiency

Various clones of tea [Camellia sinensis (L.) O. Kuntze] such as TTL-1, TTL-2, TTL-4, TTL-5, TTL-6, UPASI-2 and UPASI-3 planted in the field were subjected to soil moisture stress conditions by withholding irrigation. A control set of the same clones were maintained by watering regularly. The soil water content of the irrigated and non irrigated plants was monitored through the soil moisture status. The extent of effect of drought on tea plants were monitored through various physiological parameters such as shoot weight, leaf water potential, chlorophyll and carotenoid content, chlorophyll fluorescence (Fv/Fm), net photosynthetic rate, transpiration rate, stomatal conductance and biochemical parameters such as extent of proline accumulation and free radical generation. These parameters were studied on the 30 d of non irrigation and on the 5 d during recovery from drought. The plants recovered when re-irrigated after 30 d of non-irrigation, which suggests that permanent wilting did not occur due to non-irrigation up to 30 d. On the 30 d of non-irrigation the clones TTL-1, TTL-6 and UPASI-2 showed lesser reduction of shoot weight, leaf water potential, chlorophyll fluorescence, photosynthetic rate, transpiration rate and stomatal conductance and increased proline and lesser lipid peroxidation as compared to the other clones. From these results it can be concluded that the clones TTL-1, TTL-6 and UPASI-2 are comparatively more drought tolerant than the clones TTL-2, TTL-4, TTL-5 and UPASI-3.     

Chemotype Diversity of Indigenous Dalmatian Sage (Salvia officinalis L.) Populations in Montenegro

To identify how many chemotypes of Salvia officinalis exist in Montenegro, the chemical composition of the essential oils of 12 wild‐growing populations was determined by GC‐FID and GC/MS analyses. Among the 40 identified constituents, the most abundant were cis‐thujone (16.98–40.35%), camphor (12.75–35.37%), 1,8‐cineol (6.40–12.06%), trans‐thujone (1.5–10.35%), camphene (2.26–9.97%), borneol (0.97–8.81%), viridiflorol (3.46–7.8%), limonene (1.8–6.47%), α‐pinene (1.59–5.46%), and α‐humulene (1.77–5.02%). The composition of the essential oils under study did not meet the ISO 9909 requirements, while the oils of populations P02–P04, P09, and P10 complied with the German Drug Codex. A few of the main essential‐oil constituents appeared to be highly intercorrelated. Strong positive correlations were observed between α‐pinene and camphene, camphene and camphor, as well as between cis‐thujone and trans‐thujone. Strong negative correlations were evidenced between cis‐thujone and α‐pinene, cis‐thujone and champhene, cis‐thujone and camphor, as well as between trans‐thujone and camphene. Multivariate analyses allowed the grouping of the populations into three distinct chemotypes, i.e., Chemotype A, rich in total thujones, Chemotype B, with intermediate contents of thujones, α‐pinene, camphene, and camphor and high borneol contents, and Chemotype C, rich in camphor, camphene, and α‐pinene. The chemotypes did not significantly differ in the total essential‐oil content and the cis/trans‐thujone ratio.     

Mechanism for the detoxification of aluminum in roots of tea plant (Camellia sinensis (L.) Kuntze)

To determine the mechanism of aluminum (Al) detoxification in the roots of tea plants (Camellia sinensis (L.) Kuntze), the amounts of Al and Al-chelating compounds (fluoride (F), organic acids and catechins) were measured and the chemical forms of Al in root cell extracts were identified by the application of 27Al-nuclear magnetic resonance (NMR) spectroscopy. Tea plants were cultivated in nutrient solutions containing 0, 4, 1.0 and 4.0 mM of Al at pH 4.2 for approximately 10 weeks. The levels of soluble Al, water-soluble oxalate and citrate, but not F, malate or catechins in young roots increased with an increase in the concentration of Al in the treatment solution. The 27Al NMR spectra of root tips and cell sap extracted from root tips that had been treated with Al were almost identical and had four signals, with two (11 and 16 ppm) apparently corresponding to the known chemical shifts of Al-oxalate complexes. In the spectra of cell sap, the resonances at 11 and 16 ppm increased with an increase in the Al contents. These results suggest that the levels of Al-oxalate complexes increased in response to an increase in the Al level, implying that oxalate is a key Al-chelating compound in the mechanism of Al detoxification in the tea root.     

A Study of the Early Growth of Cuttings of Tea (Camellia sinensis (L) O. Kuntze)

The changes in dry matter, leaf area, and moisture contentsof tea cuttings in two situations, a veranda and an outsidenursery under two watering régimes are described. Theestimates of the changes in dry matter and moisture from monthto month were obtained by sampling and adjusting the sampledweights according to the linear measurements made on the cuttingsdesignated for the final sampling. The greatest rate of increasein dry matter occurred within 51 days of planting in all exceptthe light watering treatment in the veranda. Most of this initialincrement in dry matter was retained by the original cuttingleaf. Subsequently, accumulation of dry matter was at a slowerrate and over the last sampling period a net loss occurred dueto the abscission of some original leaves. The greatest rateof increase was under the heavy watering treatment in the nursery. Shoot growth commenced after the 51st day and reached its maximumrate between the third and fourth samplings. The leaf area andshoot dry weight of cuttings with shoots existent prior to the8ist day did not increase in the last two sampling periods,as the shoots had completed a flush cycle and had become banji(dormant). Root growth was first noted at the third sampling.The growth of both root and shoot was greatest under the heavywatering nursery treatment. The percentage of moisture in the cuttings was not increasedby the watering treatment. The data are discussed with reference to the modifying influenceof the treatments on the growth type.     

Somatic embryogenesis and plant regeneration from the immature cotyledonary tissues of cultivated tea (Camellia sinensis (L).O. Kuntze)

Embryogenic callus development, plant regeneration, and plant recovery were achieved from immature cotyledon explants of cultivated tea, when cultured on MS basal medium. The somatic embryo induction frequency was influenced when the medium was supplemented with 1 M auxin (NAA, NOA, 2,4-D, TPB, and PBOA) in combination with cytokinin (0.5 M BA, KIN) or 10% CM. The highest somatic embryo induction frequency was obtained using PBOA + BA or PBOA + KIN treatments. All auxins except 2,4-D stimulated rhizogenesis using 0.8% and l.5% agar concentrations, and differentiation of a characteristic swelling and friable callus from the exposed surface of the explant that remained nonembryogenic. Conversely, the novel auxins TPB and PBOA at 1 M concentration with 3% or 6% agar, produced somatic embryo induction, while at 0.8% and 1.5% produced nonembryogenic callus. Explants isolated proximal to the zygotic embryonal axis showed a greater somatic embryo induction frequency than did the distal explants. The embryogenic competence was maintained through repetitive embryogenesis for a period of over 18 months. The somatic embryos developed into plantlets when incubated on hormone-free medium. The conversion frequency was increased by 50% in MS medium containing 1 M Brassin and 0.8% agar. Concentration of agar at 3% and 6% decreased the conversion frequency and promoted anomalous plantlet development. The normal plantlets were treated with 1 M IAN, 1 M Brassin and 10 Phloroglucinol in liquid MS medium for 15 d, where profuse lateral roots were induced favoring a high rate of plant recovery.