茶树CsSNAT基因的克隆与表达分析

为明确茶树SNAT基因的序列特征和表达特点,在茶树转录组测序的基础上以茶树(Camellia sinensis)品种舒茶早为试验材料,通过SMART^TM RACE技术克隆茶树褪黑素合成途径中关键限速酶5-羟色胺-N-乙酰转移酶CsSNAT基因的cDNA全长序列,并分析其基因结构和表达模式。结果表明,CsSNAT基因序列全长1 014 bp,其中包含742 bp的完整开放阅读框(ORF),编码247个氨基酸,预测等电点7.64,蛋白分子量27.36 kDa。氨基酸序列比对显示,CsSNAT蛋白与其他高等植物的SNAT蛋白具有较高同源性,与葡萄 VvSNAT(CBI31163)同源性最高,为66.19%。诱导蛋白最佳条件为温度28℃,异丙基-β-D-硫代半乳糖苷(IPTG)浓度0.5 mg·L^-1,诱导时间6 h,在上清液中可获得大量分子量为66 kDa的可溶性融合蛋白。实时定量 PCR 分析表明,CsSNAT在褪黑素处理6 h后表达量最高,为对照的3倍;茶尺蠖取食6 h后,CsSNAT表达显著上调。不同激素处理结果显示,CsSNAT受脱落酸(ABA)、茉莉酸甲酯(MeJA)和水杨酸(SA)诱导表达。本研究结果为5-羟色胺-N-乙酰转移酶功能研究奠定了一定的基础。     

茶树咖啡碱合成酶CRISPR/Cas9基因组编辑载体的构建

CRISPR/Cas9技术是一门新兴的基因组定点编辑技术,具有操作简单、高效的优点,可轻松实现对目标基因的敲除、替换和定点突变等操作。该技术刚诞生,就受到了全球生命科学领域研究者的关注,不到3年的时间就已经成功应用于多种动、植物当中。然而CRISPR/Cas9技术在茶树中的应用面临载体构建问题,本文以茶树咖啡碱合成酶为例,联合采用常规PCR、Overlapping PCR和Golden Gate Cloning技术,构建了包含茶树咖啡碱合成酶双靶点的CRISPR/Cas9基因编辑载体,为CRISPR/Cas9介导的基因组编辑技术在茶树中的应用奠定了坚实基础。     

不同激素对金花茶圈枝繁殖生根的影响

分析NAA、IBA及2种激素等体积混合溶液对金花茶圈枝繁殖生根成活率的影响。结果表明:采用萘乙酸和吲哚丁酸的等体积混合激素溶液1 000~1 500 mg/L能明显提高金花茶圈枝繁殖苗的生根率,达到95%以上。该技术简化了操作程序,可节省人力,在基质方面也节约了生产成本,可直接应用于金花茶苗木大规模生产。     

Changes in peroxidase activity and lignin content of cultured tea cells in response to excess manganese

Abstract

The present study was undertaken to identify the effects of manganese (Mn) on the activity of peroxidase (PO) and the amount of ascorbic acid (AsA) and lignin, as well as the cell viability of suspension-cultured tea cells (Camellia sinensis L. cv. Yabukita). Cells were grown in B5 medium (containing 0.06 mmol L^?1 Mn) and cultured for 24 h in medium with a Mn concentration of 0.9 mmol L^?1 as an excess treatment. No significant difference was observed between cellular growth and the viability of the cultured tea cells after treatment with excess Mn compared with cells grown under control conditions, although the content of Mn in cells in the excess Mn treatment was 12-fold higher than that of the control cells. The amount of total AsA was also not affected by the Mn treatment. The activity of ionically wall-bound peroxidase (IPO) increased in the presence of excess Mn, unlike the content of lignin. Conversely, the activities of soluble PO and covalently wall-bound PO decreased with excess Mn. These findings suggested that IPO might contribute to Mn tolerance in tea cells. However, its role in the mechanism(s) of Mn tolerance has not been elucidated.     

不同激素对碧云茶树丛生芽增殖培养的影响实验

为了获得碧云茶树的组培技术,加快其优良品种繁殖速度,2010～2011年进行了不同浓度的细胞分裂素6-BA和生长素NAA对碧云茶树丛生芽增殖培养的影响。结果表明,以MS为基本培养基,当6-BA为2.0 mg/L,NAA为0.2 mg/L时,对碧云茶树芽的诱导和增殖最有利,增殖率可达2.84;其中NAA差异不显著,6-BA差异显著。     

Armillaria species on tea in Kenya identified using isozyme and DNA sequence comparisons

The aim of this study was to identify seven Armillaria isolates obtained from diseased tea bushes in Kenya using pectic enzyme profiles, PCR-RFLP and IGS-I DNA sequence data. The combination of these identification methods confirmed the presence of three distinct Armillaria groups. One of these groups resembled Zimbabwean group I ( A. fuscipes ). The second group was phylogenetically closely related to A. mellea ssp. nipponica . The third group was different from all other African isolates examined, but had isozyme patterns, especially of pectin methylesterases (PMEs), similar to those of isolates related to A. mellea ssp. nipponica. Analyses of sequence data suggested that this group is phylogenetically closely related to A. hinnulea from Australia and New Zealand.     

Alternaria Brown Spot of Minneola in Greece; Evaluation of Citrus Species Susceptibility

During the last three years, a new disease was observed in northwestern Greece on Minneola trees, hybrid of mandarin and grapefruit. On May small brown necrotic leaf spots surrounded by yellow halo areas of various sizes appeared and covered a major portion of the leaves with extension of necrosis into the veins. On young fruits small, slightly depressed black spots were the first symptoms, which later became 2–7 mm in diameter. Brown spots were observed on the leaves and fruits in several orchards in the same area, causing leaves and fruits to drop. In some orchards over 50% of the fruits were affected. From the fruit and leaf spots the typical small-spore species Alternaria alternata was isolated. Pathogenicity tests were performed by artificially inoculating fruits of Minneola, common mandarin and Clementine. The symptoms of the disease were reproduced only on fruits of Minneola hybrids by the specific strain of the fungus Alternaria alternata pv. citri. Different citrus susceptibility tests indicated that mandarins Minneola, Nova and Page were very susceptible to tested isolates while Clementine SRA and Poros Clementine were not. All lemons and lime Seedless were not susceptible. Grapefruit New Hall was not susceptible, while the Star Ruby was. Orange Lane Late, Navel Late, Oval Poros, Olinda, Navel Athos were not susceptible and only Moro showed reaction being slightly susceptible only to one isolate.     

山茶花红色素的提取及其性质初探

 初步探索了山茶花红色素的提取条件和理化性质, 结果表明, 用料液比1∶10 ( g/mL) 、pH 1(10%盐酸调) 的95%乙醇作提取剂, 在80℃恒温提取6 h, 提取效率较好。山茶花红色素属花色素苷类,pH值对色素影响明显, 在酸性条件下色泽具有热稳定性。光照能加快色素降解, 但与紫外光无关。金属离子Na⁺ 、Ca^{2 +} 、A1^{3 +} 、Cu^{2 +} 、Zn^{2 +}对色泽无影响, Fe^{3 +} 、Pb^{2 +}有不良影响。色素的抗氧化能力较差, 耐还原性较好。蔗糖、葡葡糖和盐等添加剂对色素无影响。     

三个山茶花种(品种) 香气成分初探

 对红山茶品种‘克瑞墨大牡丹’、‘香神’、物种攸县油茶的鲜花香气成分进行了GC /MS分析, 鉴定了‘克瑞墨大牡丹’香气成分37种, ‘香神’香气27种, 攸县油茶34种, 主要以醇、醛、酯、烯、烷及芳樟醇氧化物为主。‘克瑞墨大牡丹’芳樟醇相对含量远高于其它成分, ‘香神’以壬醛和芳樟醇含量最高, 而攸县油茶却以顺- 芳樟醇氧化物Ⅱ和苯乙醇占优势。两个品种在开花前期醇类含量较高, 后期酯类成分含量增加, 攸县油茶在开花前期醇类物质含量高, 越到后期含量越低。     

Susceptibility of citrus species to Alternaria alternata, the causal agent of the Alternaria brown spot

Alternaria alternata, the causal agent of Alternaria brown spot (ABS), causes necrosis on leaves, twigs, and fruit, reducing the productivity and quality of fruits. Tangerines and their hybrids are highly susceptible to the disease. Species, hybrids, and cultivars of Citrus from the germplasm bank of the Estação Experimental de Citricultura de Bebedouro, São Paulo, Brazil, were evaluated in 2004 and 2005 with respect to their resistance to A. alternata, both through natural infection and by inoculation. Detached leaves were also used to demonstrate susceptibility or resistance to the disease. Ten cultivars of Satsumas (Citrus unshiu), and 14 cultivars of Clementine mandarin (C. clementina) did not show any symptoms of the disease in their leaves, either through natural infection or when inoculated in the field. The Burguess SRA-412, Wallent SRA-438, Carvalhais, Ampefy SRA-459, Ananas SRA, and Macaque SRA-426 mandarin hybrids (C. reticulata) did not show symptoms of the disease under natural or artificial infection in the field. Some cultivars of C. deliciosa, C. tangerina, C. erythrosa, and C. temple showed symptoms of the disease, even though no previous record of their susceptibility to Alternaria brown spot had been previously reported. The hybrids Fairchild, Nova, Page, Fortune, and Sunburst were susceptible to the disease. However, Fremont mandarin (a crossing between C. clementina and C. reticulata), Encore (C. nobilis × C. deliciosa), and Fallglo (C. reticulata × C. paradisi) did not show symptoms in field, and few symptoms were verified in detached leaves. These materials are promising for the cultivation of tangerines, and will enable genetic improvement for the development of cultivars resistant to Alternaria brown spot.