黄花蒿rbcL基因电子克隆、生物信息学及适应性进化分析

[目的]运用电子克隆的方法获得黄花蒿中rbc L基因,并对该基因进行生物信息学及适应性进化分析,获得氨基酸正选择位点及其空间结构特征。[方法]以短葶飞蓬rbc L基因为种子序列(探针,Genbank登录号为:KF482865.1),对黄花蒿的EST数据库进行搜索,应用相关的生物软件进行拼接、组装,利用PAML程序检测其rbc L基因的适应性进化。[结果]获得一个rbc L基因的c DNA序列重叠群,该重叠群长为1 832bp,包含一个完整的长为1 461bp的开放阅读框,共编码486个氨基酸,且与菊科的其他植物的rbc L基因具有较高的同源性,适应性进化分析在rbc L蛋白上有两个氨基酸正选择位点(249S和449T)。[结论]电子克隆获得的c DNA序列为完整的黄花蒿rbc L基因全长c DNA,黄花蒿对生境的适应可能与rbc L蛋白大亚基正选择位点空间结构有关。     

秃房茶嘌呤生物碱组成特点及生化品质成分的研究

该研究采用分光光度法和高效液相色谱法,分不同叶位对秃房茶(Camellia gymnogyna)的嘌呤生物碱组成特点以及茶多酚、儿茶素组分、游离氨基酸、黄酮、茶氨酸等生化品质成分进行了测定。结果表明:秃房茶的嘌呤生物碱组成及配比显著区别于茶叶植物凤凰单从(C.sinensis),同时具有可可碱、咖啡碱和苦茶碱三种组分,而且可可碱含量最多,为13.46~39.72 mg·g~(-1),咖啡碱含量最低,为0.51~2.02 mg·g~(-1),苦茶碱含量介于两者中间并随芽叶成熟度增加而升高。茶叶植物只存在咖啡碱和可可碱,含量变化分别为22.22~53.13 mg·g~(-1)和0.47~12.82 mg·g~(-1)。在相同叶位中,秃房茶儿茶素组分含量变化规律为EGCGCECGEGCECGCCGGCG,且儿茶素总量、酯型儿茶素含量均低于茶叶植物,而非酯型儿茶素总量接近,保持为40~50 mg·g~(-1)。除黄酮含量在各叶位变化趋势不大外,其他品质成分含量变化基本符合第1叶芽第2叶第3叶第4叶,一芽二叶的含量介于第1叶和第2叶之间的规律,而茶多酚、黄酮、茶氨酸等其它品质成分含量均低于茶叶植物。该研究首次明确了秃房茶主要生化品质成分变化规律,特别是嘌呤生物碱的组成及配比特点,且含有特征性成分—苦茶碱。该研究结果为生物碱代谢机理、特异茶加工、功能成分开发、低咖啡碱资源、选育种等提供了优良材料。     

一种用高效液相色谱-电喷雾/串联质谱(HPLC-ESI/MSn)定量检测植物组织中微量1-氨基环丙烷-1-羧酸(ACC)含量的方法

介绍一种用高效液相色谱碳18柱(HPLC-C18)分离.电喷雾串联质谱(ESI-MSn)鉴定和定量检测植物组织中微量1-氨基环丙烷-1-羧酸(ACC)含量的方法,其最低检测限达0.7 pmol,标准曲线线性符合系数为0.9992.建立了从20～100 mg微量植物样品中提取ACC和固相萃取(SPE)预纯化的方法,加样回收率为95.1%±4.2%.此种提取方法结合HPLC-ESI/MSn分析与定性定量检测苹果'2001富士'中ACC含量为306.6 ng·g-1(FW),表明此法适合于定性定量检测植物样品中的微量ACC.     

加工产品中转基因玉米Bt11成分实时荧光PCR定量(性)检测

实验在玉米自身基因和外源基因的边界序列之间设计了具有品种和品系特异性的引物和探针 ,并以实时荧光PCR技术 ,建立了加工产品中转基因玉米Bt1 1成分品系鉴定检测和定量检测的方法。实验对加热条件和时间对检测转基因成分的影响作了探讨 ,并检测了部分市售食品和饲料。检测结果发现 ,加热时间温度越高、时间越长 ,对转基因成分定量检测的影响越大 ;在所检测的样品中可以检测出转基因玉米Bt1 1成分 ,有些样品还同时检出其他转基因成分。本研究实验建立的方法 ,可以用于加工产品中转基因成分的定量检测 ,也可以用于定性检测 ,或作为常规PCR定性检测后的确证实验方法。     

五种金花茶组植物类黄酮成分及其与花色关系

以金花茶、小果金花茶、扶绥金花茶、龙州金花茶和陇瑞金花茶等五种金花茶组植物为试验材料,按照CIE L*a*b*表色系法测量其花色,利用超高效液相色谱-四极杆-飞行时间质谱(UPLC-Q-TOF-MS)联用技术定性定量分析其花中类黄酮成分与含量,运用多元线性回归方法研究花色与类黄酮成分之间的关系。结果表明:5种金花茶组植物花中共检测到8种类黄酮成分,其中天竺葵素-3-O-葡萄糖苷(Pg3G)、木犀草素-7-O-芸香糖苷(Lu7R)、芸香柚皮苷和圣草素为金花茶组植物中首次发现;槲皮素-3-O-葡萄糖苷(Qu3G)、槲皮素-7-O-葡萄糖苷(Qu7G)、槲皮素-3-O-芸香糖苷(Qu3R)和山柰酚-3-O-葡萄糖苷(Km3G)为扶绥金花茶和小果金花茶中首次发现;金花茶花中类黄酮成分总量最高,其次是扶绥金花茶和小果金花茶,陇瑞金花茶和龙州金花茶较低;金花茶和小果金花茶主要类黄酮成分为Qu3G、Qu3R和Pg3G,扶绥金花茶为Qu3G和Qu7G,陇瑞金花茶和龙州金花茶为圣草素和芸香柚皮苷; Qu3G和Qu3R是决定金花茶组植物花瓣呈现黄色的主要成分,圣草素与花瓣红晕显著正相关,Pg3G影响花色鲜艳程度。     

弯枝藻属rbcL基因的适应性进化分析

为探讨淡水红藻的叶绿体基因及其适应性进化特征,选取弯枝藻属(Compsopogon)及相近外类群的rbc L基因共17条,利用PAML 4.9软件,对弯枝藻属rbc L基因编码蛋白进行生物信息学分析,并分别采用分支模型、位点模型以及分支-位点模型对基因的选择位点进行检测。结果表明,弯枝藻属rbc L基因编码蛋白的二级结构主要由α螺旋和β折叠构成,结构稳定。采用最大似然法构建的系统发育树表明,内类群为单一物种,分为3个小分支,具有一定地理分布规律。在3种进化模型中均未检测到统计上显著的正选择位点,表明绝大多数位点处于负选择压力下。因此,弯枝藻属rbc L基因未发生适应性进化。     

我国西南元江大理茶的挥发性成分及其抗氧化活性

大理茶(Camellia taliensis)为山茶科山茶属茶组植物,主要分布于云南横断山脉澜沧江至伊洛瓦底江流域,即从云南的西部及西南部至缅甸北部.在其分布区,大理茶亦被称为野生大茶树,常用于加工制作茶叶.采用水蒸气蒸馏法、GC及GC/MS联用技术,首次对大理茶的鲜幼叶和鲜幼叶及老叶分别制成的绿茶中的挥发性成分进行提取和分析,共鉴定出91个化合物.研究结果表明,大理茶鲜幼叶的主要香气成分为棕榈酸(30.52％),亚油酸(19.82％),植醇(8.75％)和亚麻酸乙酯(2.54％)等有机酸及其酯和二萜类,而制成绿茶后,其主要香气成分则为芳樟醇(28.43％),脱氢芳樟醇(1.13％),α-松油醇(11.68％),橙花醇(4.92％)和香叶醇(12.34％)等单萜醇类成分.从大理茶鲜叶到由其制成的绿茶,香气成分发生了较大变化,形成了28种原鲜叶中未检测到的香气成分,其中,(Z,Z,Z)-9,12,15-十八烷三烯-1-醇的含量分别达到1.21％(幼叶绿茶)和11.2％(老叶绿茶),是大理茶制作的绿茶的特征香气成分.DPPH和ABTS+自由基清除实验结果显示大理茶鲜叶及其制成的绿茶的挥发性成分均具有一定的抗氧化活性,但均弱于茶多酚的抗氧化活性.     

基于三种叶绿体基因序列的证据分析阴石粉背蕨和金粉背蕨的关系

选择阴石粉背蕨(Aleuritopteris humatifolia)和金粉背蕨(A.chrysophylla)及其相关类群共27种植物,提取基因组DNA,通过聚合酶链式反应(PCR)扩增其叶绿体rbc L,trn L-F和mat K序列,所得序列利用Clustal X软件进行对位排列,用MEGA6.0软件计算种内及种间遗传距离,并采用最大简约法(MP)构建系统发育树。结果显示:阴石粉背蕨1个样品和金粉背蕨2个样品的rbc L,trn L-F和mat K序列间差异很小,最大遗传距离均小于其与其它近缘种的种间最小遗传距离,结合两个种叶片形态、孢子、囊群盖和根状茎鳞片等形态学特征的对比研究,作者认为阴石粉背蕨应该作为金粉背蕨的异名处理。     

我国西南元江大理茶的挥发性成分及其抗氧化活性(英文)

大理茶 ( Camellia taliensis) 为山茶科山茶属茶组植物,主要分布于云南横断山脉澜沧江至伊洛瓦底江流域,即从云南的西部及西南部至缅甸北部。在其分布区,大理茶亦被称为野生大茶树,常用于加工制作茶叶。采用水蒸气蒸馏法、GC 及 GC/MS 联用技术,首次对大理茶的鲜幼叶和鲜幼叶及老叶分别制成的绿茶中的挥发性成分进行提取和分析,共鉴定出 91 个化合物。研究结果表明,大理茶鲜幼叶的主要香气成分为棕榈酸 ( 30. 52%) ,亚油酸 ( 19. 82%) ,植醇 ( 8. 75%) 和亚麻酸乙酯 ( 2. 54%) 等有机酸及其酯和二萜类,而制成绿茶后,其主要香气成分则为芳樟醇 ( 28. 43%) ,脱氢芳樟醇 ( 1. 13%) ,α-松油醇( 11. 68%) ,橙花醇 ( 4. 92%) 和香叶醇 ( 12. 34%) 等单萜醇类成分。从大理茶鲜叶到由其制成的绿茶,香气成分发生了较大变化,形成了 28 种原鲜叶中未检测到的香气成分,其中,( Z,Z,Z) -9,12,15-十八烷三烯-1-醇的含量分别达到 1. 21% ( 幼叶绿茶) 和 11. 2% ( 老叶绿茶) ,是大理茶制作的绿茶的特征香气成分。DPPH 和 ABTS+自由基清除实验结果显示大理茶鲜叶及其制成的绿茶的挥发性成分均具有一定的抗氧化活性,但均弱于茶多酚的抗氧化活性。     

比较不同DNA条形码对中国海岸带耐盐植物的识别率

海岸带耐盐植物是一个生态和经济价值独特的庞杂类群,人们对其DNA条形码特性的了解甚少。本文对我国从辽宁到海南10个沿海省(市)大陆及岛屿海岸带耐盐植物广泛采样,从采集获得的样品中筛选出53科97属116个物种共562个样品进行DNA条形码研究。对3个叶绿体片段(mat K、rbc L、trn H-psb A)和1个核基因片段(ITS)进行了扩增和测序,统计各个片段的引物通用性和序列获得率,并检验了物种识别率。从序列获得率来看,mat K和trn H-psb A片段表现最好,ITS较差,ITS和mat K的引物通用性比其他2个片段差。序列相似性分析表明,单个片段ITS物种识别率最高(73.36%),其次为mat K(64.03%)和trn H-psb A(61.21%),rbc L的物种识别率最低,仅为46.41%。系统发育树分析显示mat K的物种识别率最高(82.3%),依据trn H-psb A片段难以获得可靠的系统发育树。多维度非度量分析(non-metric multidimensional scaling,NMDS)表明在进行海岸带区域性植物的DNA条形码研究时,叶绿体片段和核基因片段均应该考虑。综合上述研究结果,推荐联合片段ITS+mat K作为中国海岸带耐盐植物DNA条形码。本文为海岸带耐盐植物研究提供了总计1,939条DNA条形码基础数据,为构建耐盐植物DNA条形码数据库奠定了基础。     

毛细管电泳四色荧光检测法分析茶树SSR标记

将毛细管电泳四色荧光栓测技术应用于茶树SSR标记分析.该方法采用三引物PCR扩增SSR位点,三引物即在5'端加有M13尾巴序列(5'-CACGACGTTGTAAAACGAC-3')的特异正向引物、特异反向引物及带有荧光标记的通用型M13引物:为了运用四色荧光检测系统使通过一次毛细管电泳能同时检测3个以上的SSR位点,采用蓝、绿、黑3种不同颜色的荧光染料分别对3个M13引物进行标记. 应用该方法对42个茶树品种(系)的16个SSR位点进行遗传分析的结果表明:此法具有简便、可靠、低成本及高通量的优点;且随着所分析SSR位点数的增加,降低成本的效果更加显著.采用建立的方法,还筛选获得了11个多态性丰富的可应用于茶树遗传研究的SSR标记.     

Productivity and biochemical properties of green tea in response to full-length and functional fragments of HpaG Xooc, a harpin protein from the bacterial rice leaf streak pathogen Xanthomonas oryzae pv. oryzicola

Harpin proteins from plant pathogenic bacteria can stimulate hypersensitive cell death (HCD), drought tolerance, defence responses against pathogens and insects in plants, as well as enhance plant growth. Recently, we identified nine functional fragments of HpaG;Xooc, a harpin protein from Xanthomonas oryzae pv.oryzicola, the pathogen that causes bacterial leaf streak in rice. Fragments HpaG;1-94'HpaG;10-42, and HpaG;62-138, which contain the HpaG;Xooc regions of the amino acid sequence as indicated by the number spans, exceed the parent protein in promoting growth, pathogen defence and HCD in plants. Here we report improved productivity and biochemical properties of green tea (Camellia sinensis) in response to the fragments tested in comparison with HpaG;Xooc and an inactive protein control. Field tests suggested that the four proteins markedly increased the growth and yield of green tea, and increased the leaf content of tea catechols, a group of compounds that have relevance in the prevention and treatment of human diseases. In particular, HpaG;1-94 was more active than HpaG;Xooc in expediting the growth of juvenile buds and leaves used as green tea material and increased the catechol content of processed teas. When tea shrubs were treated with HpaH;Xooc and HpaG;1-94 compared with a control, green tea yields were over 55% and 39% greater, and leaf catechols were increased by more than 64% and 72%, respectively. The expression of three homologues of the expansin genes, which regulate plant cell growth, and the CsCHS gene encoding a tea chalcone synthase, which critically regulates the biosynthesis of catechols, were induced in germinal leaves of tea plants following treatment with HpaG;1-94 or HpaG;Xooc. Higher levels of gene expression were induced by the application of HpaG;1-94 than HpaG;Xooc. Our results suggest that the harpin protein, especially the functional fragment HpaG;1-94, can be used to effectively increase the yield and improve the biochemical properties of green tea, a drink with medicinal properties.     

DNA barcoding of arid wild plants using rbcL gene sequences

DNA barcoding is currently gaining popularity due to its simplicity and high accuracy as compared to the complexity and subjective biases associated with morphology-based identification of taxa. The standard chloroplast DNA barcode for land plants recommended by the Consortium for the Barcode of Life (CBOL) plant working group needs to be evaluated for a wide range of plant species. We therefore tested the potential of the rbcL marker for the identification of wild plants belonging to diverse families of arid regions. Maximum likelihood tree analysis was performed to evaluate the discriminatory power of the rbcL gene. Our findings showed that using rbcL gene sequences enabled identification of the majority of the samples (92%) to genus level and only 17% to species level.     

茶树诱导抗虫性的研究进展

为了抵御植食性昆虫的为害,植物在进化过程中形成了包括组成抗性和诱导抗性在内的复杂防御体系.在通过受体识别茶树害虫为害后,茶树会启动早期信号事件,继而激活茉莉酸、水杨酸、乙烯和赤霉素等植物激素信号通路,从而引起次生代谢物的积累,最终对害虫产生直接和间接抗性.基于近年来茶树害虫为害诱导的茶树防御反应及其相关调控机理的研究进...     

Amplification of the rbcL gene from dissolved and particulate DNA from aquatic environments

The carboxylation of ribulose biphosphate by the enzyme ribulosebisphosphate carboxylase/oxygenase is the mechanism for CO2 fixation and primary production in nearly all ecosystems on this planet. Although certain algal isolates and higher plants contain conserved nucleotide sequences in the large subunit of the gene (rbcL) for this enzyme, such genes from natural microbial assemblages have not been heretofore examined. Using oligonucleotide primers designed for conserved regions of the rbcL gene of a Synechococcus sp. (Anacystis nidulans), we have amplified rbcL from DNA preparations from planktonic samples from a Florida reservoir and from algal isolates by the polymerase chain reaction. We have also detected rbcL by gene amplification in the extracellular DNA fraction of this reservoir, indicating that phytoplankton can be a source of dissolved DNA. These results suggest that gene amplification can be applied for the detection of conserved genes encoding enzymes involved in important ecological functions in aquatic environments.     

Amplification of the rbcL gene from dissolved and particulate DNA from aquatic environments.

The carboxylation of ribulose biphosphate by the enzyme ribulosebisphosphate carboxylase/oxygenase is the mechanism for CO2 fixation and primary production in nearly all ecosystems on this planet. Although certain algal isolates and higher plants contain conserved nucleotide sequences in the large subunit of the gene (rbcL) for this enzyme, such genes from natural microbial assemblages have not been heretofore examined. Using oligonucleotide primers designed for conserved regions of the rbcL gene of a Synechococcus sp. (Anacystis nidulans), we have amplified rbcL from DNA preparations from planktonic samples from a Florida reservoir and from algal isolates by the polymerase chain reaction. We have also detected rbcL by gene amplification in the extracellular DNA fraction of this reservoir, indicating that phytoplankton can be a source of dissolved DNA. These results suggest that gene amplification can be applied for the detection of conserved genes encoding enzymes involved in important ecological functions in aquatic environments.     

Detection of plant genes using a rapid, nonorganic DNA purification method

We have developed a simple procedure for the preparation of plant genomic DNA using FTA paper. Plant leaves were crushed against FTA paper, and the genomic DNA was purified using simple, nonorganic reagents. The 18S rRNA gene and the gene encoding the ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit (rbcL) from the chloroplast genome were detected by PCR amplification of DNA on FTA paper. DNA amplification was successful using extracts from 16 dicot and monocot plants. Studies of specific plant extracts revealed that extracts of leaf samples could be collected and stored at room temperature on FTA paper without a decrease in the DNA amplification success rate for more than a month. Both the 18S RNA gene and the rbcL gene were detected in the genomic DNA isolated from various soybean cultivars stored in this manner. Furthermore, by modestly increasing the number of cycles of DNA amplification, we were able to detect the uidA gene in transgenic tobacco and rice leaves as well as a single copy gene linked to the resistance gene of cyst nematode race 3 using genomic DNA isolated on FTA paper. These results demonstrate that genomic DNA isolated using FTA paper can be used for the detection of plant genes, from a wide range of plants with either high or low gene copy number and of either nuclear or cytoplasmic origin.     

中国某些野生和栽培茶的核型研究

研究了茶的4个变种和广东野生毛叶茶等共12个材料的核型。所有材料的染色体数目均是2n=30,为二倍体。所有中国大叶变种(越南大叶除外)(Cametlia sinensis var. macrophylla)和阿萨姆大叶变种(C. sinensis var. assamica)均具比较对称或原始的“2A”核型;中国小叶变种(C.sinensis var.bohea)(“铁观音”品种除外),掸部变种(C.sinensis var. shan form)和广东野生毛叶茶(C. ptilophylla)均具较不对称或较进化的“2B”核型。根据核型特征,植物习性和地理分布,作者认为中国四川和云南可能是茶的起源中心,向东或北迁移,演变为中国小叶变种;向南移则演变为阿萨姆变种和掸部变种。     

Testing four proposed barcoding markers for the identification of species within Ligustrum L.(Oleaceae)

DNA barcoding is a biological technique that uses short and standardized genes or DNA regions to facilitate species identification. DNA barcoding has been used successfully in several animal and plant groups. Ligustrum (Oleaceae) species occur widely throughout the world and are used as medicinal plants in China. Therefore, the accurate identification of species in this genus is necessary. Four potential DNA barcodes, namely the nuclear ribosomal internal transcribed spacer (ITS) and three chloroplast (cp) DNA regions (rbcL, marK, and trnH-psbA),were used to differentiate species within Ligustrum. BLAST, character-based method, tree-based methods and TAXONDNA analysis were used to investigate the molecular identification capabilities of the chosen markers for discriminating 92 samples representing 20 species of this genus. The results showed that the ITS sequences have the most variable information, followed by trnH-psbA, matK, and rbcL. All sequences of the four regions correctly identified the species at the genus level using BLAST alignment. At the species level, the discriminating power of rbcL, matK, trnH-psbA and ITS based on neighbor-joining (NJ) trees was 36.8%, 38.9%, 77.8%, and 80%,respectively. Using character-based and maximum parsimony (MP) tree methods together, the discriminating ability of trnH-psbA increased to 88.9%. All species could be differentiated using ITS when combining the NJ tree method with character-based or MP tree methods. Overall, the results indicate that DNA barcoding is an effective molecular identification method for Ligustrum species. We propose the nuclear ribosomal ITS as a plant barcode for plant identification and trnH-psbA as a candidate barcode sequence.