拟耧斗菜昼夜差异表达ESTs的分离与分析

应用磁珠介导的抑制消减杂交方法,以青藏高原白马雪山流石滩地区植物拟耧斗菜为实验材料,分别在白天与夜晚采样,以白天样品为目标,晚上样品为驱动样品,分离昼夜差异表达的基因。随机挑选了96个差异ESTs克隆并进行测序,测序结果进行blastx网上数据库比对、Blast2go功能注释和KEGG代谢路径分析。结果表明：绝大部分ESTs功能与物质能量代谢、生物合成、蛋白和核酸结合、转移酶活性相关,还有少数与逆境胁迫相关等。这为进一步研究高山植物逆境适应的分子机制奠定了基础。     

黄瓜芽黄突变体抑制消减杂交文库的构建及初步分析

利用抑制消减杂交技术(suppression subtractive hybridization,SSH)分离了黄瓜芽黄突变体及其野生型之间差异表达的cDNA片段.以突变体和野生型分别作检测子和驱赶子,建立正向和反向两个消减杂交cDNA文库;经阳性克隆鉴定,在正向文库中获得特异表达的阳性克隆有133个,在反向文库中得到的阳性克隆有73个.测序后将所得到的159条非重复且非黄瓜的ESTs(登录号:GH270133～GH270291)进行序列同源性比对分析,发现这些ESTs分别与叶绿素合成、光合系统、信号转导、转录因子、氨基酸代谢、糖类代谢、脂类代谢等相关酶及蛋白基因高度同源.     

宽果苁菔适应昼夜变化ESTs的分离与分析

以流石滩地区植物广布种宽果苁菔为实验材料,应用磁珠介导的抑制消减杂交方法,分离昼夜差异基因。随机挑选了136个差异ESTs克隆并进行测序,测序结果使用Blast2go程序进行功能注释和分析。结果表明绝大部分ESTs功能与稳定细胞状态和抗性响应相关,其次与物质能量代谢和信号传导相关,宽果苁菔适应昼夜变化过程的机制非常复杂,本研究为进一步研究高山植物适应流石滩恶劣环境的机制奠定了基础。     

flg22诱导的拟南芥转录组分析及芥子油苷代谢途径的变化

flg22是细菌鞭毛蛋白N端的一段保守性极高的区域,能够诱导植物天然的免疫反应,为全面了解植物在受到细菌性病原菌侵害后的系统响应,利用Illumina Hiseq2000对flg22处理和未处理的拟南芥幼苗进行转录组测序。对两组数据进行差异表达分析,共获得1 200个差异表达基因,包括290个下调基因和910个上调基因。对差异表达基因进行GO富集分析和KEGG pathway富集分析,结果显示,flg22处理后,拟南芥在能量代谢、氨基酸代谢及次生代谢产物的生物合成等方面产生了巨大变化。芥子油苷是一类在植物防御病原菌的天然免疫反应中起重要作用的次生代谢产物,因此对芥子油苷代谢途径的变化进行了深入分析。根据测序结果,Flg22处理后吲哚族芥子油苷合成途径的基因表达水平显著提高,而脂肪族芥子油苷代谢途径几乎没有变化,进一步对吲哚族芥子油苷合成途径的关键酶基因进行Real Time RT-PCR的分析,验证了测序结果的正确性,证明了吲哚族芥子油苷在植物抗病防御反应中的重要作用。这为深入理解病原菌诱导的植物防御性反应及吲哚族芥子油苷的抗病机制提供了大量参考数据。     

镰刀菌诱导的泸定百合SSH文库构建及抗病相关基因筛选

该研究以泸定百合(Lilium sargentiae Wilson)为材料,构建其组培苗经百合尖孢镰刀菌侵染后的叶片SSH文库,从中筛选镰刀菌枯萎病抗病相关基因。从正向SSH文库中随机挑取300个单克隆测序后得到280条ESTs,进行功能比对分析后,除去未知功能、沉冗蛋白以及无同源序列,得到有功能的ESTs共168条,其功能涉及信号传导、蛋白质合成与代谢、抗病与防御、物质与能量代谢、转录相关等多种途径,其中有31条ESTs与抗病防御相关。从抗病防御相关基因中选取8条ESTs:过氧化氢酶、ATP结合盒转运蛋白(ABC transporter)、Kunitz型胰蛋白酶抑制剂4(Kunitz trypsin inhibitor 4)、丝氨酸乙醛酸氨基转移酶、多聚泛素、脂氧合酶I(Lipoxygenase I)、丝氨酸/苏安酸蛋白激酶(Serine/Threonine-protein kinase)、抗坏血酸过氧化物酶(Arabidopsis thaliana),通过RTPCR对其表达情况进行分析,发现经镰刀菌诱导后均为上调表达,推测它们可能参与了泸定百合镰刀菌枯萎病的抗病反应途径。     

人参植物与皂苷生物合成相关的差减cDNA文库构建及基因差异表达分析

应用抑制差减杂交技术,分别以源于4年和1年生人参根组织cDNA群体作为检测子(tester)与驱赶子(driver),成功构建了与人参植物皂苷生物合成相关的差减cDNA文库,并时从中筛选的阳性cDNA克隆进行DNA测序及其序列分析、PCR及Northern印迹杂交鉴定．结果显示,获得的13个克隆为新基因序列．其中6个差减克隆系人参植物根生长发育阶段差异表达基因．目前,6个差异表达新基因的结构与功能仍在进一步研究中．     

中国蛇岛蝮蛇毒腺cDNA文库ESTs序列测定及生物信息学分析

前期我们构建了中国蛇岛蝮蛇(Gloydius shedaoensis shedaoensis,GSS)毒腺(GSSG)的cDNA(GSSG-cD-NA)文库。本文从构建的GSSG-cDNA文库阳性重组子中随机挑选了216个单克隆进行5’端表达序列标签(EST)单向测序,获得了211条高质量的ESTs。生物信息学序列比对分析结果表明84个克隆为已知功能基因,29个克隆为未知功能基因,98个克隆为新基因,分别占总ESTs的39.8%、13.7%和46.5%。成功获得了GSSG的部分ESTs序列,为GSS蛋白活性组分基因的克隆、表达和功能研究奠定了一定基础。     

西藏八角莲与桃儿七根及根茎SSH文库的建立

以西藏八角莲(Dysosma tsayuensis Ying)和桃儿七[Sinopodophyllum hexandrum(Royle)Ying]为材料,构建其根及根茎的SSH文库,从中筛选鬼臼类植物属种间与鬼臼毒素生物合成相关的差异表达基因。从文库中随机挑取201个阳性克隆测序后得到183条ESTs。去除载体序列和冗余序列,聚类拼接得到17个西藏八角莲的unique ESTs。经BLAST同源比较和功能查寻,有功能注释的unique ESTs共12个,占70.6%,所编码的蛋白涉及光合作用、合成代谢、转录调控等功能;无功能注释和匹配结果的共5个,占29.4%。该研究成功构建了西藏八角莲和桃儿七SSH文库,为进一步揭示鬼臼毒素生物合成途径及其调控机制奠定了基础。     

狭果葶苈高温响应表达序列标签的分离与初步分析

目的:分离与分析白马雪山流石滩地区植物狭果葶苈高温响应基因,研究高山植物高温耐受机制。方法:应用磁珠介导的抑制消减杂交分离差异表达序列标签(EST),通过测序与Blastx比对,用Blast2go程序进行功能注释和代谢路径分析。结果:大部分EST功能与物质能量代谢、生物合成、蛋白和脂肪结合、转移酶活性相关,还有一些胁迫响应的基因。结论:狭果葶苈高温胁迫应答机制是非常复杂的,需要众多基因的参与。     

柔嫩艾美耳球虫新基因的生物信息学分析与克隆表达

为了研究柔嫩艾美耳球虫(Eimeria tenella)的两个主要入侵发育阶段——子孢子和裂殖子的差异基因,根据已报道的子孢子与裂殖子差异的ESTs序列,应用RACE技术克隆获得了子孢子阶段差异表达的新基因全长cDNA序列,命名为ZL583.该基因全长为862 bp,开放阅读框(ORF)为486 bp,编码161个氨基酸,编码蛋白的分子量约为16.9 kD,利用生物信息学分析软件,分析新基因所编码蛋白的性质、定位、结构等特征.利用荧光定量PCR对柔嫩艾美耳球虫不同发育阶段该基因的表达量进行分析显示,子孢子阶段的表达高于其他发育阶段.将ZL583克隆于pET-28a中构建重组质粒,转化大肠杆菌BL21(DE3)后经IPTG诱导表达,获得重组蛋白分子量约23 kD.免疫印迹分析显示该重组蛋白可与兔抗子孢子血清发生特异性反应,表明该蛋白具有较好的反应原性.该结果为进一步研究该基因的生物学功能奠定基础.     

Isolation and Analysis of Differential Expressed ESTs from Stem Trichomes of Lycopersicon esculentum (Solanaceae)

Glandular trichomes are special organs involved in plant defense response and synthesis of volatile secondary metabolites, analyzing trichome specific expressed sequence tags will help us further understand the specific function of plant trichomes. In this paper, suppression subtractive hybridization（SSH） based on magnetic beads technology was used to isolate differential expressed genes of the glandular trichomes in Lycopersicon esculentum. The differential expressing cDNA library was constructed using the glandular trichomes cDNA as tester and the cDNA from the stem without glandular trichomes as driver. After randomly sequencing 108 differential ESTs, Blast2go program was used to do blastx, functional annotation and metabolism analysis. The results show that most ESTs are related to substance metabolism, response to stress, biotic or abiotic stimulus, and have binding and catalytic function. These differential genes lay the foundation for further research on defense mechanism of the tomato trichomes.     

A proteome approach defines protective functions of tobacco leaf trichomes

The leaf surface of most terrestrial plants is covered with plant hairs called trichomes. These epidermal appendages are thought to contribute to many aspects of plant defense against biotic and abiotic stresses in a variety of species. Trichome development has been intensively studied in Arabidopsis, and the phytochemical composition of trichomes was analyzed in a number of plant species. However, comparatively little is known of the proteins expressed. We therefore initiated a proteome approach to better define the cellular mechanisms operating in plant trichomes using two-dimensional gel electrophoresis to separate proteins of whole leaves and isolated trichomes. Tobacco was chosen due to the presence of glandular trichomes involved in the secretion of defense compounds. Comparative image analysis of the protein patterns indicated a number of spots, which were highly enriched in trichomes relative to leaves. These spots were excised for identification by mass spectrometry. The results showed that among the proteins specifically enriched in trichomes, the components of stress defense responses were strongly represented. The high expression of stress-related proteins was verified by Western blotting. Superoxide dismutase isoforms were additionally analyzed by activity staining. Our results demonstrate feasibility of the proteome approach to elucidate the cell biology of plant trichomes.     

Bidirectional Secretions from Glandular Trichomes of Pyrethrum Enable Immunization of Seedlings

Glandular trichomes are currently known only to store mono- and sesquiterpene compounds in the subcuticular cavity just above the apical cells of trichomes or emit them into the headspace. We demonstrate that basipetal secretions can also occur, by addressing the organization of the biosynthesis and storage of pyrethrins in pyrethrum (Tanacetum cinerariifolium) flowers. Pyrethrum produces a diverse array of pyrethrins and sesquiterpene lactones for plant defense. The highest concentrations accumulate in the flower achenes, which are densely covered by glandular trichomes. The trichomes of mature achenes contain sesquiterpene lactones and other secondary metabolites, but no pyrethrins. However, during achene maturation, the key pyrethrin biosynthetic pathway enzyme chrysanthemyl diphosphate synthase is expressed only in glandular trichomes. We show evidence that chrysanthemic acid is translocated from trichomes to pericarp, where it is esterified into pyrethrins that accumulate in intercellular spaces. During seed maturation, pyrethrins are then absorbed by the embryo, and during seed germination, the embryo-stored pyrethrins are recruited by seedling tissues, which, for lack of trichomes, cannot produce pyrethrins themselves. The findings demonstrate that plant glandular trichomes can selectively secrete in a basipetal direction monoterpenoids, which can reach distant tissues, participate in chemical conversions, and immunize seedlings against insects and fungi.     

The molecular cloning of artemisinic aldehyde Delta11(13) reductase and its role in glandular trichome-dependent biosynthesis of artemisinin in Artemisia annua

At some point during biosynthesis of the antimalarial artemisinin in glandular trichomes of Artemisia annua, the Delta11(13) double bond originating in amorpha-4,11-diene is reduced. This is thought to occur in artemisinic aldehyde, but other intermediates have been suggested. In an effort to understand double bond reduction in artemisinin biosynthesis, extracts of A. annua flower buds were investigated and found to contain artemisinic aldehyde Delta11(13) double bond reductase activity. Through a combination of partial protein purification, mass spectrometry, and expressed sequence tag analysis, a cDNA clone corresponding to the enzyme was isolated. The corresponding gene Dbr2, encoding a member of the enoate reductase family with similarity to plant 12-oxophytodienoate reductases, was found to be highly expressed in glandular trichomes. Recombinant Dbr2 was subsequently characterized and shown to be relatively specific for artemisinic aldehyde and to have some activity on small alpha,beta-unsaturated carbonyl compounds. Expression in yeast of Dbr2 and genes encoding four other enzymes in the artemisinin pathway resulted in the accumulation of dihydroartemsinic acid. The relevance of Dbr2 to trichome-specific artemisinin biosynthesis is discussed.     

Tobacco NtLTP1, a glandular-specific lipid transfer protein, is required for lipid secretion from glandular trichomes

Glandular trichomes are the phytochemical factories of plants, and they secrete a wide range of commercially important natural products such as lipids, terpenes and flavonoids. Herein, we report that the Nicotiana tabacum LTP1 (NtLTP1) gene, which is specifically expressed in long glandular trichomes, plays a role in lipid secretion from trichome heads. NtLTP1 mRNA is abundantly transcribed in trichomes, but NtLTP3, NtLTP4 and NtLTP5 are not. In situ hybridization revealed that NtLTP1 mRNAs accumulate specifically in long trichomes and not in short trichomes or epidermal cells. X-gluc staining of leaves from a transgenic plant expressing the NtLTP1 promoter fused to a GUS gene revealed that NtLTP1 protein accumulated preferentially on the tops of long glandular trichomes. GFP fluorescence from transgenic tobacco plants expressing an NtLTP1-GFP fusion protein was localized at the periphery of cells and in the excreted liquid droplets from the glandular trichome heads. In vitro assays using a fluorescent 2-p-toluidinonaphthalene-6-sulfonate probe indicated that recombinant NtLTP1 had lipid-binding activity. The overexpression of NtLTP1 in transgenic tobacco plants resulted in the increased secretion of trichome exudates, including epicuticular wax. In transgenic NtLTP1-RNAi lines, liquid secretion from trichomes was strongly reduced, but epicuticular wax secretion was not altered. Moreover, transgenic tobacco plants overexpressing NtLTP1 showed increased protection against aphids. Taken together, these data suggest that NtLTP1 is abundantly expressed in long glandular trichomes, and may play a role in lipid secretion from long glandular trichomes.     

Localization of salvinorin A and related compounds in glandular trichomes of the psychoactive sage, Salvia divinorum

BACKGROUND AND AIMS: Salvia divinorum produces several closely related neoclerodane diterpenes. The most abundant of these, salvinorin A, is responsible for the psychoactive properties of the plant. To determine where these compounds occur in the plant, various organs, tissues and glandular secretions were chemically analysed. A microscopic survey of the S. divinorum plant was performed to examine the various types of trichomes present and to determine their distribution. METHODS: Chemical analyses were performed using thin layer chromatographic and histochemical techniques. Trichomes were examined using conventional light microscopy and scanning electron microscopy. KEY RESULTS: It was found that neoclerodane diterpenes are secreted as components of a resin that accumulates in peltate glandular trichomes, specifically in the subcuticular space that exists between the trichome head cells and the cuticle that encloses them. Four main types of trichomes were observed: peltate glandular trichomes, short-stalked capitate glandular trichomes, long-stalked capitate glandular trichomes and non-glandular trichomes. Their morphology and distribution is described. Peltate glandular trichomes were only found on the abaxial surfaces of the leaves, stems, rachises, bracts, pedicles and calyces. This was consistent with chemical analyses, which showed the presence of neoclerodane diterpenes in these organs, but not in parts of the plant where peltate glandular trichomes are absent. CONCLUSIONS: Salvinorin A and related compounds are secreted as components of a complex resin that accumulates in the subcuticular space of peltate glandular trichomes.