Effects of drought on non-structural carbohydrates and C,N,and P stoichiometric characteristics of Pinus yunnanensis seedlings

To study non-structural carbohydrate character-istics and nutrient utilization strategies of Pinus yunnanen-sis under continuous drought conditions,2-year-old seed-lings were planted in pots with appropriate water,light and moderate and severe drought treatments[(80±5),(65±5),(50±5),and(35±5)%of field water-holding capacity].Non-structural carbohydrates,carbon(C),nitrogen(N),and phosphorus(P)concentrations were measured in each plant component.The results show that:(1)With increasing drought,non-structural carbohydrates gradually increased in leaves,stems,and coarse roots,while gradually decreased in fine roots;(2)C concentrations of all were relatively stable under different stress levels.Phosphorous utilization of each component increased under light and moderate drought conditions,while N and P utilization efficiency of each plant component decreased under severe drought.Growth was mainly restricted by N,first decreasing and then increasing with increased drought;(3)There was a correlation between the levels of non-structural carbohydrates and C,N,and P in each component.Changes in N concentration affected the interconversion between soluble sugar and starch,which play a regulatory role in the fluctuation of the concentration of non-structural carbohydrates;and,(4)Plasticity analysis showed that P.yunnanensis seedlings responded to drought mainly by altering starch concentration,the ratio of soluble sugar to starch in leaves and stems,and further by alter-ing N and P utilization efficiencies.Overall,these results suggest that the physiological activities of all organs of P.yunnanensis seedlings are restricted under drought and that trade-offs exist between different physiological indicators and organs.Our findings are helpful in understanding non-structural carbohydrate and nutrient adaptation mechanisms under drought in P.yunnanensis seedlings.

The Identification of Phenylalanine Ammonia-Lyase(PAL)Genes from Pinus yunnanensis and an Analysis of Enzyme Activity in vitro

Phenylalanine ammonia lyase(PAL)is the rate-limiting and pivotal enzyme of the general phenylpropanoid path-way,but few reports have been found on PAL genes in Pinus yunnanensis.In the present study,three PAL genes were cloned and identified from P.yunnanensis seedlings for thefirst time,namely,PyPAL-1,PyPAL-2,and PyPAL-3.Our results indicated that the open-reading frames of PyPAL genes were 2184,2157,and 2385 bp.Phylogenetic tree analysis revealed that PyPALs have high homology with other known PAL genes in other plants.In vitro enzymatic analysis showed that all three PyPAL recombinant proteins could catalyze the deamination of L-phenylalanine to form trans-cinnamic acid,but only PAL1 and PAL2 can catalyze the conversion of L-tyrosine toρ-coumaric acid.Three PyPAL genes were expressed in different tissues in 1-year-old P.yunnanensis,and such genes had different expression patterns.This study lays a foundation for further understanding of the biosynthesis of secondary metabolites in P.yunnanensis.

Productions of Taxol and Related Taxanes by Cell Suspension Cultures of Taxus yunnanensis

A high taxol yield cell line of Taxus yunnanensis Cheng et L. K. Fu keeps a high taxol_producing level after successive subcultures for more than eight years. In this study, eight taxanes were isolated from the suspension cell cultures of this cell line. Based on NMR and MS analyses, and comparison with literature data and standards, their structures were determined to be 2α,5α,10β_triacetoxy_14β_propionyloxy_4(20),11_taxadiene (1), 2α,5α,10β_triacetoxy_14β_(2′_methyl)_butyryloxy_4(20),11_taxadiene (2), 2α,5α,10β_14β_tetra_acetoxy_4 (20),11_taxadiene (3, taxuyunnanine C), 2α,5α,10β_triacetoxy_14β_(2′_methyl_3′_hydroxy)_butyryloxy_4(20),11_taxadiene (4, yunnanxane) and its 3′_epimer (5), baccatin Ⅳ (6), baccatin Ⅲ (7) and taxol (8), respectively. Among those compounds, 3, 5, 6 and 7 were reported to be isolated from the suspension cell cultures of T. yunnanensis for the first time. TLC and HPLC analyses indicated that the chemical constituents of the culture solution were similar to those of cultured cells. Moreover, the highest taxol content of this cell line reached 0.3% and the cell line could be applied for a large_scale culture.

The Chemical Constituents of Amoora yunnanensis

Two new sterols, 3 beta, 7 alpha, 16 beta -trihydroxy-stigmast-5,22-diene (1), 3 beta, 7 alpha, 16 beta -trihydroxy-stigmast-5-ene (2), were isolated together with six known compounds, ergosta-5, 24(28)-dien-3 beta, 7 alpha -diol (3), ergosta-5,24(28)-dien-3 beta, 7 beta, 16 beta -triol (4), beta -amyrone (5), beta -amyrin (6), 11 alpha, 12 alpha -epoxy-14-taraxeren-3-one (7), and 6-guaiene-4 alpha, 10 alpha -diol (8) from the EtOH extract of the bark of Amoora yunnanensis (H. L. Li) C. Y. Wu. Their structures were deduced on the basis of spectral data.

Study and Identification of Gaultheria yunnanensis Leaves from Different Regions

[Objective] In order to identify and comparatively study leaves of ethnic medicine Gaultheria yunnanensis from different regions of Guizhou.[Method] Characteristics,microscopic,physical and chemical identification were studied on Gaultheria yunnanensis.[Result] There was no obvious difference in characteristics and microscopic sections among Gaultheria yunnanensis leaves from different regions.Blue fluorescence spots were observed clearly by TLC（Thin Layer Chromatography） test.[Conclusion]The study provided a basis for improvement of the quality standard of the Gaultheria yunnanensis.

云南红豆杉(Taxus Yunnanensis)内生真菌中产紫杉醇真菌的筛选

报道了从云南红豆杉 (TaxusYunnanensis)树皮和枝条中分离得到的 5 2株内生真菌中 ,有 19株菌的发酵产物经TLC分析和HPLC检测验证后产紫杉醇或紫杉烷类物质。其中紫杉醇含量在 1 以上的菌株有 8株。TAX - 4 7(茎点霉属 )和TAX - 4 9(组丝核菌属 )的紫杉醇得率分别为 4 7 30 2 μg L和 31 0 6 μg L ,TAX - 2 5 (茎叶核菌属 )巴可亭Ⅲ得率为 74 4 2 μg L 。

Olfactory Responses of Tomicus yunnanensis to Different Non-host Plants

[ Objective ] With Cupressustorulosa, Cinnamomum camphora and Cyclobalanopsis Oerst as test materials, the paper studied the effects of different non- host plants on olfactory responses of Tomicus yunnanensis. [ Method ] The needles of Pinus yunnanensis were mixed with the leaves of Cupressustondosa, C. cam- phora and C. Oerst according to the ratios of 0 g ： 6 g, 1 g ： 5 g, 2 g ： 4 g, 3 g ： 3 g, 4 g ： 2 g, 5 g ： 1 g and 6 g ： 0 g, and the mixtures were put in the re- spanse arm of Y-tube olfactometer as odor source to observe the olfactory behavior of T. yunnanensis, the empty arm was set as control. [ Result ] When the needles of P. yunnanensis were mixed with the leaves of non-hest plants according to the ratio of 1 g ： 5 g and 2 g ： 4 g, they had less difference on attractive rate to T. yun- nanensis compared with complete P. yunnanensis needles in mixture （ratio： 6 g ： 0 g）, and the maximum difference was 14%. When the needles ofP. yunnanen- sis were mixed with non-host leaves according to the ratio of 1 g ： 5 g, 2 g ： 4 g, the attractive rate to T. yunnanensis decreased compared with complete P. yun- nanensis needles in mixture （ ratio： 6 g ： 0 g）, and the decrease value in maximum was 40%. [ Conclusion] When the ratio of non-host plants was relatively small in mixed leaves, non-host had less impact on olfactory responses of T. yunnanensis. As the proportion of non-hest leaves gradually increased, the attractive rate of leaf mixtures to T. yunnanensis was gradually small. The results could provide reference for determination of mixed ratio in construction of mixed forest and the de- velopment of botanical attractive and repellent of T. yunnanensis.

滇桑(Morusyunnanensis)的染色体核型分析

染色体核型分析可为研究植物的系统演化、亲缘关系以及物种的起源和遗传特性提供重要的细胞学依据。以从云南省大围山国家级自然保护区收集野生滇桑(Morus yunnanensis)的愈伤组织为材料,采用去壁低渗法制备染色体标本,对其染色体核型进行分析。结果表明,大围山滇桑体细胞的染色体数目为14,核型公式为K(2n)=2x=2St+12m,14条染色体组成7对。该结果在不久前发现川桑(Morus notabilis)染色体基数为7的基础上,验证了桑树染色体基数为7条的推论,对于进一步研究滇桑的起源与进化具有重要意义。

云南松(Pinus yunnanensis)林外生菌根真菌的时空分布

2000年至2005年，调查了滇中及其附近云南松林下外生菌根真菌的生态分布，共采集、鉴定标本834号，计有27科39属211种（含变种、变型）。结果表明，红菇属（Russula）、牛肝菌属（Boletus）、乳菇属（Lactarius）、乳牛肝菌属（Suillus）、口蘑属（Tricholoma）、鸡油菌属（Cantharellus）和革菌属（Thelephora）等为云南松林下的主要外生菌根菌类群。它们的发生与分布受到气候（如：气温和降水）、植被（如：林龄、林地郁闭度和草本植被）、地形特征（如：海拔、坡向和坡度）、土壤条件（如：pH值、地表腐殖质和枯枝落叶层等）和人为干扰（比如：商业化采集、林木采伐、火烧和地表物清理）诸多因素的影响。总结为如下：（1）5a的调查结果显示，云南松外生菌根菌的分布表现出季节性变化的规律;其中以每年1、2、3月份的物种多样性为最低，雨季期间急剧增加，至中夏和秋末达到顶峰，种类最为繁多。（2）在海拔1500-2100m，云南松外生菌根菌种类随着海拔的升高而逐渐增加，至顶峰后，又呈缓慢下降趋势。海拔因素不但对其物种多样性，而且对于类群的组成也具有重要的影响。特定的类群往往发生在特定海拔范围。（3）随着云南松林龄的增加，外生菌根菌呈现由少至多的演替过程。外生菌根菌多样性随云南松林生长而逐渐增加的演替方式，可能与宿主光合作用产物、根部分泌物和土壤条件的逐渐变化有关。（4）人类干扰是云南松外生菌根菌物种多样性和类群组成的主要负影响因子。大规模的商业化采集可破坏或枯竭地下菌丝体，打破各物种之间的竞争平衡，减少孢子释放影响资源再生能力，进而直接影响到子实体的产生。外生菌根菌物种多样性的减少趋势会随林木砍伐和火烧强度的增加而加剧。地表枯枝落叶层与杂草密度也会影响子实体的产生，其中枯枝落叶层的厚度与云南松外生菌根菌子实体的发生呈负相关性，而被紫茎泽兰覆盖的云南松林地内也很少会发现相应的子实体。

云南红豆杉(TaxusYunnanensis)皮下真菌类群初步研究

分离纯化了与云南红豆杉 (TaxusYunnanensis)内共生的皮下真菌 ,获得了 52株内生真菌。对 52株菌进行了分类研究 ,结果是 :无孢类各属是优势菌群 (共 2 9株 ) ,其次是木霉属 ( 5株 )和茎点霉属 ( 4株 )。首次报道了红豆杉属植物皮下真菌的类群和区系特点。

利用扦插和组织培养技术繁殖特异种质资源滇桑(Morus yunnanensis Koidz.)

滇桑(Morus yunnanensis Koidz.)属于桑树特异种质资源,是研究桑属植物进化的重要桑种。解决滇桑嫁接不亲和性而不易繁殖的技术难题,对促进滇桑的科学研究和开发利用有重要意义。以云南省屏边大围山国家自然保护区采集的滇桑冬芽及枝条为材料,利用扦插和植物组织培养技术进行无性繁殖试验,结果表明:滇桑的冬芽能够在添加3.0 mg/L 6-BA和0.5 mg/L GA3的MS培养基中萌发,其萌发的小芽在添加1.0 mg/L 6-BA和0.2 mg/L GA3的MS培养基中能正常生长并木质化,继续在添加0.5 mg/L IBA的1/2 MS培养基中培养生根后能够移栽成活,其萌发率、生根率和移栽成活率分别达到95%、55%、90%;扦插能够诱导含冬芽的滇桑枝条生根获得完整植株并移栽成活,但其扦插生根率较低,仅30%左右。试验结果提示,进一步优化组织培养和扦插条件,有望建立滇桑的无性繁殖技术体系。

滇桑(Morus yunnanensis)的组织培养繁殖

滇桑(Morus yunnanensis)作为我国特有的桑树种质资源,具有重要的科学研究和应用开发价值。为了解决滇桑不易无性繁殖的难题,以从云南省大围山国家级自然保护区采集野生滇桑枝条上的侧芽为外植体,通过组织培养的方法初步建立滇桑的快速繁殖技术体系。在MS培养基中添加2.0 mg/L 6-BA和0.2 mg/L NAA有利于侧芽的萌发;添加1.0 mg/L 6-BA能促进幼苗的生长;茎部木质化的幼苗在添加0.25 mg/L IBA和0.25 mg/L NAA以及0.4%活性炭的WPM培养基上能分化出健壮的根系并移栽成活。试验结果显示细胞分裂素和生长素在大围山滇桑的组织培养繁殖中发挥了重要作用,低浓度活性炭对滇桑组培幼苗根系的生长和发育有促进作用。

Determination of 10-Deacetyl baccatin Ⅲ and BaccatinⅢ in Taxus yunnanensis

报道了一种在Ｃ＿（１８）高效液相色谱柱上分离紫杉醇（Ｔａｘｏｌ）的前体化合物ＢａｃｃａｔｉｎⅢ和１０－去乙酰ＢａｃｃａｔｉｎⅢ的分析方法。所用流动相为甲醇－水（５２：４８），必要时可使用水－乙腈－四氢呋喃（６７：２５：８），流速１．０ｍｌ／ｍｉｎ，检测波长２２７ｎｍ。运用此方法对云南红豆杉不同部位（干皮、枝、叶、心木）的若干样品进行了测定，发现ＢａｃｃａｔｉｎⅢ和１０－去乙酰ＢａｃｃａｔｉｎⅢ在各部位间含量差异较大，即使同一部位也有很大差异。

滇榛(Corylus yunnanensis)SSR引物的筛选

滇榛是我国西南地区的一个重要榛属资源,本文成功筛选到滇榛9个SSR标记,并对滇榛群体遗传多样性与遗传结构进行了初步分析,结果显示滇榛群体具有中等程度的遗传多样性与遗传分化,这为滇榛遗传改良研究奠定了基础,也有助于推动本地区榛属资源的保护与可持续利用。

Isolation of the Endophytic Fungi of Paris polyphylla var. yunnanensis and Their Effects on the Embryo Development of P. polyphylla var. yunnanensis Seeds

[Objective] This study aimed to isolate the endophytic fungi of Paris polyphylla var. yunnanensis and investigate their effects on the embryo development of P. polyphylla var. yunnanensis seeds. [Method] The endophytic fungi of P. polyphylla were isolated and identified morphologically, and their effects on the embryo development of P. polyphylla var. yunnanensis seeds were studied by using paraffin sectioning and microphotography. [Result] Nine endophytic fungi, i.e. P. polyphylla var. yunnanensis endophytic fungi PPYEF-1, PPYEF-2, PPYEF-3, PPYEF-4, PPYEF-5, PPYEF-6, PPYEF-7, PPYEF-8 and PPYEF-9 belonging to seven genera in five families, three orders were isolated from the rhizomes. Except PPYEF-4 (Cladosporium sp.), other fungi could promote the embryo development of the P. polyphylla var. yunnanensis seeds, mostly reaching the extremely significant or significant level. PPYEF-9 (Trichoderma sp.) resulted in the highest embryo length and embryo-emerging ratio. [Conclusion] This paper could provide a reference for the application of the endophytic fungi of P. polyphylla var. yunnanensis in the dormancy-breaking of P. polyphylla var. yunnanensis seeds.

滇西北玉龙雪山不同海拔云南松(Pinus yunnanensis)径向生长对气候因子的响应

云南松(Pinus yunnanensis)是重要的造林树种,在我国西南地区广泛分布。研究不同海拔云南松径向生长对气候变化的响应,有助于了解气候变化背景下云南松的敏感性和适应性。在滇西北丽江玉龙雪山不同海拔采集了云南松树木年轮样品,采用传统的树木年轮方法制作了不同海拔云南松树轮宽度标准化年表,并分析了不同海拔云南松径向生长与气候因子的相关性。结果表明:1)低海拔样点云南松具有较快的年平均生长速率。2)不同海拔云南松对气候因子的响应模式一致,树轮宽度与当年5—6月的降水量、帕尔默干旱指数(PDSI)和相对湿度呈正相关,与同期温度呈负相关。3)不同海拔的云南松径向生长对气象因子的响应程度不一样,即低海拔样点云南松树轮宽度与当年5月份的干旱指数、相对湿度、降水量相关系数较高;而高海拔样点的云南松树轮宽度与5—6月的降水、相对湿度、干旱指数的相关系数较低。研究表明春末夏初的水分条件是玉龙雪山云南松径向生长的主要限制因子,且低海拔地区云南松生长受水分限制更为严重,区域气候变暖和干旱化趋势可能对低海拔地区云南松的生长产生持续的负面效应。研究结果可为探讨气候变化下云南松的适宜分布区、以及云南松人工林的经营和可持续管理提供参考。

New Macrocyclic Diamide from Rauvolfia Yunnanensis Tsiang

A new macrocyclic diamide, 22-membered macrocyclic diamide, named cyclodicaprylamide（2）, and five known compounds, bis（2-ethylhexyl） phthalate（1）, ethyl 3,4,5-trimethoxybenzoate（3）, ethyl 3,4,5-trimethoxycinnamate （4）, （＋）-syringaresinol（5）, loliolide（6）, were isolated from the roots of Rauvolfia yunnanensis Tsiang. Their structures were elucidated based on NMR, 2D NMR, and MS spectrum, respectively. They were obtained from it for the first time.

A novel phenylpropanoid glycosides and a new derivation of phenolic glycoside from Paris Polyphylla var. yunnanensis

A novel phenylpropanoid glycosides 1, named parispolyside E and a novel derivation of phenolic glycoside 2, named parispolyside G, as well as two known flavonoid glycosides were isolated from the rhizome of Paris polyphylla var. yunnanensis. Their structures were elucidaed by spectroscopic methods.

RNA-seq analysis of Paris polyphylla var.yunnanensis roots identified candidate genes for saponin synthesis

Paris polyphylla Smith var.yunnanensis(Franch.) Hand.-Mazz.is a rhizomatous,herbaceous,perennial plant that has been used for more than a thousand years in traditional Chinese medicine.It is facing extinction due to overharvesting.Steroids are the major therapeutic components in Paris roots,the commercial value of which increases with age.To date,no genomic data on the species have been available.In this study,transcriptome analysis of an 8-year-old root and a 4-year-old root provided insight into the metabolic pathways that generate the steroids.Using Illumina sequencing technology,we generated a high-quality sequence and demonstrated de novo assembly and annotation of genes in the absence of prior genome information.Approximately 87,577 unique sequences,with an average length of 614 bases,were obtained from the root cells.Using bioinformatics methods,we annotated approximately 65.51% of the unique sequences by conducting a similarity search with known genes in the National Center for Biotechnology Information's non-redundant database.The unique transcripts were functionally classified using the Gene Ontology hierarchy and the Kyoto Encyclopedia of Genes and Genomes database.Of 3082 genes that were identified as significantly differentially expressed between roots of different ages,1518(49.25%) were upregulated and 1564(50.75%) were downregulated in the older root.Metabolic pathway analysis predicted that 25 unigenes were responsible for the biosynthesis of the saponins steroids.These data represent a valuable resource for future genomic studies on this endangered species and will be valuable for efforts to genetically engineer P.polyphylla and facilitate saponin-rich plant development.

Levels of rhizome endophytic fungi fluctuate in Paris polyphylla var.yunnanensis as plants age

Paris polyphylla var. yunnanensis is an important medicinal plant with abundant saponins that are widely used in the pharmaceuticals industry. It is unclear why the levels of active ingredients increase as these plants age. We speculated that the concentrations of those components in the rhizomes are mediated by fungal endophytes. To test this hypothesis, we took both culture-dependent and-independent(metagenomics) approaches to analyze the communities of endophytic fungi that inhabit those rhizomes in plants of different age classes(four, six, and eight years old). In all, 147 isolates representing 18 fungal taxa were obtained from 270 segments(90 per age class). Based on morphological and genetic characteristics, Fusarium oxysporum(46.55% frequency of occurrence) was the predominant endophyte,followed by Leptodontidium sp.(8.66%) and Trichoderma viride(6.81%). Colonization of endophytic fungi was maximized in the eight-year-old rhizomes(33.33%) when compared with four-year-old(21.21%) and six-year-old(15.15%) rhizomes. Certain fungal species were present only at particular ages. For example,Alternaria sp., Cylindrocarpon sp., Chaetomium sp., Paraphaeosphaeria sporulosa, Pyrenochaeta sp., Penicillium swiecickii, T. viride, and Truncatella angustata were found only in the oldest plants. Analysis of(metagenomics) community DNA extracted from different-aged samples revealed that, at the class level,the majority of fungi had the highest sequence similarity to members of Sordariomycetes, followed by Eurotiomycetes and Saccharomycetes. These results were mostly in accord with those we obtained using culture methods. Fungal diversity and richness also changed over time. Our investigation is the first to show that the diversity of fungi in rhizomes of P. polyphylla var. yunnanensis is altered as plants age, and our findings provide a foundation for future examinations of useful compounds.