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.

Effects of Arbuscular Mycorrhizal Fungi on the Physiology and Saponin Synthesis of Paris polyphylla var. yunnanensis at Different Nitrogen Levels

Arbuscular mycorrhizal fungi(AMF)are important members of the plant microbiome and affect the uptake and transfer of mineral elements by forming a symbiotic relationship with plant roots.Nitrogen(N),as an important min­eral element,can directly affect plant growth and development at different N levels.It has been confirmed that inoc­ulation with AMF can improve the efficiency of N utilization by plants.However,there are still fewer reports on the dynamic relationship between arbuscular mycorrhizal and plant secondary metabolites at different nitrogen levels.In this experiment,the physiological indexes and genes related to saponin synthesis were determined by applying differ­ent concentration gradients of nitrogen to the medicinal plant P.polyphylla var.yunnanensis infested by AMF as the test material.It was found that nitrogen addition increased the biomass,chlorophyll content,and nutrient content of above-and below-ground plant parts and increased the content of saponin content of P.polyphylla var.yunnanensis to some extent,but AMF inoculation increased the saponin content of P.polyphylla var.yunnanensis more significantly.AMF inoculation also promoted the expression of genes related to the saponin synthesis pathway,including 3-hy­droxy-3-methylglutaryl coenzyme A synthase(HMGS),squalene epoxidase 1(SE1),and cycloartenol synthase(CAS),which is in according with the accumulation of saponin in plants.It also may increase the saponin content of AMF plants by altering the expression of P450s and UGTs related to saponin synthesis.

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.

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.

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.

Mycorrhizas Affect Polyphyllin Accumulation of Paris polyphylla var.yunnanensis through Promoting PpSE Expression

Paris polyphylla var.yunnanensis is a traditional Chinese medicinal plant,in which polyphyllin as the main medicinal component is an important secondary metabolite with bioactivity.Arbuscular mycorrhizal fungi(AMF)have multiple positive effects on plants,while it is not clear whether AMF increase the content of medicinal components in medicinal plants.In this study,a total of nine AMF treatments were laid to analyze the mycorrhizal effect on polyphyllin accumulation and PpHMGR and PpSE expression of P.polyphylla var.yunnanensis.AMF increased the content of polyphyllin in the cultivated variety with low relation to the increase of inoculation intensity.Polyphyllin I,II,and VII were identified and partly improved by AMF inoculation,dependent on AMF treatments and culture environments.Similarly,the PpHMGR and PpSE expression was induced by mycorrhization,dependent on AMF species,whilst the induction was more obvious in PpSE than in PpHMGR after mycorrhization.It concluded that the symbiotic relationship between P.polyphylla var.yunnanensis and AMF increased polyphyllin content level in the plant,which was associated with the up-regulation of PpSE transcripts.

Determination of 10 Kinds of Inorganic Elements in Paris polyphylla Smith var. yunnanensis(Franch.) Hand.-Mazz.(and Polygerm Varieties)

[Objectives] To determine the content of 10 kinds of inorganic elements(Pb,Cu,As,Cd,Hg,Zn,Fe,Mn,Sr and Cr) in samples of Paris polyphylla Smith var. yunnanensis( Franch.) Hand.-Mazz.( and polygerm varieties) produced in different areas. [Methods] The wet digestion and technique of inductively coupled plasma optical emission spectrometry( ICP-OES) were used. [Results] Under the experimental conditions,elements were not related to each other,and several kinds of elements could be measured at the same time. The content of Pb exceeding the standard only in sample S4 of P. polyphylla Smith var. yunnanensis( and polygerm varieties),Hg was not detected in all samples,and the rest heavy metals did not exceed the limit. [Conclusions] This study was expected to provide a certain reference for the development and utilization of P. polyphylla Smith var. yunnanensis( and polygerm varieties).

Mycorrhizal Fungal Effects on Growth,Antioxidant Capacity,and Medicine Quality of Paris polyphylla var.yunnanensis

A field experiment was conducted to determine the effects of two commercial strains composed of mulple arbuscular mycorrhizal fungi(AMF)species on plant growth,antioxidant capacity,and medicine quality of Paris polyphylla var.yunnanensis in three subtropical soils from Wanzhou,Anshun and Baoshan in fields.The results showed that AMF inoculation enhanced the fungal colonization rate and activities of both succinate dehydrogenase and alkaline phosphatase,thereby,enhancing the mycorrhizal viability of P.polyphylla var.yunnanensis.The concentrations of photosynthetic pigments(chlorophyll a,b,a+b and carotenoids),soluble sugar,soluble protein and photosynthetic capacity were higher in AMF-inoculated plants than in non-AMF-treated plants in field.AMFtreated plants recorded higher activities of catalase,peroxidase and superoxide dismutase,and caused the reduction in malondialdehyde content,indicating lower oxidative damage,compared with non-AMF plants.Polyphyllin I,Polyphyllin II,Polyphyllin III,Polyphyllin IV and total polyphyllin contents were increased by AMF treatment.In conclusion,AMF improved the plant growth,antioxidant capacity and medicinal quality of P.polyphylla var.yunnanensis seedlings.Hereinto,AMF effects on the soil from Wanzhou was relatively greater than on other soils.

Identification of Paris polyphylla Smith var. yunnanensis(Franch.) Hand.-Mazz.(Polygerm Varieties) Produced in Gaoligong Mountain Areas

[Objectives] To make original plant and microscopic identification and study the traits of Paris polyphylla Smith var. yunnanensis( Franch.) Hand.-Mazz.( polygerm varieties) produced in Gaoligong Mountain areas,and provide experimental data for their identification and application. [Methods] The original plant powder was used,and paraffin sections and physicochemical properties were identified. [Results]P. polyphylla Smith var. yunnanensis( polygerm varieties) produced in Gaoligong Mountain areas showed obvious microscopic features and physicochemical properties. [Conclusions] This study was expected to provide certain reference for identification of original plant and medicine and formulation of quality standard for P. polyphylla Smith var. yunnanensis( polygerm varieties) produced in Gaoligong Mountain areas,and provided basis for their further research and development.

Epigenetic Factors Altered in Dehisced Anther Correlated to Seed Dormancy in <i>Paris polyphylla</i>var. Yunnanensis

Paris polyphylla var. yunnanensis (Franch.), one of the best-known medicinal plants in China, has a dehiscent anther which physiologically work in pollination, however, the dehiscent anther always closes in response to darkness every day, and watering or raining every time. To explore this frequently closing and its unkown physiology, next-generation sequencing was performed, and the transcriptome was de novo assembled. RNA-sequencing was carried out in 15 samples including seven openning samples, four closed samples owing to darkness or watering, and tissue samples (leaf, petal, calyx, and stigma) were used for control. We obtained 72.75 GB data, assembled into 79,815 unigenes. Differentially expressed unigenes (DEGs) between opened and closed anther samples were 6231 and the DEGs between anther and control samples were 2831. Comparation between the two DEGs by KEGG enrichment showed that “plant hormone signal transduction” pathway is the most significant pathway for DEGs from closing anther vs. opening anther, and expression model of DEGs in the pathway might elicit change in germination and seed dormancy. Further examination of the action of the signal pathway on physiology showed “chromatin binding” function was prominent in “DNA binding” function of annotated DEGs between opened and closed anthers, of the 215 “chromatin binding” unigenes, 120 were involved in epigenetic silencing, and 50 of the epigenetic unigenes were directly related to germination or seed dormancy, strongly correlating anther closing to epigenetic modification and seed dormancy. These results were verified that at least three auxins involved in seed dormancy showed same expression patterns occurred in abnormal closing anther and seed embryo in Paris polyphylla var. yunnanensis. In conclusion, the information from transcriptome point out that frequent abnormal closing of dehiscent anthers possibly transfer the impact on seed dormancy, and epigenetic modification happened in closing may be the cause.

Investigation on the Resources of Paris Polyphylla var.yunnanensis

In this study,the resource distribution,morphological characteristics,artificial cultivation and market circulation of Paris Polyphylla var.yunnanensis and its polygerm varieties in Yunnan were investigated by referring to the research literature,field investigation and our planting experience.It is found that P.polyphylla var.yunnanensis is widely distributed in Yunnan Province,realizing large-scale artificial cultivation,while the wild resources of polygerm varieties are almost on the verge of extinction and are in urgent need of protection.

滇重楼的psbA-trnH条形码分子鉴定研究

为探讨滇重楼药材鉴定新方法,本研究通过对重楼样品提取基因组DNA,PCR扩增叶绿体psb A-trn H序列并进行双向测序,所得序列经Codon Code Aligner拼接后,采用MEGA5.0软件进行序列比对,计算种内和种间遗传距离(K2P),构建邻接数(neighbor-joining tree)进行结果分析。研究表明,叶绿体psb A-trn H片段在滇重楼种内变异较小,平均K2P遗传距离为0.007,而滇重楼与其它重楼种间平均K2P遗传距离为0.025,滇重楼种间最大K2P遗传距离明显小于滇重楼与重楼属其它种内的最小K2P遗传距离。中药材DNA条形码鉴定系统比对和NJ树鉴定结果均表明psb A-trn H序列可区分滇重楼及其同属物种,且具有较好的稳定性和准确性,为保障临床安全用药提供了新的技术手段。

不同产地滇重楼HPLC指纹图谱及化学模式识别研究

采用高效液相色谱法,建立了滇重楼的HPLC指纹图谱,使用SPSS 17.0软件对19批不同产地滇重楼根茎样品的HPLC指纹图谱进行了主成分分析,在主成分得分系数矩阵的基础上,对其进行了系统聚类分析;并使用相似度评价软件进行了相似度评价验证。结果表明,19批滇重楼根茎样品共提取出3个主成分,被分为3大类;与共有峰直接聚类相比,主成分—聚类分析更符合相似度评价结果。本研究所建立的模式识别方法,操作简便,统计结果具有可靠性,可对滇重楼化学计量分类及其质量评价提供参考。

Parisaponin Ⅰ和Parisvietnaside A的NMR数据分析

从滇重楼中分离得到2个甾体皂苷,利用1H NMR、13C NMR、1H-1H COSY、HSQC、HMBC、1D和2D TOCSY等多种核磁共振方法鉴定其结构分别为(25R)-26-O-β-D-吡喃葡萄糖基-3β,22α,26-三羟基-呋甾-5-烯-3-O-α-L-吡喃鼠李糖基-(1→2)-[α-L-呋喃阿拉伯糖基-(1→4)]-β-D-吡喃葡萄糖苷(1,Parisaponin I)和(25R)-3β,5α,6β-三羟基-△7-螺甾烯-3-O-β-D-吡喃葡萄糖基-(1→3)-[α-L-吡喃鼠李糖基-(1→2)]-β-D-吡喃葡萄糖苷(2,paris-vietnaside A).对化合物1和2的1H NMR和13C NMR信号分别进行了归属和详细分析,并纠正了文献中的核磁数据归属错误.化合物2为首次从滇重楼中分离得到.

一种侵染滇重楼的Potexvirus病毒分离物分子鉴定及其3′末端序列分析

从云南武定的滇重楼上得到一个病毒分离物Paris-YN,病毒粒体为弯曲线状。利用RT-PCR扩增获得一条1074bp的片段,序列比较分析发现其与马铃薯X病毒属(Potexvirus)病毒3′末端的结构最为相似,且与属内的白三叶草花叶病毒等20个不同分离物3′末端有36.7%～58.9%的同源性;该病毒cp基因长639个核苷酸,编码212个氨基酸(22.8kDa),与20个Potexvirus病毒分离物的CP氨基酸序列比较发现,Paris-YN与白三叶草花叶病毒的CP氨基酸同源性最高(60.1%)。证据表明,该分离物可能为Potexvirus的新成员,暂命名为重楼X病毒(Paris polyphylla virus X)。

运用聚类分析与TOPSIS法综合评价云南重楼种质资源

为筛选不同类群云南重楼的优势种源,为其育种提供理论依据,采用TOPSIS法与聚类分析法对23份云南重楼种质资源的亲缘关系进行综合评价。结果表明:经TOPSIS法综合评价有12份云南重楼资源具有优势种源的特性,且其在4个聚类群中均有分布。

滇重楼皂苷部位HPLC指纹图谱的研究

[目的]建立滇重楼皂苷有效部位HPLC指纹图谱分析方法,研究不同产地滇重楼药材的质量。[方法]采用HPLC等度洗脱的方法进行色谱分离,使用"相似度评价软件"进行数据处理,对不同产地的滇重楼药材皂苷部位进行指纹图谱的研究。[结果]确立了滇重楼药材皂苷部位指纹图谱的高效液相分析条件,确定8个共有峰。不同产地的滇重楼药材皂苷部位指纹图谱有一定差异。[结论]采用HPLC法制定滇重楼药材皂苷部位的指纹图谱,其皂苷类成分得到较好的分离,方法重现性好,可用于滇重楼的质量评价。

滇重楼(Paris polyphylla var.yunnanensis)的内生细菌的分离与鉴定

滇重楼(P. polyphylla var. yunnanensis)是中国传统药用植物,多年生草本植物。很多研究已经证明,植物内生菌在植物次生代谢过程中起着十分关键的作用。鉴于此,本研究利用栽培于云南大理的滇重楼为实验材料,对其全株组织(叶,茎,根状茎)内生细菌进行了分离、纯化,并通过测序方法对其种类进行鉴定。结果显示滇重楼叶中内生细菌含有8属11种,茎中含有2属5种,根茎中含有9属11种。滇重楼不同组织中的内生细菌种属分布差异大,叶与根状茎中的内生细菌种类只共有鞘氨醇杆菌属,其余都不共有,而茎与叶、根状茎中的内生细菌都不相同,表明滇重楼内生细菌在不同组织中所体现的多样性及不同内生菌在分布上的差异性。本研究结果为后续滇重楼内生菌的多样性开发利用提供了科学依据,为未来滇重楼内生细菌深度挖掘与利用提供研究基础。

滇重楼(Paris polyphylla var. yunnanensis)中产皂苷内生真菌的分离及筛选

药用植物内生真菌在植物生长发育、抵御不良环境甚至繁育生殖过程中都扮演着重要的角色,尤其在宿主的次生代谢产生中具有十分重要的作用。内生真菌的代谢产物具有多种生理活性,部分内生真菌发酵液还可产生与宿主植物中相同的活性成分。鉴于此,本研究以传统药用植物滇重楼(P.polyphylla var.yunnanensis)为实验材料,通过组织分离法及分子测序鉴定法,共鉴定出18种内生真菌,并对这些真菌进行了物理发泡及薄层层析色谱法(TLC)实验,目的是筛选出能够产重楼皂苷的内生真菌。通过对菌丝菌液皂苷的提取,最终获得产皂苷类物质的菌株数共有3株,且皂苷类物质主要存在于内生真菌的发酵液中。

滇重楼新病毒ParV1的分子变异分析及致病性测定

为明确云南省引起滇重楼(Paris polyphylla var.yunnanensis)病毒病的病原,以昆明市发现的花叶、斑驳症状的滇重楼叶片为样品,进行透射电镜观察、高通量测序和反转录(RT-PCR)检测。结果表明,观察到大小约(700~1000)nm×13 nm的线状病毒粒子;基于高通量测序结果设计特异性引物进行RTPCR扩增并克隆,得到1条全长为10066个核苷酸(nt)的病毒序列,将该病毒暂命名为Paris virus 1(ParV1);BLAST比对分析表明,ParV1与马铃薯Y病毒属(Potyvirus)中的浙贝母花叶病毒(Thunberg fritillary mosaic virus,TFMV)具有最高为65.1%、68.9%的核苷酸及氨基酸(aa)序列同源性;多序列比对分析显示,ParV1多聚蛋白与potyviruses具有49.6%~65.1%的nt序列同源性及47.1%~68.9%的aa序列同源性;基于多聚蛋白氨基酸序列构建的系统发育树表明,该病毒与TFMV亲缘关系较近且聚为一个单独的分支。综上所述,确定ParV1属于Potyvirus的一种新病毒。将含ParV1的病毒汁液以机械摩擦的方法接种到心叶烟、本氏烟上对ParV1进行致病性初步测定。接种后的心叶烟植株叶片表现出明显病毒病症状,经RT-PCR及分子克隆在接种的心叶烟中检测到ParV1的存在,表明心叶烟植株受到ParV1的侵染,而接种的本氏烟上暂未检测到该病毒的存在。