Distribution and diversity of endophytic fungi in Gentiana rigescens and cytotoxic activities

Objective:In the present study,Gentiana rigescens was screened for fungi communities to clarify their diversity and community assemblage in hosts.Meanwhile,the identification and activity assays of the strains were also conducted.Methods:By culture-dependent(endophytic fungi isolations from plant sections)and culture-independent(metagenomic library and cloning from plant sections)techniques,fungi communities were studied.The metagenomic library was generated using direct DNA isolation of whole plants,plant radixes,plant stems,plant leaves,plant flowers and soils around the plant.Meanwhile,endophytes were isolated from all parts ofG.rigescens plants.After fermentation of the fungi isolations,all the isolates were evaluated for their cytotoxicity against four kinds of human cancer cell lines(HCT116,BEL7404,A549,MDA-MB-231).Results:Eventually,200 strains were isolated and 103 strains were further identified through the internal transcribed spacer(ITS,ITS1 and ITS2 regions)sequence by using the universal primers ITS5 and ITS4.A total of 59,106 fungal sequences corresponding to 374 putative operational taxonomic units(OTU)were identified by 454 pyrosequencing.Through 454 pyrosequencing,the main fungal genera were Sebacina,Botrytis,Mycosphaerella,Boletus and Gibberella,and the major fungal genera which were directly isolated were Aspergillus,Fusarium,Penicillium and Alternaria.Activity assays showed strains 5-26(Aspergillus sp.)and 6-2(Fusarium avenaceum)had the outstanding cytotoxicity to all the tested cell lines with IC5o values<5μg/mL.Conclusion:This study revealed the abundance of endogenetic fungal resources and a variety of genetic information in G.rigescens by high-throughput 454 sequencing technology and fungi isolation methods.Activity assays indicated that endophytes were a promising natural source of potential anticancer agents.

海拔对苍山地区野生滇龙胆品质的影响

目的:本论文旨在研究海拔对苍山地区野生滇龙胆(Gentiana rigescens Franch.)品质的影响。方法:采用高效液相色谱(HPLC)法对滇龙胆根茎叶中龙胆苦苷的含量进行测定,并将各部分龙胆苦苷含量与海拔高度进行相关性分析。结果:苍山地区滇龙胆品质优良,根茎叶三部分龙胆苦苷含量均超过《中国药典》规定,根部龙胆苦苷含量在3.9%~8.2%之间。此外,根部龙胆苦苷含量与海拔高度有显著线性相关性,其线性拟合方程为y = −0.2752 + x * 1.35048E−4。结论:苍山地区野生滇龙胆品质优良;在海拔2325.3~2599.7 m区间内,滇龙胆品质随海拔上升而升高;苍山地区海拔2400~2700 m范围内,是潜在的优良人工滇龙胆种植区。

不同生长年限滇龙胆中阴离子含量变化

目的:建立滇龙胆中阴离子含量的快速测定方法,同时为滇龙胆合理施肥和规范化栽培提供依据。方法:采用超声辅助提取-离子色谱(UAE-IC)法快速、准确测定了滇龙胆不同年限、不同部位中F-、Cl-、NO2-、NO3-、PO43-5种离子。结果:所测定的滇龙胆样品中除NO2-未检测出外,其余离子含量NO3->PO34->F->Cl-;NO3-、PO34-、F-、Cl-4种离子在1.5年时含量最高,2年生含量最低,2～3年生呈上升趋势。NO 3-和Cl-离子含量随根、茎、叶、花4个部位呈先增加后下降的趋势;PO43-和F-在四个部位中呈先下降后增加的趋势。各离子加标回收率为96.47%～117.23%。结论:该方法操作简便、准确性好、结果可靠,测定滇龙胆中无机阴离子含量获得满意结果。

滇龙胆中细胞色素P450还原酶基因的克隆及生物信息学分析

细胞色素P450还原酶在体内可催化许多反应,通过FAD和FMN辅基将NAD(P)H的电子传递至细胞色素P450酶,从而使细胞色素P450酶同底物产生氧化还原反应,起到电子供体的作用。本研究对药用植物滇龙胆(Gentiana rigescen Franch.)的细胞色素还原酶基因GrCPR1进行克隆、生信分析和异源表达。结果表明,GrCPR1基因cDNA全长2078 bp,相对分子质量77.10 kD,含691个氨基酸,等电点为5.01,含73个极性氨基酸残基,104个非极性氨基酸残基,平均疏水性为-0.330,脂溶系数为82.56,分子式C3449H5349N917O1046S22,半衰期为30 h。不稳定指数42.25,属于不稳定亲水蛋白。GrCPR1蛋白为非分泌蛋白,不具有信号肽识别功能。第27~46位氨基酸为该蛋白跨膜区,同时亚细胞定位预测分析发现GrCPR1蛋白位于内质网上。CPR是为细胞色素单加氧酶CYP的催化过程提供电子,在P450系统中起重要作用。本研究结果可以为今后研究龙胆苦苷的生物合成途径的CYP基因功能提供重要的数据支持。