基于高通量测序技术研究药食两用薏苡仁中污染真菌多样性

目的:调查药食两用薏苡仁中污染真菌多样性,为其安全使用提供参考依据。方法:收集薏苡仁样品18批,提取真菌DNA并扩增ITS2序列,基于Illumina MiSeq PE250平台进行高通量测序。结果:共检测到4门18纲44目99科149属的真菌,子囊菌门Ascomycota是最优势菌门,镰刀菌属Fusarium(3.05%~60.32%)是属水平最优势属,其次是曲霉属Aspergillus(2.20%~45.44%)、白僵菌属Beauveria(0.07%~63.21%)、链格孢属Alternaria(0.80%~11.92%)、Arachnomyces(0.03%~39.36%)和青霉属Penicillium(0.24%~8.03%)。此外,共检测到5种潜在产毒真菌,分别是烟曲霉A.fumigatus、土曲霉A.terreus、梨孢镰刀菌F.poae、囊状青霉P.capsulatum和展青霉P.paxilli。结论:高通量测序技术可以快速有效地检测薏苡仁中污染真菌种类,为薏苡仁污染真菌毒素提供风险预警。     

金银花nrDNA ITS区聚合酶链反应条件的研究

目的:金银花nrDNA ITS区序列G C含量为68%,用常规的PCR方法扩增效果差.本研究通过改变扩增程序及使用改性剂的方法显著提高了金银花nrDNA ITS区的扩增效率.方法:分别从金银花叶及药材中提取总DNA,通过优化扩增条件,对nrDNA ITS区进行扩增,并比较了两种优化方法的差别及适应性.方法1:提高变性温度至97℃;方法2:添加混合改性剂(DMSO 4%-甘油10%).结果:与方法1相比,方法2可降低变性温度5 ℃,升高退火温度9 ℃,降低镁离子浓度至0.5 mmol/L,降低Taq DNA聚合酶用量至0.1 U(30 μl体系),PCR产物量显著提高,且可消除植物DNA粗提物中杂质的干扰.结论:通过提高退火温度及添加改性剂可克服高G C含量金银花nrDNA ITS区序列扩增的困难,该方法的建立有助于解决植物来源DNA模板的PCR扩增问题.     

Ribosomal DNA internal transcribed spacer sequences do not support the species status of Ampelomyces quisqualis, a hyperparasite of powdery mildew fungi

Phylogenetic relationships among Ampelomyces isolates, pycnidial hyperparasites and biological control agents of powdery mildews, were inferred from internal transcribed spacer (ITS) sequences of the ribosomal DNA (rDNA). Currently, these hyperparasites are considered to be a single species, A. quisqualis, despite observed morphological and cultural differences. Ten Ampelomyces isolates, representing seven previously defined ITS RFLP groups, were sequenced and analyzed. Sequence-divergence values among isolates belonging to different RFLP groups ranged from 4.3 to 22.4%, suggesting that these isolates may represent different taxa. When Ampelomyces ITS sequences were analyzed by cladistic methods with the sequences of other ascomycetous fungi, they formed two lineages in the Dothideales. Slow-growing Ampelomyces isolates formed a clade with Leptosphaeria microscopica and L. nodorum, whereas fast-growing Ampelomyces isolates formed a clade with Epicoccum nigrum. Sequence-divergence values between these two clades ranged from 17.3 to 22.4%, suggesting that the taxa in the two clades are not closely related and possibly not congeneric. The data presented here indicate that the identification of `A. quisqualis' isolates used in biological control experiments should be re-evaluated. Received: 10 March 1997 / Accepted: 13 February 1998     

Sequence variation of the rDNA ITS regions within and between anastomosis groups in Rhizoctonia solani

Sequence analysis of the rDNA region containing the internal transcribed spacer (ITS) regions and the 5.8s rDNA coding sequence was used to evaluate the genetic diversity of 45 isolates within and between anastomosis groups (AGs) in Rhizoctonia solani. The 5.8s rDNA sequence was completely conserved across all the AGs examined, whereas the ITS rDNA sequence was found to be highly variable among isolates. The sequence homology in the ITS regions was above 96% for isolates of the same subgroup, 66 – 100% for isolates of different subgroups within an AG, and 55 – 96% for isolates of different AGs. In neighbor-joining trees based on distances derived from ITS-5.8s rDNA sequences, subgroups IA, IB and IC within AG-1 and subgroups HG-I and HG-II within AG-4 were placed on statistically significant branches as assessed by bootstrap analysis. These results suggest that sequence analysis of ITS rDNA regions of R. solani may be a valuable tool for identifying AG subgroups of biological significance. Received: 11 February /16 May 1997     

基于ITS2序列的翻白草与委陵菜分子鉴定

目的 应用DNA条形码技术对翻白草及易混品委陵菜进行分子鉴定,保障药材质量和临床用药安全。方法 提取翻白草及委陵菜的基因组DNA,扩增内转录间隔区2（ITS2）序列并双向测序,所得序列用SeqMan软件校对、拼接;在线双序列比对翻白草对照药材（F_R）及委陵菜对照药材（W_R）的ITS2序列,并预测其二级结构;从GenBank下载部分相关序列,运用MEGA X软件进行多序列比对,分析序列变异位点,计算种内、种间遗传距离,采用邻接法（NJ）构建系统进化树。结果 所有样品均成功扩增出ITS2序列,翻白草和委陵菜的ITS2序列长度均为210 bp。双序列比对结果表明,F_R与W_R的ITS2序列相似性为92.9%,二级结构存在明显差异。多序列分析结果表明,翻白草ITS2序列平均鸟嘌呤（G）+胞嘧啶（C）占比为63.0%,存在2个变异位点,种内遗传距离为0~0.009 6;委陵菜ITS2序列平均G+C占比为64.4%,存在5个变异位点,种内遗传距离为0~0.019 3;种间遗传距离为0.054 8~0.075 8。NJ树结果显示,翻白草、委陵菜聚为不同分支,可明显区分。结论 利用ITS2序列可以准确鉴别翻白草与委陵菜,为保障其安全用药提供了新的技术手段。     

长江中下游地区菱属植物的DNA分子鉴别

目的 对野生菱和栽培菱种rDNA ITS片段进行分析，探讨该片段在两大群体中的系统学及鉴别研究意义。方法 对菱的rDNA ITS区进行了PCR扩增、测序，运用clustal、Mega 2．0等软件对ITs区进行序列分析鉴别。结果 获得rDNAI TS1、ITS2和5．8S rDNA完整序列，10个居群菱的ITS1与ITS2序列的长度分别为234～236 bp和220～221 bp，5．8S区均为164bp，不同居群的碱基差异为0．22％～2．94％。变异位点数为16个，信息位点数6个。用NJ法根据ITS序列数据构建系统发生树。结论 尽管菱属各居群间rDNA ITS的差异百分率较小，其亲缘关系较近，但根据ITS序列的特征可以很好地鉴别野生菱、栽培菱，菱属植物有可能都起源于同一种野生类居群。     

PCR技术在赫坎按蚊复合体近缘种鉴别中的应用

目的应用聚合酶链反应（PCR）技术分析湖北省不同地区媒介按蚊的种型.方法采用传统的形态学方法和新建立的基因鉴别技术对现场捕获的按蚊分别进行形态特征鉴别和基因鉴别及比较.结果现场捕获181只按蚊,形态学确认176只为中华按蚊,而PCR鉴定172只为中华按蚊,另4只为八代按蚊;经形态学特征鉴别的5只嗜人按蚊中,PCR鉴别有4只为嗜人按蚊,另1只为八代按蚊.结论采用PCR基因鉴别技术能准确鉴别赫坎按蚊种团内中华按蚊、嗜人按蚊、八代按蚊等近缘种按蚊,较传统的按蚊形态学鉴别方法准确,适用于复合媒介地区的疟疾媒介调查和监测.     

花椒及其混淆品的rDNA ITS区序列分析与鉴别

目的研究不同居群的花椒及其混淆品的rDNA ITS区碱基序列的特征及其差异，为花椒的鉴别提供可靠的分子标记。方法运用PCR产物直接测序和克隆测序法对甘肃、陕西、四川、河北等7个花椒居群及3个混淆种的rDNA ITS区(包括ITS1，5.8S，ITS2)碱基序列进行序列测定。结果首次报道花椒ITS区的碱基序列，序列总长度为619-620 bp，长度变异较少，与混淆种长度仅相差4 bp。花椒各居群中，rDNA ITS区碱基序列有15个变异位点、12个信息位点、3个特异性识别位点。与混淆品间的碱基差异则较为显著，多达71个变异位点，有4个花椒特异性识别位点。结论依据花椒ITS区的序列特征可准确鉴别各居群的花椒及其混淆品；亲缘关系密切的花椒居群在地理位置上也非常靠近；rDNA ITS序列特征可作为花椒种内和种间鉴别的有效分子标记。