根际土壤微生物DNA提取及纯化方法的比较

本文采用了四种方法对红松和长白赤松土壤根际土壤微生物DNA进行了提取,同时分别采用了三种方法对所提取的根际土壤微生物粗DNA进行了纯化,并且对不同的提取和纯化方法进行了比较和评价。结果表明:对根际土壤微生物总DNA提取效果最好的是,以SDS为基础的含有1.0%(w/v)的高浓度NaCl的蛋白酶K法。这个方法可有效地去除腐殖酸和其它杂质。由于透析法能够积极有效地去除粗DNA中的棕色物质和腐殖酸,所以非常适合纯化根际土壤微生物DNA,而且纯化产物进行PCR扩增时效果很好。此外,挤胶法由于其经济有效的优点,所以也是一个很好的纯化根际土壤微生物粗DNA的方法。     

一种适合AFLP分析的油茶DNA提取方法

以油茶的成熟叶片为材料提取DNA,40个样品用改良的提取纯化方法成功提取出适用于AFLP分析的高质量DNA,OD260/OD280在1.7-2.0之间,可以用于AFLP反应。该方法打破了仅能用幼嫩叶片作为提取材料的限制,具有更广泛的适用性和重要的实用性。     

14个油茶良种遗传多样性的SRAP分析

对14个油茶良种进行SRAP标记(序列相关扩增多态性)的遗传多样性分析。采用改良CTAB法提取的油茶叶片基因组DNA为模板,利用优化的SRAP-PCR反应体系,从16个正向引物和20个反向引物中筛选出条带特异、多态性好、稳定性高的8对引物组合。利用这些引物组合对14个油茶良种扩增出65条清晰可重复的条带,其中22条是多态性条带,多态性条带百分率为33.85%。通过生物学软件对所得数据进行遗传学分析,计算出遗传相似系数,绘制出分子聚类图,为油茶良种的后续分子鉴定及分子育种提供了科学基础。     

高质量丝栗栲基因组DNA的提取方法

为了促进丝栗栲分子生物学的研究,采用尿素法、柠檬酸钠法、改良CTAB法和SDS法4种方法提取丝栗栲叶片基因组DNA,并对提取效果进行了比较和分析。结果表明,改良CTAB法较适合丝栗栲基因组DNA提取,其提取的DNA产率高,凝胶检测条带整齐清晰,杂质少、无明显降解,OD260/OD280比值在1.8左右;而尿素法难以提取基因组DNA,SDS法提取的DNA得率较低,柠檬酸钠法有蛋白质、多糖等杂质污染均不太适用丝栗栲基因组DNA提取。采用改良CTAB法提取得到丝栗栲幼叶、老叶、嫩芽和幼茎基因组DNA,产率分别为177.3、167.1、242.0和156.7μg/g。酶切分析也证明,改良CTAB法提取的不同组织DNA适用于进行后续分子生物学研究。     

红檵木DNA提取及其检测

用改良的异丙醇沉淀法对红木 (Loropetalumchinensevar .rubrum)叶片总DNA进行了提取。结果表明 ,OD2 60 /OD2 80 值接近 1 80 ,符合RAPD分析的要求 ,且方法简便 ,成本低 ,速度快。     

芒果属植物叶绿体DNA提取方法的研究

为了探究不同提取方法对芒果属植物叶绿体DNA提取质量的影响,以芒果和扁桃的成熟叶片为材料,比较植物叶绿体DNAout试剂盒法和高盐-低p H法分离叶绿体及提取叶绿体DNA效果。结果表明:叶绿体DNAout试剂盒法能够较好地去除蛋白质、酚类、多糖等代谢物质,OD260/OD230大于2.000,OD260/OD280在1.800~1.900之间。高盐-低p H法提取叶绿体DNA产率高达75.8 ng/μL,远大于叶绿体DNAout试剂盒法提取叶绿体DNA最高产率42.8 ng/μL,2种方法所得模板电泳图和SSR标记图谱谱带完整性皆好。显微镜下观察叶绿体,2种方法都可以得到杂质少、背景清晰的叶绿体显微成像效果,但高盐-低p H法提取的叶绿体细胞器密度更高。2种方法获得的叶绿体DNA均可满足后续研究工作的需要。     

桉属植物叶片DNA抽提

对植物组织DNA抽提是在分子水平上对植物进行系统分析与研究的基础性工作。我们使用改良CTAB法抽提桉属植物叶片中总的DNA获得了成功，其纯度和得率完全能满足常规分子生物学操作的要求。与DNA得率有关的几个问题在此一并探讨。     

油茶叶乙醇提取物清除DPPH自由基作用的研究

用二苯基苦基肼自由基(DPPH·)法测定了油茶叶乙醇提取物的抗氧化活性,并与芦丁、二丁基羟基甲苯(BHT)进行了比较,在波长517 nm、反应时间40 min的条件下测定了DPPH·的清除率为50%时抗氧化剂的质量浓度(EC50)值.结果表明,油茶叶乙醇提取物时DPPH·具有明显的清除作用,且随着油茶叶乙醇提取物纯化程度的提高,其清除能力也相应增强.测得油茶叶粗提物、油茶叶精提物、芦丁和BHT清除DPPH·的EC50值分别为12.19、6.765、9.481和37.53 mg/L,清除DPPH·能力的大小顺序依次为油茶叶精提物＞芦丁＞油茶叶粗提物＞BHT.     

漾濞核桃叶片基因组DNA的两种提取方法效果比较

以漾濞核桃不同生长期的叶片为材料，采用高盐低pH法和改良CTAB法作提取其基因组DNA的效果比较实验。通过琼脂糖凝胶电泳、紫外分光光度计和RAPD扩增对所提取的DNA样品进行检测，比较所得DNA的产量、质量，认为从漾濞核桃不同发育时期叶片中获得DNA的提取效果不同。4种叶样以冬芽和新叶的效果为好，老叶最差；用两种提取方法从叶片中得到的DNA产量和质量有所不同，高盐低pH法提取的DNA纯度高，但是产量较低；而改良CTAB法则相反，产量高，纯度低。     

刺五加AFLP分子标记技术体系的建立

用改良的的试剂盒法提取刺五加叶片的基因组DNA,可以获得高质量的刺五加基因组DNA,DNA降解较少、污染低、电泳条带清晰,可以用于PCR扩增反应,可以被内切酶EcoRⅠ和MseI彻底消化。通过优化酶切反应、引物连接、预扩增、选择扩增,建立了优化的刺五加AFLP分析技术体系,从64对引物中筛选到5对高效扩增引物,并利用这5对引物在6个刺五加种源中获得了89个多态性AFLP分子标记。建立了利用红外荧光检测技术和Li-Cor4300DNA分析系统,进行刺五加AFLP分析的分子标记技术。     

桢楠DNA提取和RAPD条件的优化

以桢楠（Phoebe zhennan）新鲜叶片、硅胶干燥叶片为材料,研究了DNA的提取方法,并对影响RAPD反应的各因素进行了优化。得到了桢楠RAPD的优化反应体系及程序,适合桢楠RAPD反应的体系总体积为25μL,DNA模板2μL,Taq DNA聚合酶用量0.3μL,引物用量1μL,Mg2＋用量5μL,dNTPs用量0.5μL,ddH2O16.2μL;适合桢楠RAPD扩增的程序是94℃预变性3 min,94℃变性1min,36℃复性1 min,72℃延伸2 min,42个循环,最后72℃延伸10 min,产物于4℃保存,同时结果表明硅胶保存的样品完全可以与传统的新鲜叶片得到同样的PCR扩增结果,完全可以满足研究需要。     

Fast methods for DNA extraction for PCR analyses from Heterobasidion annosum mycelium and Endocronartium pini aeciospores

The extraction of DNA from mycelium of Heterobasidion annosum or aeciospores of Endocronartium pini could be done rapidly by homogenizing the fungal material in PCR-compatible buffer in Eppendorf tubes with a disposable pestle mounted on an electric drill. The DNA yield with this method was relatively low but sufficient for PCR analysis with ITS-specific primers. For large series of fungal samples this protocol provides a quick and cheap method of determining the intersterility groups of H. annosum or for specific primer studies on E. pini populations. The time needed for DNA isolation from E. pini aeciospores and H. annosum mycelium could be reduced by nearly 85% compared to the mortar-and-pestle protocol. By using a disposable pestle mounted on an electric drill and phenol-chloroform as extraction solution, the yield of DNA was higher than with the mortar-and-pestle method with phenol-chloroform as extraction solution. The quality of DNA was sufficient for both specific-primer and RAPD analyses, but the more complicated extraction method with phenol was needed.     

鲜(干)薄荷叶、薄荷花及花苞挥发油的GC-MS比较分析

比较分析新鲜、干燥的薄荷叶、薄荷花及花苞的挥发油化学成分.采用挥发油提取法提取薄荷叶、薄荷花及薄荷花苞的挥发油,用GC-MS进行分析鉴定.实验结果表明:从薄荷叶、薄荷花、薄荷花苞中共检测出28种化学成分,含有许多烯、醇及酮类物质,主要由1-薄荷醇、胡薄荷酮、萜品烯、β-石竹烯和桉叶油醇组成.薄荷花与花苞同薄荷叶的化合物基本相同,干燥薄荷样品特征化合物如1-薄荷醇、胡薄荷酮和异薄荷酮的相对成分较新鲜的含量更多,α-律草烯和反式松香芹醇为花苞独有.     

杉木DNA的简便提取方法

杉木ＤＮＡ的简便提取方法＊尤勇洪菊生关键词杉木针叶ＤＮＡ巯基乙醇次生物质杉木（Ｃｕｎｎｉｎｇｈａｍｉａｌａｎｃｅｏｌａｔａ（Ｌａｍｂ）Ｈｏｏｋ．）是我国的特有树种,其分子生物学研究目前在国内外开展的较少,其近缘种的研究也较少。为了开展杉木分子生物学的...     

无患子总DNA提取方法研究

针对无患子富含多酚、多糖、蛋白质及其他次生代谢物质的特点,以无患子叶片为材料,分别采用改良的CTAB法和CTAB与SDS相结合法提取不同种源无患子的总DNA。结果显示,改良的CTAB法提取的总DNA,得率较高,纯度好,可用于酶切、PCR扩增等后续试验,并能获得清晰、稳定的扩增产物。而采用CTAB与SDS相结合的方法得到的基因组DNA效果不稳定,无法检测出DNA。改良的CTAB法具有简单、经济的特点,可用于无患子总DNA的提取,成功地进行SRAP分子标记分析,为研究无患子遗传资源多样性奠定基础。     

樟树不同组织总RNA提取方法比较及RT-PCR检测

采用Invitrogen Total RNA Purification Kit直接提取法,Trizol试剂盒法和本实验室改良的CTAB法,分别提取樟树不同组织(根、茎、叶和花)的总RNA,并对提取效果进行了检测和比较分析。结果表明:(1)采用本实验室改良的CTAB法提取的RNA完整性好,无杂质污染且得率高,稳定性好,可满足半定量RT-PCR等试验需要;而Invitrogen Total RNA Purification Kit直接提取法和Trizol试剂盒法提取的樟树不同组织总RNA,结果均不理想,存在RNA 28S rRNA谱带模糊不清、有DNA污染、降解严重和得率低等问题,这两种方法均不适用于樟树不同组织RNA的提取。(2)樟树不同组织总RNA提取的得率不同,其中叶片的总RNA得率最高,达到782.34 mg/kg;茎的得率最低,为514.33 mg/kg。(3)以Actin作为内参基因,对樟树根、茎、叶和花中的Fad2基因片段进行半定量RT-PCR检测,结果表明,Fad2基因片段在樟树叶片中的表达量相对较高,而在花中的表达量较低。     

贵州枇杷炭疽病病原菌的鉴定及rDNA-ITS序列分析

2009—2010年,在贵州遵义、罗甸(苗圃)枇杷树叶片上发现疑似炭疽病害,且有暴发之势。为了确诊该病害,用组织分离法分离病原菌,纯化培养后用菌碟回接到健康枇杷树叶片上,10 d后叶片出现典型病斑,自发病组织再分离所得病原菌形态特征与初分离一致;以枇杷炭疽病菌总DNA为模板,采用真菌通用引物(ITS1和ITS4),通过PCR扩增到约550 bp的片段,测序结果表明片段长为538个核苷酸,通过GenBank上BLAST分析,表明该病原菌为胶孢炭疽菌Colletotrichum gloeosporioides Penz.。     

翠雀花主要性状之间的关系分析

运用灰色系统理论和方法,对影响翠雀花观赏性状的几个主要数量性状的相互关系进行了分析。结果表明,对花蕾数量影响的关联度由大到小的次序是分枝数、茎粗、叶长、叶宽、花冠直径、株高、基叶数。其中分枝数对花朵数量的影响是最大的,二者的关系为线性。对单朵花径影响的关联度由大到小的次序是株高、叶长、叶宽、茎粗、分枝数、叶数、花蕾数量,回归分析中株高与花径呈抛物线关系。     

Foliar nematode,Litylenchus crenatae ssp. mccannii,population dynamics in leaves and buds of beech leaf disease‐affected trees in Canada and the US

A foliar nematode, Litylenchus crenatae ssp. mccannii, is associated with beech leaf disease (BLD) symptoms. Information about the types of tissues parasitized and how nematode populations fluctuate in these tissues over time is needed to improve surveys as well as understand the nematodes role in BLD. During this study, the nematode was detected throughout the known range of BLD by researchers at both Canadian and US institutions using a modified pan method to extract nematodes. Monthly collections of symptomatic and asymptomatic leaves during the growing season (May–October), and leaves and buds between growing seasons (November–March), revealed that nematodes were present in all tissue types. Progressively larger numbers of nematodes were detected in symptomatic leaves from Ohio and Ontario, with the greatest detections at the end of the growing season. Smaller numbers of nematodes were detected in asymptomatic leaves from BLD‐infected trees, typically at the end of the growing season. The nematode was detected overwintering in buds and detached leaves. The discovery of small numbers of nematodes in detached leaves at one location before BLD was detected indicates that nematodes may have been present before disease symptoms were expressed. Other nematodes, Plectus and Aphelenchoides spp., were infrequently detected in small numbers. Our findings support the involvement of the nematode in BLD and indicate that symptoms develop only when certain requirements, such as infection of buds, are met. We also found that the nematode can be reliably detected in buds and leaves using the modified pan extraction method.     

Beech leaf disease symptoms caused by newly recognized nematode subspecies Litylenchus crenatae mccannii (Anguinata) described from Fagus grandifolia in North America

Symptoms of beech leaf disease (BLD), first reported in Ohio in 2012, include interveinal greening, thickening and often chlorosis in leaves, canopy thinning and mortality. Nematodes from diseased leaves of American beech (Fagus grandifolia) sent by the Ohio Department of Agriculture to the USDA, Beltsville, MD in autumn 2017 were identified as the first recorded North American population of Litylenchus crenatae (Nematology, 21, 2019, 5), originally described from Japan. This and other populations from Ohio, Pennsylvania and the neighbouring province of Ontario, Canada showed some differences in morphometric averages among females compared to the Japanese population. Ribosomal DNA marker sequences were nearly identical to the population from Japan. A sequence for the COI marker was also generated, although it was not available from the Japanese population. The nematode was not encountered in Fagus crenata (its host in Japan) living among nematode‐infested Fagus grandifolia in the Holden Arboretum, nor has L. crenatae been reported in American beech in Japan. The morphological and host range differences in North American populations are nomenclaturally distinguished as L. crenatae mccannii ssp. n. from the population in Japan. Low‐temperature scanning electron microscopy (LT‐SEM) demonstrated five lip annules and a highly flexible cuticle. Females, juveniles and eggs were imaged within buds with a Hirox Digital microscope and an LT‐SEM. Nematodes swarmed to the tips of freshly cut beech buds, but explants could not be maintained. Inoculation of fresh nematodes from infested leaves or buds to buds or leaves of F. grandifolia seedlings resulted in BLD leaf symptoms. Injuring dormant buds prior to nematode application, in fall or spring, promoted the most reliable symptom expression. The biogeography and physiology of anguinid nematode leaf galling, and potential co‐factors and transmission are discussed.