茶树叶片总RNA提取方法的比较研究

针对茶树叶片中富含多酚类物质的特点,以陕西地方茶树代表种质紫阳槠叶种的叶片为材料,比较了Trizol法、异硫氰酸胍法和改良SDS-酸酚法三种不同的总RNA提取方法。结果表明改良SDS-酸酚法能够从新鲜叶片、冷冻叶片和干燥叶片中提取到质量高、完整性好的总RNA,经检测28SrRNA亮度为18SrRNA的2倍,A260/A280值介于1.83～2.01之间。以鲜叶、冻叶和干燥叶片的总RNA为材料,进一步用于DDRT-PCR分析,均可获得清晰稳定的多态性结果。说明改良的SDS-酸酚法能抑制茶树叶片中多酚类物质对总RNA提取的影响,是一种适于茶树叶片总RNA提取的有效方法。     

两种适用于葡萄不同组织RNA提取方法的比较

以不同葡萄组织为材料对目前常用的2种总RNA提取方法一改良SDS法和CTAB-LiCl法进行研究。2种RNA提取方法中均不使用酚。采用这两种方法从葡萄不同组织中均成功地提取到RNA,琼脂糖凝胶电泳结果显示28s和18SrRNA条带完整清晰。检测A260/A280 值分布在1．7-2．0之间,A260/A230值分布于1．9-2．3之间,说明RNA质量较高。管家基Actin和ACS5基因的检测表明2种方法所得RNA能够满足RT-PCR和基因克隆等研究需要。改良SDS法的RNA得率是CTAB-LiCl法的RNA得率的2～3倍,而CTAB-LiCl法获得的RNA纯度高。可以根据原料的数量和对RNA质量的要求来选取最佳提取方法。     

Trizol RNA提取法在雪貂组织的流感病毒H3N2定量PCR检测中优于RNeasy mini kit（Qiagen）柱子法

目的比较RNeasy mini kit（Qiagen）柱子法和Trizol法提取RNA在雪貂肺和肠组织H3N2 SYBRGreen PCR定量中的去干扰作用,从而找到适合该定量体系的RNA提取方法。方法比较Trizol法,RNeasy minikit柱子法,改良RNeasy mini kit柱子法1和改良RNeasy mini kit柱子法2提取肺肠组织RNA的质量,RNA逆转录成cDNA后,利用SYBR Green PCR方法检测样品中H3N2的载量,比较产物的特异性,综合评价RNA提取方法。结果 4种方法提取的肺和肠组织的RNA质量不相同,紫外分光光度仪测得RNeasy mini kit柱子法提取的RNA的A260/280低于1.8,其余3种方法的A 260/280在1.8～2.1之间,A 260/230只有Trizol法能达到2.0左右,其余3种方法均远低于2.0。电泳可见Trizol法提取的RNA有3条带：28 s、18 s和5 s,而RNeasy mini kit柱子法的RNA除了有28 s、18 s外,还有基因组DNA,改良RNeasy mini kit柱子法1和2提取的RNA只有28 s和18 s带。RNA逆转录后进行荧光定量PCR,从溶解曲线看,不论是鼻甲刮取物还是肺肠组织的样品模板,4种方法获得的样品与标准品均为单一峰溶解曲线峰,并且波峰位置重叠。而鼻甲刮取物定量的产物跑琼脂糖凝胶电泳均为单一的特异性条带,而肺肠组织的产物电泳发现：Trizol法获得的模板定量产物与标准品一致,为单一的特异性条带,而其它3种方法获得的模板则均有非特异性条带。结论鼻甲刮取物的病毒定量选用RNeasy mini kit柱子法提取定量结果与Trizol法一样可靠,说明对于简单样品该体系及引物非常适用,结果可信。对于组织样品肺和肠,Trizol法获得的样品定量结果比其它3种方法可靠。     

改良Trizol法提取血浆游离RNA

[目的]建立一种成本低、得率高的血浆游离RNA的提取方法。[方法]分别采用传统Trizol法、改良Trizol法、试剂盒法提取200μL健康成年人血浆中游离RNA,并比较三种方法所得RNA的浓度、纯度及大小片段RNA得率的差异。[结果]改良Trizol法所得RNA浓度是常规Trizol法的21. 7倍(P 0. 01),是试剂盒法的6. 4倍(P 0. 01);三种方法所得RNA纯度(A_(260)/A_(280))无显著差异;采用RT-q PCR方法检测血浆游离RNA大小片段的回收率,外参秀丽线虫miR-39-3p(cel-miR-39-3p)代表小片段RNA,GAPDH代表大片段RNA,改良Trizol法检测Cq平均值分别为15. 64和33. 34,平均低于常规Trizol法2. 05～3. 62个Cq,低于试剂盒法0. 32～3. 34个Cq。[结论]改良Trizol法提取血浆游离RNA相较于常规Trizol法提高了10～20倍,增加了得率。     

改良Trizol法快速提取棉叶片总RNA

棉叶片组织中存在含量较高的棉酚、多糖等次生代谢物质,使用单纯的Trizol很难获得高质量的RNA。首次采用改良Trizol法成功从棉叶片中快速的提取了总RNA,其所提的总RNA完整性好、纯度高,能进一步满足分子生物学的试验要求。     

山药组织总RNA提取方法的比较与分析

采用异硫氰酸胍-巯基乙醇联合变性法、Trizol法和CTAB-LiCl法等3种方法,分别对山药叶片和块茎进行总RNA提取效果进行了比较.结果表明,Trizoi法难以提取RNA,异硫氰酸胍-巯基乙醇联合变性法提取RNA效果不理想,存在DNA污染,这2种方法不适合于富含多糖类物质的山药组织总RNA的提取;CTAB-LiCl法提取叶片和块茎组织总RNA质量高、完整性好、成功率高,可作为山药类植物总RNA提取的首选方法.     

淡水育珠蚌外套膜提取总RNA的改良方法

通过对Trizol法加以改进,提取淡水珍珠蚌外套膜组织中的总RNA。经预处理后,在异丙醇沉淀RNA时加入高浓度的盐溶液,用75%的酒精2次洗涤RNA。用紫外分光光度法和1%琼脂糖凝胶电泳鉴定所提取的RNA。结果表明,改良法获得的总RNA完整、纯度高,改良Trizol法是一种从淡水育珠蚌外套膜组织中提取总RNA的高效、便捷、可靠的方法。     

檀香心材总RNA提取方法的比较研究

为筛选檀香心材总RNA提取方法,对5种提取方法进行比较研究,包括Trizol法、改良CTAB法、SDS酸酚法、异硫氰酸胍-CTAB法、异硫氰酸胍-SDS法。结果表明,Trizol法和异硫氰酸胍-CTAB法不能提取出檀香心材总RNA,而SDS酸酚法、改良CTAB法和异硫氰酸胍-SDS法均能提取檀香心材总RNA。SDS酸酚法的A260 nm/A230 nm小于2.0,且RNA产率低,仅为(27.94±1.06)μg g–1,不能满足后续实验要求。而改良CTAB法和异硫氰酸胍-SDS法提取的总RNA带型清晰,完整性好,A260 nm/A280 nm为1.8~2.0,A260 nm/A230 nm大于2.0,RNA产率分别为(79.06±4.22)和(107.00±1.36)μg g–1。分别以改良CTAB法和异硫氰酸胍-SDS法提取的总RNA为模板,通过RT-PCR反应,扩增檀香Actin基因片段,结果二者扩增产物大小相同且条带单一,说明改良CTAB法与异硫氰酸胍-SDS法为檀香心材总RNA提取的较好方法。     

枣不同器官和组织RNA提取方法的研究

以田间幼嫩叶片为试材,建立枣RNA改良CTAB提取法,改良之处包括利用巯基乙醇和PVP联合去除多酚、CTAB与异硫酸氰胍联合促进RNA释放及加入糖原提高产率等.改良CTAB法提取液组成:2% CTAB,4mol/L异硫酸氰胍,100mmol/L Tris-HCl(pH8.0),20mmol/L EDTA(pH8.0),2% PVP,4% β-巯基乙醇,250μg/ml糖原.进而采用Trizol试剂法、改良CTAB法、改进SDS-酚法、热硼酸法和异硫氰酸胍法5种方法分别对枣组培苗、幼叶、幼嫩枝皮、根皮和枣果5种器官和组织进行了总RNA提取.结果表明,组培苗RNA提取以Trizol试剂法为最佳,田间幼叶和成熟果实宜采用改良CTAB法,幼嫩枝皮和根皮宜采用改良CTAB法或改进SDS-酚法;本研究建立的改良CTAB法可作为枣不同器官和组织RNA提取的通用方法;根据季节可从不同器官和组织中获得高质量RNA;枣组织RNA含量相差较大,以幼嫩叶片含量最高,其次为组培苗、枝皮、枣果和根皮.     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.