分子信标在活细胞内的应用

分子信标(molecular beacon,MB)可实时成像观察活细胞内多个特异性靶分子的相互作用,在肿瘤诊断和治疗上有潜在的应用前景。由于核酸酶降解、核酸结合蛋白质结合等非特异性因素,生物稳定性差、转染效率低等成为MB活细胞内应用的瓶颈。稳定的MB并结合高效的转运载体有望成为实时观察活细胞生物学过程及肿瘤诊断和治疗新的工具。     

用于HBV病毒快速诊断的分子信标探针设计

分子信标是一种高灵敏度、高特异性的新型荧光核酸探针.它在与互补DNA或RNA靶序列杂交时放出荧光.利用Genebank中调出已知HBV病毒ayr亚型基因组信息,通过BeaconDesigner4.0软件进行分子信标探针设计,共设计出6条分子信标探针,以便于为目前HBV病毒快速诊断提供参考.     

C型利钠肽基因调控斑马鱼胚胎血管发育

本研究旨在探索C型利钠肽前体蛋白基因(natriuretic peptide precursor C,nppc)在斑马鱼胚胎期的表达及其在血管发育过程中的功能。利用胚胎整体原位杂交技术检测nppc基因在斑马鱼胚胎期的表达。同时使用转基因斑马鱼系Tg(flk1:GFP)和Tg(fli1a:n GFP),显微注射nppc特异性吗啉基反义寡核苷酸(morpholino)和nppc m RNA调控nppc基因表达,利用激光共聚焦显微镜观察分析斑马鱼节间血管(intersegment vessel,ISV)表型,并统计内皮细胞数目。结果显示,在受精后24 h和48 h,nppc基因在斑马鱼脑、心脏、血管系统中都有表达。下调nppc基因表达导致ISV发育缺陷,ISV内皮细胞数目减少。以上结果表明下调nppc基因表达可能通过抑制内皮细胞增殖和内皮细胞迁移调控斑马鱼胚胎血管发育。     

2012年第9期《遗传》封面说明

microRNA(又称miRNA)作为细胞内源表达的一类小分子非编码RNA,主要通过降解mRNA或抑制翻译调控靶基因表达。miRNA在个体发育中具有特异的时空表达模式,其功能跟它的表达位置和时间密切相关。但是目前缺乏在活体与个体水平稳定、持续地实时观察miRNA动态表达的方法。斑马鱼体外发育且胚胎透明,非常适于观察荧光蛋白的表达。本研究建立了一个称为"miRNA Tracer"的双荧光报告系统,用于在斑马鱼活体胚胎中追踪miRNA的表达及动     

斑马鱼胚胎胚盾特异表达基因的鉴定

目的由于原肠期细胞的剧烈运动,在斑马鱼胚胎的背侧汇聚形成了称为胚盾(shield organizer)的结构,是胚胎发育的信号组织中心,在背腹轴建立和胚层诱导过程中具有关键作用。系统鉴定在胚盾特异表达的基因,可为进一步探讨胚盾形成的机制及其指导胚胎早期发育的分子机理提供参考。方法 Tg(gsc:GFP)转基因鱼在胚盾表达特异的绿色荧光。通过流式细胞分选技术分离富集GFP阳性细胞并提取总RNA进行RNA深度测序,检测可能在胚盾高水平表达的基因。然后利用荧光实时定量PCR(quantitative real-time PCR,qRT-PCR)和原位杂交技术鉴定若干在胚盾特异表达的基因。结果从Tg(gsc:GFP)转基因鱼胚胎中分离富集到了纯度超过96%的GFP阳性细胞,RNA深度测序的结果显示有657个基因的表达水平比整胚细胞或GFP阴性细胞高2倍以上。最后,确认了KIAA1324、ripply1、twist2、isthmin1、nme4、zgc174153、rrbp1b等7个基因在胚盾特异表达。结论系统鉴定到了斑马鱼胚胎胚盾特异表达基因,为下一步研究这些基因的发育生物学功能奠定了基础。     

分子信标核酸检测技术研究进展

介绍了分子信标设计和分子信标核酸检测原理、技术特性和在基因突变大规模自动化检测中的应用. 分子信标是一种基于荧光共振能量转移现象设计的发卡型寡核苷酸探针,空间结构上呈茎环结构, 环序列是与靶核酸互补的探针,茎序列由与靶序列无关的互补序列构成,茎的一端连上荧光分子,另一端连上淬灭分子.通过空间结构改变决定分子信标发射荧光特性,从而对核酸进行定量检测. 分子信标技术具有操作简单、敏感、特异、可对核酸进行液相实时检测和对活体内核酸动态进行检测等特点,已应用于HIV辅助受体基因等基因突变的大规模自动化检测,是一种新型核酸定量检测技术.     

新型分子信标技术的研究进展

分子信标是一种设计巧妙的新型荧光标记核酸探针。特殊的发夹结构使分子信标具有很强的特异性识别靶标序列的能力,目前已成为生物学中一种强有力的研究工具。本文介绍了近年来出现的各种新型的分子信标(MB)的结构及工作原理。简要概述了MB技术在生命科学领域中的应用,展望了MB技术的发展趋势。     

Baff转基因斑马鱼的构建及相关基因表达检测

通过RT-PCR法由斑马鱼脾脏克隆B细胞刺激因子baff基因,构建过表达斑马鱼baff且携带有绿色荧光标记蛋白的重组质粒pIRES2-GFP-baff;胚胎显微注射获得转基因斑马鱼胚胎;通过GFP荧光标记跟踪并筛选转基因阳性鱼;Western blot法鉴定Baff-GFP融合蛋白表达情况;qPCR检测baff,GFP及baff下游相关基因bcl-2,il-4mR-NA表达情况。结果表明细胞、胚胎及幼鱼baff和GFP均高表达,baff下游基因bcl-2激活和il-4基因抑制表达。通过胚胎显微注射法可成功获得过表达baff的转基因斑马鱼,此研究为建立红斑狼疮转基因斑马鱼模型及高通量筛选Baff拮抗剂奠定了基础。     

MicroRNA靶向病毒携带的microRNA模拟靶序列对病毒的抑制作用

Micro RNA模拟靶序列(target mimic,TM)通过竞争性结合miRNA,从而干扰miRNA对靶标m RNA的调控.我们前期工作发现:以黄瓜花叶病毒(Cucumber mosaic virus,CMV)作为载体在植物体内表达TM序列有效地抑制了miRNA的活性或稳定性,从而消减了miRNA对靶基因的调控.但是,miRNA与CMV携带的TM序列的结合在一定程度上抑制了病毒的积累.研究分析了miRNA靶向病毒携带的TM序列对病毒抑制作用的内在原因.a.通过RNA印迹分析CMV携带不同miRNA TM序列对病毒积累的影响,进一步明确miRNA靶向病毒携带的TM序列对病毒的抑制作用;b.利用GFP作为报告基因,通过荧光显微镜、蛋白质印迹以及RNA印迹分析TM序列对重组病毒积累的影响;c.以GFP作为报告基因,利用荧光显微镜观察和免疫印迹方法分析模拟靶序列对GFP翻译的影响;d.利用CMV病毒的反式复制系统分析miRNA模拟靶序列对病毒负链RNA合成的影响.结果表明,多种植物内源的miRNA靶向CMV基因组携带的miRNA TM序列,在不同程度上抑制了病毒的积累,miRNA与其TM序列的结合抑制GFP蛋白的翻译和负链的合成.植物内源的miRNA通过与病毒基因组携带的miRNA模拟靶序列结合,通过抑制病毒蛋白的翻译以及病毒负链RNA的合成,从而降低了病毒的积累水平.基于该论文的研究结果有可能建立一种抗病毒的新方法.     

应用双荧光报告系统检测斑马鱼microRNA的动态表达

microRNA(miRNA)是一类细胞内源表达的小分子非编码RNA, 主要通过降解靶基因的mRNA或者抑制靶基因的翻译, 在动植物的发育以及其他重要的生理过程中起调控作用。miRNA的功能跟它的表达位置与时间密切相关, 但是目前尚缺乏一个能够在活体与个体水平稳定、持续地实时观察miRNA动态表达的方法。文章以斑马鱼为模式, 建立了一个双荧光报告系统(我们称之为miRNA Tracer), 用于在斑马鱼整体胚胎中追踪特定miRNA的表达谱及动态变化过程。该系统以Tol2转座子为基础, 采用来自斑马鱼hsp70基因的热激启动子分别驱动eGFP和mRFP1荧光报告基因, 同时在其中一个报告基因的3′-UTR区连接待测miRNA的互补序列, 构成Tracer质粒。该互补序列与斑马鱼胚胎中相应的内源miRNA结合后能够使对应报告基因的荧光信号强度减弱, 通过比较两个报告基因在表达谱上的差异辨别miRNA的表达区域, 检测斑马鱼胚胎中miRNA起作用的位置和时间。文章选择在肌肉系统特异表达的miR-206以及在神经系统特异表达的miR-219, 分别在显微注射瞬时表达和转基因稳定整合等两个层次上验证了上述Tracer系统。结果表明, 所用的方法能够如实地在单细胞水平和整体水平检测到目标miRNA的时空表达动态变化。miRNA Tracer系统为在斑马鱼发育过程中对miRNA进行活体、实时的时空定位提供了一个独特而有效的方法, 也为对miRNA进行功能与作用机制等更深入的研究奠定了基础。

补充资料

s219mRFP1-dF转基因胚胎的3-D图像 [视频]     

六种藓类植物茎的比较解剖学研究

通过对6种藓类植物,即褶叶青藓(Brachythecium salebrosum(Web.et Mohr.)B.S.G.)、湿地匐灯藓(Plagiomnium acutum(Lindb.)Kop.)、侧枝匐灯藓(Plagiomnium maximoviczii(Lindb.)Kop.)、大凤尾藓(Fissidensnobilis Griff.)、大羽藓(Thuidium cymbifolium(Doz.et Molk.)B.S.G.)和大灰藓(Hypnum plumaeforme Wils.)嫩茎和老茎的石蜡切片和显微观察发现,同一藓类植株的嫩茎和老茎,茎结构稳定,不同种藓类植物茎横切面具有不同特征.植物体茎横切面形状、表层细胞的层数、细胞大小和细胞壁厚薄、皮层细胞大小和形状、中轴的有无以及比例等特征可以作为藓类植物的分科分类依据之一.     

Cause of Senescence of Nine Sorts of Flowers

The levels of endogenous phytohormones and respiratory rate in nine sorts of flowers such as Cymbidium faberi Rolfe, Nopalxochia ackermannii Kunth and others were investigated both at full bloom and senescence and meanwhile the effect of exogenous phytohormones on prolonging the blossoms and promoting ethylene production were tested. There is a high content of endogenous ethylene in all the long-lived flowere, about 3–16 folds higer than the short-lived ones. There is a high level of ABA at full blooming flowers of short-lived flowers, in which there is no or only some cytokinins in it, but the ratio of CTK (6BA+zeatin)/ABA is smaller(l.7). The endogenous ABA reached a much higher level at senescence in all nine sorts of flowers, so it is reasonable to consider that it is ABA which plays an important role of regulation in controlling flower's senescence. There is a much higher level of GA3 and zeatin in the long-lived flowers which is not demonstrated in the shortlived ones. The respiratory rate is one of the factors controtling the longevity of flowers, but it does not play a decided role. Application of 6BA and zeatin prolongs distinctly orchid’s longevity, however exogenous IAA through the promotive action on ethylene production, evidently extends the longevity of the flowers of the Nopalxochia ackermannii Kunth.     

龙胆科药用植物化学成分的研究现状

龙胆科植物在我国的分布范围很广,且多数为药用植物,其多数种属的药用植物,至今其化学成分尚未被系统研究。综述了目前龙胆科药用植物的化学成分的研究现状及一般提取方法,对近年来发现的环烯醚萜及裂环烯醚萜类化合物进行了总结,为本科药用植物的更深入研究提供了参考。     

The effects of glutamine of the maintenance of embryogenic cultures of Cryptomeria japonica

Summary Embryogenic tissues of sugi (Cryptomeria japonica) were induced on a modified Campbell and Durzan (CD) medium containing 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 600 mg l⁻¹ glutamine, and subcultured in the medium of the same composition for over 1 yr. This resulted in a mixed culture of embryogenic and non-embryogenic cells. When embryogenic cells were isolated and cultured independently, their capacity to form embryogenic aggregates was lost. Thus, the non-embryogenic cells present within a mixed culture system were essential to the formation of embryogenic aggregates. When embryogenic tissues were isolated and cultured independently on a high glutamine-containing (2400 mg l⁻¹) medium, dry weights and endogenous levels of glutamine increased, and the tissue could generate a large number of embryogenic aggregates. Amino acid analysis of embryogenic and non-embryogenic cells from the maintenance culture indicated a higher level of glutamine was present in the latter. The high endogenous level of glutamine in the non-embryogenic portion of mixed cell masses may be the supplier of glutamine for maintaining the embryogenic property of the tissues.     

Specificity of action of piperidylcholine derivatives as substrates of cholinesterases of various origin

The review deals with study of enzymologic properties of a novel highly specific acetylcholinesterase substrate, N-(β-acetoxyethyl) piperidinium iodomethylate (“piperidylcholine”), and its 30 derivatives that were tested as effectors of cholinesterases of mammals and various species of Pacific squids. It was proven for the first time that responsible for specificity of action was structure of cyclic ammonium grouping of the alcohol part of molecule of the ester substrate. Analysis of specificity is performed based on enzymatic hydrolysis parameters—activity of catalytic center of cholinesterases and bimolecular constant of the reaction rate that are determined at optimal and low substrate concentrations. Among the specially synthesized group of thioester compounds there is revealed one more highly specific acetylcholinesterase substrate—N-(β-acetoxyethyl) piperidinium.     

真菌类遗传学分析的知识结构教学

本文以认知结构理论为指导,讨论了真菌类遗传分析与高等动植物遗传分析的内在联系,认为利用这种内在联系进行教学可收到好的效果并说明了作者的具体教学过程。 Abstract:In the paper, the relationship between genetic analysis of Fungi and genetic analysis of high animal and plant was discussed.A good results were obtained when we adopted this method in the teaching.     

Photoregulation of seed germination of wild-type and of an aurea-mutant of tomato

Seed germination of an aurea mutant of tomato ( Lycopersicon esculentum Mill.) is promoted by continuous irradiation with red, far-red or long-wavelength far-red (758 nm) light as well as by cyclic irradiations (5 min red or 5 min far-red/25 min darkness). Far-red light applied immediately after each red does not change the germination behaviour. Seed germination of the isogenic wild-type, cv. UC-105, is promoted by continuous and cyclic red light while it is inhibited by continuous and cyclic far-red light and by continious 758 nm irradiation. Far-red irradiation reverses almost completely the promoting effect of red light. The promoting effect (in the aurea mutant) and the inhibitory effect (in the wild-type) of continuous far-red light do not show photon fluence rate dependency above 20 nmol m⁻² s⁻¹. It is concluded that phytochrome controls tomato seed germination throgh low energy responses in both the wild type and the au mutant. The promoting effect of continuous and cyclic far-red light in the au mutant can be attributed to a greater sensitivity to P_fr.     

The mechanism of hatching of eggs of Haemonchus contortus

Fluid collected from hatching eggs of Haemonchus contortus contained a lipase which hydrolysed 2-naphthyl laurate (about 0·7 μmol naphthol freed /h/10⁶ eggs). The fluid also hydrolysed l-leucinamide (about 2·3 μmol leucine freed/h/10⁶ eggs). The fluid when added to normal or heated eggs caused ‘hatching’. ‘Hatching’ also occurred in exsheathing fluid from infective juveniles and in a preparation of pancreatic lipase containing leucine aminopeptidase. A purified mammalian leucine aminopeptidase in combination with several different lipases did not attack egg shells.The ‘spontaneous’ hatching of eggs of H. contortus was strongly inhibited by 1,10-phenanthroline, 10^?3M, and this inhibition was reversed by Zn²⁺. However, the inhibition of ‘hatching’ of eggs in externally applied hatching fluid, or the hydrolysis of leucinamide in hatching fluid was generally less marked.