家蚕吡哆醛激酶基因干扰降低转氨酶基因的转录表达

【目的】维生素B₆在氨基酸代谢中是多种酶的辅酶,维持氨基酸代谢的正常运行。磷酸吡哆醛（pyridoxal-5′-phosphate, PLP）是维生素B₆的主要辅酶形式,吡哆醛激酶（pyridoxal kinase, PLK）是PLP的重要生成酶,本研究试图明确PLK基因与PLP依赖酶之间转录水平的调节关系。【方法】本研究采用RNA干扰（RNA interference, RNAi）方法对家蚕 Bombyx mori 的PLK基因进行干扰,通过体外合成PLK基因的3个干扰片段（siRNA1, siRNA2和siRNA3）,将siRNA从体腔注入5龄第3天的家蚕幼虫体内诱导RNAi。利用荧光定量PCR测定不同干扰片段、不同时间点及不同组织中PLK基因表达量的变化;并测定家蚕体内磷酸丝氨酸转氨酶（phosphoserine aminotransferase, SerB）和天门冬氨酸氨基转移酶（asparate aminotransferase, AST）基因的表达量。【结果】注射干扰片段后48 h干扰效果达到最佳。3个干扰片段干扰效果从高到低依次为siRNA1, siRNA2和siRNA3。RNAi效果最好的是中肠组织,其PLK基因的相对表达量下降了55%。RNA干扰PLK基因后,后部丝腺中SerB和AST基因相对表达量分别下降了90%和29%。【结论】本研究通过RNAi实现了家蚕PLK基因干扰,并进一步证明了家蚕PLK基因和SerB基因及AST基因存在联动调节关系。     

小干扰RNA对庚型肝炎病毒基因在人Huh-7细胞中表达的抑制作用

RNA干扰(RNAi)是由小干扰RNA(siRNA)引发的生物细胞内同源基因的转录后基因沉默现象, 是近年来兴起的一项研究生物基因调控与功能的崭新技术. 庚型肝炎病毒(HGV)是一单正链RNA病毒, 复制时不与宿主细胞基因组整合, 尤其适合用于RNAi的研究. 构建了含HGV完整结构基因并携带筛选标志潮霉素基因的真核表达载体pVAX.EH, 转染Huh-7细胞后, 筛选获得稳定表达HGV 结构蛋白的Huh-7细胞株(Huh-7-EH). RT-PCR和Western blot检测证实, HGV结构基因能在Huh-7-EH细胞中转录、表达, 并能进行剪切和翻译后修饰. 以体外转录法制备了2对靶向HGV E2基因的siRNA(1-E2 siRNA和2-E2 siRNA), 将其导入Huh-7-EH细胞中, 采用Western blot和克隆形成实验证实, HGV 1-E2 siRNA和2-E2 siRNA均能特异性抑制HGV结 构蛋白的表达, 抑制作用可维持1周以上. 其中2-E2 siRNA的抑制作用更强, 转染后对Huh-7-EH细胞潮霉素抗性克隆形成的抑制率达到了99%. Huh-7-EH细胞转染siRNA后对潮霉素敏感, 说 明HGV E2 siRNA不仅使HGV E2区的mRNA降解, 还可使融合在HGV E2区下游的潮霉素mRNA降解. 综上所述, 本实验建立的稳定表达HGV结构蛋白的Huh-7-EH细胞株, 能作为用于研究HGV复制和RNAi的细胞模型; HGV结构基因区的siRNA可同时抑制HGV结构蛋白及其下游的潮霉素基因的表达, 证明RNAi在真核细胞Huh-7-EH内可能存在放大作用.     

RNA干扰对离体大鼠神经干细胞NgR基因表达的影响

目的观察RNA干扰（RNA interference,RNAi）对离体大鼠神经干细胞的Nogo-66受体（Nogo-66 reeeptor,NgR）基因表达的影响。方法设计并合成3条大鼠NgR基因mRNA的小分子干扰RNA（small interfering RNA,siRNA）转染体外培养的神经干细胞,免疫荧光及Western blot检测NgR表达。结果siRNA可以成功的转染神经干细胞,3条siRNA不同程度的阻断了神经干细胞的NgR基因表达,阻断效率随时间延长逐渐下降。阻断效果最好的1条siRNA在转染后第5d仍能保持（85．22±3．1）％高阻断效率。结论应用RNAi可以高效率的使大鼠神经干细胞的NgR基因表达沉默。     

RNA干扰及其在动物繁殖研究中的应用

RNA干扰(RNAi)是指小分子双链RNA通过特异性降解与其同源的mRNA,而在mRNA水平上高效阻断体内特异性基因表达的现象,属于转录后水平基因沉默。利用该技术进行基因功能研究,经济有效,通用性好,已成为反向遗传学研究中最重要的工具之一。在动物繁殖领域,RNAi技术主要应用于体外研究哺乳类和禽类卵母细胞发育、胚胎发育和精子形成中重要基因的功能及其作用机制。随着该技术不断发展完善,RNAi必将在动物繁殖生产实践中发挥巨大的作用。     

RNA干扰研究进展

RNA干扰(RNA interference,RNAi)是指由双链RNA(double-strandedRNA,dsRNA)启动的序列特异的转录后基因沉默现象,广泛存在于真菌、植物和动物中。它是细胞内由双链RNA诱导降解与其配对的特定mRNA的过程。细胞内双链RNA在酶的作用下,形成20-25碱基大小的小干扰RNA(siRNAs),由siRNAs进一步掺入多组分核酸酶并使其激活,从而精确降解与siRNAs序列相同的mRNA,抑制该基因在细胞内的翻译表达。RNAi技术是近年来迅速发展起来的高效、特异、易操作的基因沉默技术。与反义寡核苷酸等传统方法相比,RNAi技术有着无可比拟的优势。本文就其近年的研究进展作一综述。     

RNA干扰技术在哺乳动物中的应用

RNA干扰(RNAi)是生物界普遍存在的一种抵御外来基因和病毒感染的进化保守机制.RNAi是由双链RNA触发的转录后基因沉默机制,具有序列特异性,在哺乳动物细胞中,RNAi由21～23个核苷酸组成的双链RNA引发.小干扰RNA(siRNA)可以在体外合成或通过表达载体在哺乳动物细胞内合成.由于RNAi技术具有快速、简单和特异性强等特点,在基因功能研究、抗病毒治疗和抗肿瘤治疗等方面有广泛的应用前景.     

外源性双链RNA对小鼠卵母细胞basonuclin基因表达的影响

在植物及低等动物线虫中, 引入外源性双链RNA(dsRNA)会导致细胞中同源mRNA降解, 从而干扰其内源基因的表达, 这可能是有机体防范病毒或转座子诱导DNA突变的一种生理机制, 称为RNA干涉(RNA interference, RNAi). 利用RNAi研究basonuclin基因在卵母细胞发生过程中的功能. 将basonuclin特异的dsRNA导入小鼠生发泡期卵母细胞, 可有效降低其mRNA的丰度, 这种降解作用与dsRNA的浓度及作用时间成正比, 但不影响非同源基因的表达, 证明basonuclin dsRNA能特异识别、降解其内源基因的转录产物. 免疫组化实验显示, dsRNA能降低卵母细胞中basonuclin蛋白的水平, 但其效率不如RNAi对tPA及cMos基因活性的抑制, 这可能是由于basonuclin蛋白的半衰期较长, 而生发泡期卵母细胞在体外存活时间较短. 结果表明, dsRNA能阻抑卵母细胞中同源基因的表达, 其作用相当于基因敲除. 质粒表达的发夹环型dsRNA也可以有效降解basonuclin转录产物, 这为研究basonuclin在卵母细胞发育早期的功能提供了新的手段.     

Gas6在猪卵母细胞及早期胚胎中表达模式与功能的初步研究

该研究通过免疫组化和QRT-PCR等方法系统分析了Gas6（growth arrest-special gene 6）基因在猪卵泡发生及早期胚胎发育过程中的表达规律,并提出了一种改良猪卵母细胞体外成熟系统的方法。研究结果显示,Gas6基因表达于猪卵巢中的卵母细胞细胞核及其周围的卵丘细胞,在卵母细胞体外成熟及早期胚胎发育过程中始终有表达,且在囊胚中表达最高。Gas6 mRNA在卵母细胞成熟过程中始终存在,但在孤雌激活后迅速消失,直到发育到囊胚时再次出现。在猪卵母细胞体外培养系统中添加不同浓度的Gas6重组蛋白培养卵母细胞,发现添加Gas6重组蛋白对卵母细胞的极体率无显著影响;但是当添加浓度为100 ng/mL时,培养的卵母细胞孤雌激活后分裂率、囊胚率及囊胚细胞数都显著增高。Gas6可能是通过改善卵母细胞细胞质的成熟质量提高卵母细胞的发育潜能,从而获得了更多、更好的胚胎。     

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.     

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     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

Entwicklungsgeschichte und Ultrastruktur von Pollenkitt und Exine bei nahe verwandten entomophilen und anemophilen Angiospermensippen derAlismataceae,Liliaceae, Juncaceae,Cyperaceae, Poaceae undAraceae

Some closely related members of the monocotyledonous familiesAlismataceae, Liliaceae, Juncaceae, Cyperaceae, Poaceae andAraceae with variable modes of pollination (insect- and wind-pollination) were studied in relation to the ultrastructure of pollenkitt and exine (amount, consistency and distribution of pollenkitt on the surface of pollen grains). The character syndromes of pollen cementing in entomophilous, anemophilous and intermediate (ambophilous or amphiphilous) monocotyledons are the same in principal as in dicotyledons. Comparing present with former results one can summarize: 1) The pollenkitt is always produced in the same manner by the anther tapetum in all angiosperm sub-classes. 2) The variable stickiness of entomophilous and anemophilous pollen always depends on the particular distribution and consistency of the pollenkitt, but not its amount on the pollen surface. 3) The mostly dry and powdery pollen of anemophilous plants always contains a variable amount of inactive pollenkitt in its exine cavities. 4) A step-by step change of the pollen cementing syndrome can be observed from entomophily towards anemophily. 5) From the omnipresence of pollenkitt in all wind-pollinated angiosperms studied one can conclude that the ancestors of anemophilous angiosperms probably have been zoophilous (i.e. entomophilous) throughout.

     

Identification and Characterization of the First Equine Parainfluenza Virus 5

正Dear Editor,Parainfluenza virus 5 (PIV5), known as canine parainfluenza virus in the veterinary field, is a negative-sense,nonsegmented, single-stranded RNA virus belonging to the Paramyxoviridae family (Chen 2018). The virus was first reported in primary monkey kidney cells in 1954 (Hsiung1972), then it has been frequently discovered in various     

Pathogenic Characterization and Full Length Genome Sequence of a Reassortant Infectious Bursal Disease Virus Newly Isolated in Pakistan

<正>Dear Editor,Infectious bursal disease (IBD) is one of the most important diseases of the poultry. The IBD virus (IBDV), a nonenveloped virus belonging to the Birnaviridae family with a genome consisting of two segments of double-stranded RNA (segments A and B), targets B lymphocytes of bursa of Fabricious leading to immunosuppression. In Pakistan,poultry farming is the second biggest industry and IBD is the second biggest disease threating the poultry sector.However, there is limited genome information of IBDV