解偶联蛋白UCP1启动子荧光素酶报告基因载体的构建

目的:构建解偶联蛋白UCP1启动子荧光素酶报告基因载体,为寻找调控UCP1表达的小分子化合物提供有效工具。方法:从小鼠基因组DNA中PCR扩增小鼠UCP1启动子上游2000 bp序列,并将该序列连接到荧光素酶报告基因载体p GL3-basic中,构建p GL3-UCP1启动子。测序正确后,提取质粒,然后将上述载体与p RL-TK载体共转染至HEK293细胞、小鼠白色脂肪前体细胞和小鼠棕色脂肪前体细胞,48 h后裂解细胞检测荧光素酶的活性。结果:通过PCR成功扩增获得了目的片段,并将其克隆至p GL3-basic中。与细胞内源UCP1表达水平相似,荧光素酶报告系统表明构建的p GL3-UCP1在棕色脂肪细胞中启动子活性最高,在白色脂肪细胞中活性较低,在HEK293细胞中基本没有活性。同时β3肾上腺素受体激动剂CL 316,243同样能够上调p GL3-UCP1的启动子活性。结论:成功构建了小鼠UCP1启动子荧光素酶报告基因载体,并证明在棕色脂肪细胞中,该启动子具有很强的启动子活性,而在白色脂肪和HEK293细胞中,启动子活性很低。该启动子报告系统有望为寻找激活UCP1的小分子化合物提供重要平台。     

IL-37基因启动子的克隆及其荧光素酶报告基因载体的构建

[目的]克隆白介素-37(IL-37)基因启动子,构建其荧光素酶报告基因载体,并分析活性。[方法]用PCR方法扩增IL-37基因5'端上游区3个不同长度的启动子片段,分别克隆入荧光素酶报告基因载体p GL3,构建IL-37基因启动子荧光素酶报告基因质粒。将所构建的质粒转染HEK-293细胞,通过双荧光素酶报告基因系统分析启动子的转录活性。[结果]754、1 017、2 043 bp等3个IL-37启动子片段正确亚克隆入荧光素酶报告基因载体,重组质粒转染HEK-293细胞后,双荧光素酶活性分析显示1 017 bp的启动子片段具有较强转录活性,约为对照组(空载体p GL3-basic)的10.9倍。[结论]成功克隆IL-37基因启动子,构建了大小为1 017 bp的IL-37基因启动子荧光素酶报告基因载体,为IL-37表达的调控机制的研究提供有效的工具。     

探讨穿心莲内酯对HIV-1感染辅助受体CXCR4启动子作用机制的研究

穿心莲内酯具有明显的抗病毒作用,对HIV-1(Human immunodeficiency virus type 1)具有明显的抑制作用,本研究探讨了穿心莲内酯影响CXCR4启动子活性的作用机制。首先构建双荧光素酶报告基因载体pFireRlucCXCR4(C-X-C chemokine receptor 4),并转染入人HEK293T细胞;利用CCK8法检测穿心莲内酯对人HEK293T细胞细胞毒性作用;双荧光素酶报告基因技术检测穿心莲内酯对CXCR4启动子活性的影响;MTT法检测穿心莲内酯对人T淋巴细胞Jurkat细胞活性影响;实时荧光定量PCR检测穿心莲内酯对人T淋巴细胞Jurkat细胞表面CXCR4 mRNA和蛋白表达的影响。PCR分别扩增了CXCR4启动子（777 bp）和Rluc(1 997 bp)表达单元,通过测序和酶切鉴定双荧光素酶报告基因载体插入正确;穿心莲内酯作用于转染pFireRluc-CXCR4HEK293T细胞,双荧光素酶结果显示：穿心莲内酯能够下调CXCR4启动子活性,差异具有显著性（P<0.05）。穿心莲内酯作用于人T淋巴细胞Jurkat细胞后,qPCR...     

大鼠SCN5A基因内含子1＋204bp～＋757bp转录调控功能分析

目的 克隆大鼠心肌SCN5A基因5’端调控区,检测目的片段在HEK293细胞中的转录活性。方法 扩增大鼠心肌SCN5A基因＋204bp-＋757bp、＋204bp-＋385bp和＋204bp-＋329bp片段,构建含目的片段的荧光素酶报告基因——pGL3-P0、pGL3-P1和pGL3-P2,比较HEK293细胞中荧光素酶活性,评价不同长度DNA片段的转录调控活性。结果 成功构建大鼠心肌SCN5A基因5’端3个片断的荧光素酶报告基因,HEK293细胞中pGL3-P1相对荧光素酶活性分别为pGL3-PO、pGL3-P2的4．3倍和2．8倍。结论SCN5A基因内含子1目的片段在HEK293细胞中具有较强的转录调控活性;SCN5A基因＋329bp～＋385bp为正调控区,软件分析MZF1、USF、C-ETs-1等因子能与这些正调控区域结合并可能参与SCN5A基因的表达调控。     

EphA3基因启动子区域定位表达及活性分析

目的构建含有不同长度EphA3基因启动子片段的报告基因载体,研究其在293T细胞和MEF细胞中的转录活性。方法以Balb／C小鼠基因组DNA为模板,扩增不同长度的EphA3基因启动子片段,并克隆进入荧光素酶报告基因质粒pGL3-Basic真核表达载体内。酶切鉴定及基因测序无误后,将重组质粒和pRL—CMV内对照质粒共转染293T和MEF细胞,分析不同长度的OhA3基因启动子片段的转录活性。结果酶切和测序鉴定表明表达载体构建成功,EphA3基因的核心启动子区域位于-279bp～＋110bp之间,在293T细胞和MEF细胞中其转录活性相似。结论成功构建了荧光素报告基因重组质粒,并确定了BphA3基因的核心启动子区域。     

含p21启动子的荧光素酶报告基因的构建和活性测定

目的:克隆p21基因的启动子,插入荧光素酶报告基因载体中,并检测其活性。方法:采用PCR技术从人乳腺癌细胞系MCF-7基因组中扩增出p21启动子,插入荧光素酶报告基因载体pGL3-basic中,确定所扩增的DNA序列,在293T细胞中检测其活性。结果:测序结果表明扩增的p21启动子序列正确,活性实验表明构建的报告基因具有启动子活性,雌激素受体(ER)α能以剂量依赖的方式升高p21报告基因的转录。结论:克隆了p21启动子,为ERα共调节子的功能研究提供了重要基础。     

人claudin-10基因启动子转录调控的初步分析

[目的]构建不同长度的人claudin-10基因上游启动子荧光素酶报告基因载体,并在LO2细胞中比较其活性。[方法]以人LO2肝细胞基因组DNA为模板,PCR扩增获得claudin-10基因5'侧翼序列,并将其插入p MD18-T载体;PCR扩增获得不同长度的claudin-10基因上游启动子区序列,构建p GL3-Basic系列荧光素酶基因报告载体。瞬时转染LO2细胞,双荧光素酶报告基因分析系统检测转录活性。[结果]成功构建6个不同长度的人claudin-10基因上游启动子区(-1 451/+100bp、-1 022/+100bp、-1 005/+100bp、-677/+100bp、-377/+100bp和-108/+100bp)的报告载体;启动子活性实验结果表明:当claudin-10启动子片段从-1 005 bp截短至-677 bp,从-108 bp截短至+100 bp时活性显著下降(P0.05);其余截短时活性无改变(P0.05)。[结论]claudin-10启动子-1 005bp~-677bp和-108bp~+100bp区是claudin-10基因的主要转录调控区。     

大鼠NKx6.1启动子克隆及特异性表达分析

旨在构建大鼠NKx6.1启动子的报告载体,验证转录因子T3R对NKx6.1启动子的调控活性。用PCR扩增大鼠脑组织NKx6.1的5'上游启动子片段2.4 kb,应用生物信息学方法预测该片段上潜在的转录因子T3R结合位点,根据不同的结合位点作系列截短,获得3段长度不等的启动子缺失片段,分别克隆到荧光素酶报告基因表达质粒(p GL3-Basic)上,构建相应的报告载体。将报告载体和T3R共转染大鼠星形胶质细胞(rat astrocytes,RA),并检测报告基因荧光素酶的活性。结果显示,成功构建大鼠NKx6.1启动子报告载体,双荧光素酶报告基因活性检测表明T3R对NKx6.1启动子有明显调控作用,其中-1 887 bp-1 507 bp活性最高,即存在关键顺式调控元件。克隆并筛选出启动子核心区域,揭示了甲状腺激素对大鼠NKx6.1的表达调控机制。     

含p27启动子的荧光素酶报告基因的构建和活性测定

目的:克隆p27基因的启动子,插入荧光素酶报告基因载体中,并检测其活性。方法:采用PCR技术从人乳腺癌细胞系MCF-7基因组中扩增出p27启动子,插入荧光素酶报告基因载体pGL3-basic中,确定所扩增的DNA序列,在293T细胞中检测其活性。结果:测序结果显示扩增的p27启动子序列正确,活性实验表明构建的报告基因具有启动子活性,雌激素受体(ER)α能以剂量依赖的方式升高p27报告基因的转录。结论:克隆了p27启动子,为ERα共调节子的功能研究提供了重要基础。     

斑马鱼tnfb启动子的克隆和报告基因载体的活性分析

本研究通过采用生物信息学软件预测细胞因子tnfb基因5'上游2 000 bp左右的序列,并预测其相关的转录因子,克隆启动子,构建双荧光素酶报告基因表达载体,经双酶切和测序鉴定,最后通过双荧光素酶报告基因系统检测重组载体的活性。研究发现tnfb的转录因子结合位点为:Oct-1、TBP、GATA-1、RSRFC4、GLO、C/EBPα、NF-资B、ER、Pit-1a、Oct-2、Oct-2.1、RAP1。tnfb的启动子区没有发现Cp G岛,其5'侧翼序列含有与转录密切相关的TATA Box和CAAT Box转录元件。将tnfb的启动子片段插入p GL3-enhancer构建p GL3-tnfb-promoter-enhancer质粒后,质粒经酶切测序显示酶切片段大小一致,序列正确;转染了p GL3-tnfbpromoter的Raw264.7细胞的相对荧光素酶活性高于对照组细胞,双荧光素酶报告基因检测系统证实构建的p GL3-tnfb-promoter-enhancer具有启动子活性。为研究tnfb参与的免疫相关的信号通路之间的调控提供了有力的研究工具。     

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