绞股蓝法呢基焦磷酸合酶基因的克隆及其序列分析

目的:克隆并分析绞股蓝法呢基焦磷酸合酶(FPS)基因的全长序列。方法:参照罗汉果法呢基焦磷酸合酶基因,设计扩增绞股蓝FPS基因的3′RACE引物,采用3'RACE和5'RACE法克隆绞股蓝FPS基因全长cDNA。结果:获得绞股蓝FPS基因全长cDNA序列共1288个核苷酸,包含一个1026核苷酸的开放读框,编码342个氨基酸残基,推断该蛋白的相对分子质量为3.94×104。NCBI Blast结果显示绞股蓝FPS基因编码蛋白的氨基酸序列与已知的植物FPS氨基酸序列的同源性为91%~74%,核酸序列的同源性为88%~78%。结论:克隆了绞股蓝FPS基因全长cDNA序列,为进一步研究绞股蓝FPS基因的表达及三萜皂苷合成通路关键酶分子的进化奠定了基础。     

法呢基焦磷酸(FPP)的生物合成及其分子调控

法呢基焦磷酸合酶作为异戊二烯途径中的重要调节酶,是许多萜类物质的合成前体。FPS的cDNA克隆在许多生物体中也已得到了分离并进行了表达特性研究。从FPP的生物合成途径入手,对FPP生物学特性、FPS酶基因调控的相关信息进行了综述,同时对FPS在基因工程方面的应用进行了展望。     

人脑红蛋白(NGB)全长cDNA序列的克隆

人脑红蛋白(neuroglobin, NGB)是新发现的神经系统特异的携氧蛋白, 然而其全长cDNA序列一直未见报道. 采用电子序列延伸技术和cDNA序列末端快速扩增技术(rapid amplification of cDNA ends, RACE)研究发现, 人NGB全长cDNA序列为1 909 bp, 5′非编码区为375 bp, 编码区(456 bp)可编码151个氨基酸, 3′非编码区为1 078 bp, 其中含27 bp的poly(A)(GenBank接受号: AF422797). 综合采用电子序列延伸技术与RACE技术是获得全长cDNA序列的有效方法, 为后续的功能研究提供了重要基础.     

大豆异戊烯焦磷酸异构酶基因的电子克隆及生物信息学分析

利用电子克隆方法获得大豆异戊烯焦磷酸异构酶基因(IPI)的cDNA序列,并采用生物信息学方法对该基因及其编码蛋白的一般理化性质、疏水性、信号肽、二级结构和亚细胞定位等方面进行了预测和分析。结果表明,大豆异戊烯焦磷酸异构酶基因的cDNA序列全长1384bp,包含一个906bp的ORF,编码301个氨基酸。大豆IPI酶为一亲水性的非分泌蛋白,α-螺旋和无规则卷曲是其主要的二级结构,该酶定位于叶绿体。     

北美鹅掌楸LtuFPPS1基因克隆与组织表达分析

法尼基焦磷酸合酶(farnesyl diphosphate synthase,FPPS)是植物萜类物质合成前体法尼基焦磷酸(farnesyl pyrophosphate,FPP)的关键合成酶。为探究北美鹅掌楸(Liriodendron tulipifera)萜类合成途径的分子机制,该文利用北美鹅掌楸转录组数据,通过设计特异性引物,采用RACE技术克隆得到LtuFPPS1基因的全长序列并进行生物信息学分析。结果表明:(1) LtuFPPS1基因全长1 460 bp,开放阅读框(ORF)长1 056 bp,编码351个氨基酸,蛋白质分子量为40 589.45 D,等电点为5.19,不稳定系数为43.50,归类为不稳定蛋白; LtuFPPS1基因所编码的蛋白不含信号肽,定位于线粒体,是一种不稳定亲水性蛋白,具有类异戊二烯类化合物合酶的特征结构域,α-螺旋为其氨基酸序列的主要二级结构。(2)进化树和同源序列分析表明,北美鹅掌楸LtuFPPS1蛋白与乐昌含笑(Michelia chapensis)的FPPS蛋白的亲缘关系更近。(3)组织表达分析结果发现,LtuFPPS1基因在北美鹅掌楸雌蕊中的表达量最高,其高低顺序为雌蕊花芽茎雄蕊萼片叶片花瓣根,据此推测萜类代谢物在花器官中的合成相对较多。综上所述,LtuFPPS1基因为萜类合成酶基因,可为从分子生物学层面探究北美鹅掌楸萜类物质的合成提供一定的理论帮助。     

三角帆蚌CypA基因的克隆及其在嗜水气单胞菌感染后的表达分析

为研究亲环蛋白A (Cyclophilin A, CypA)在三角帆蚌(Hyriopsis cumingii)免疫系统中的作用,研究利用PCR与RACE技术克隆获得三角帆蚌CypA基因全长cDNA序列(基因序列登录号:MN121742)。三角帆蚌CypA基因cDNA序列全长1237 bp,其5′非编码区(UTR)长度为57 bp, 3′非编码区长度为688 bp,三角帆蚌CypA基因的开放阅读框(Open Reading Frame, ORF)长度为492 bp,编码164个氨基酸, CypA蛋白质理论等电点为8.9,相对分子量为17.29 kD。在GenBank中进行同源序列检索,三角帆蚌CypA氨基酸序列与其他贝类CypA的相似性依次为池蝶蚌(Hyriopsis schlegelii)(99.39%)、青蛤(Cyclina sinensis)(77.44%)、菲律宾蛤仔(Ruditapes philippinarum)(78.05%)、近江牡蛎(Crassostrea ariakensis)(78.66%)和大竹蛏(Solen grandis)(75.05%)。通过SWISS-MO...     

穿心莲ent-柯巴基焦磷酸合酶基因的克隆与生物信息学分析

旨在克隆穿心莲CPS的编码基因,并进行序列分析.通过RT-PCR和cDNA末端快速扩增的技术从穿心莲叶片中获得目的基因;并利用生物信息学的方法对其编码蛋白进行功能分析.结果显示,获得了全长2 499 bp的cDNA序列,编码一个833aa的蛋白.序列同源性比较表明,该蛋白与野甘草、咖啡等其他植物来源的CPS具有较高的同源性.保守功能结构域分析显示,该蛋白包含一个富含Asp残基的植物萜类合酶的共有保守功能域“DXDD”,归属于萜类生物合成酶Ⅰ类超级家族和顺式聚异戊二烯焦磷酸合酶超级家族.初步推测克隆得到了穿心莲CPS的编码基因(命名为ApCPS,GenBank登录号为JN216843.1).     

铁皮石斛蔗糖磷酸合成酶基因的克隆与原核表达

应用同源序列克隆法克隆了铁皮石斛蔗糖磷酸合成酶(SPS)基因cDNA全长,并进行了原核表达分析,为进一步研究该基因的时空表达、功能分析及多糖合成机理提供理论依据。结果表明:(1)铁皮石斛SPS基因cDNA全长3 502bp,编码区3 186bp,GenBank登录号JF423929。该基因编码1 061个氨基酸,与文心兰的SPS基因氨基酸序列的一致性最高为93%,与其他科植物SPS基因的氨基酸序列的一致性均高于60%。(2)原核诱导表达结果显示,SPS基因在大肠杆菌中的重组蛋白分子质量约为118.7kD,其表达与序列分析推测的结论一致。(3)生物信息学分析表明,铁皮石斛SPS基因的二级结构包括了螺旋、β-折叠和无规则卷曲,是非跨膜结构的亲水性不稳定蛋白,有2个功能结构域,分别是蔗糖合成功能域及糖基转移功能域。     

西瓜牻牛儿基牻牛儿基焦磷酸合成酶基因的克隆及表达分析

目的:了解牻牛儿基牻牛儿基焦磷酸合成酶基因(GGPS)在4种不同瓤色西瓜果实发育过程中的表达变化,将为西瓜类胡萝卜素基因操作提供工具。方法:通过RT-PCR和RACE技术从西瓜果实中克隆GGPS的cDNA全长,用生物信息学方法分析其序列及推测氨基酸序列,并用实时定量PCR技术检测GGPS在不同瓤色果实发育过程的表达水平。结果:GGPS基因cDNA全长1 445 bp(GenBank登录号为KF914758),其开放阅读框为1 092 bp,编码363个氨基酸。预测其氨基酸序列N末端存在转运肽信号序列。西瓜GGPS与甜瓜和黄瓜GGPS的序列同源性高达90%以上。GGPS在红瓤西瓜中的表达量最高,白瓤西瓜中最低。结论:克隆获得的GGPS可能对西瓜果实中类胡萝卜素的积累具有重要影响。     

菘蓝牦牛儿基牦牛儿基焦磷酸合成酶基因(IiGGPPS1)的克隆及其表达特性分析

本研究通过分析菘蓝转录组数据库得到一条牦牛儿基牦牛儿基焦磷酸合成酶(GGPPS)的编码序列,利用RT-PCR扩增得到其全长,命名为Ii GGPPS1,利用生物信息学方法对该基因及其编码的蛋白进行分析,并对该基因的功能、表达特性进行了研究。研究结果表明:Ii GGPPS1的c DNA序列全长为1 086 bp,开放阅读框长1 080 bp,编码359个氨基酸。Ii GGPPS1蛋白与其它植物GGPPSs高度保守,蛋白N端含有叶绿体信号肽,二级结构主要由螺旋和无规则卷曲组成,同源建模结果显示Ii GGPPS1蛋白是个同源二聚体。组织特异表达分析表明该基因在叶片中的表达量最高,根中次之,茎中最低,且受虫害颜色浅的叶片中该基因表达量显著低于正常叶片中的表达量,推测该基因可能与二萜化合物叶绿素的合成相关。该结果将为进一步研究菘蓝牦牛儿基牦牛儿基焦磷酸合成酶基因(Ii GGPPS1)的功能及其表达调控奠定理论基础,也可为菘蓝的生长、发育调节与品质改良积累研究资料。     

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.     

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.     

Enterovirus 71 3C proteolytically processes the histone H3 N-terminal tail during infection

Highlights
1. The N-terminal tail of histone H3 is specifically cleaved during EV71 infection.
2. Viral protease 3C is identified as a protease responsible for proteolytically processing the N-terminal H3 tail.
3. Our finding reveals a new epigenetic regulatory mechanism for Enterovirus 71 in virus-host interactions.     

Detection of EBV and HHV6 in the Brain Tissue of Patients with Rasmussen’s Encephalitis

Rasmussen’s encephalitis (RE) is a rare pediatric neurological disorder, and the exact etiology is not clear. Viral infection may be involved in the pathogenesis of RE, but conflicting results have reported. In this study, we evaluated the expression of both Epstein-Barr virus (EBV) and human herpes virus (HHV) 6 antigens in brain sections from 30 patients with RE and 16 control individuals by immunohistochemistry. In the RE group, EBV and HHV6 antigens were detected in 56.7% (17/30) and 50% (15/30) of individuals, respectively. In contrast, no detectable EBV and HHV6 antigen expression was found in brain tissues of the control group. The co-expression of EBV and HHV6 was detected in 20.0% (6/30) of individuals. In particular, a 4-year-old boy had a typical clinical course, including a medical history of viral encephalitis, intractable epilepsy, and hemispheric atrophy. The co-expression of EBV and HHV6 was detected in neurons and astrocytes in the brain tissue, accompanied by a high frequency of CD8⁺ T cells. Our results suggest that EBV and HHV6 infection and the activation of CD8⁺ T cells are involved in the pathogenesis of RE.     

Perinatal Vertical Transmission of Chikungunya Virus in Ruili,a Town on the Border between China and Myanmar

正Dear Editor,Chikungunya virus (CHIKV), an arbovirus in the family of Togaviridae, genus Alphavirus, is transmitted by the A.aegyptii or A. albopictus mosquito, and causes disease in humans characterized by fever, rash, and arthralgia (Silva and Dermody 2017; Suhrbier 2019). It was first reported in 1953 in Tanzania, and caused only a few outbreaks and sporadic cases in Africa and Asia in last century. However, in the epidemic in 2004, CHIKV acquired mutations that conferred enhanced transmission by the A. albopictus mosquito(Schuffenecker et al. 2006). Since then, it has successively caused outbreaks in Africa, the Indian Ocean, South East Asia, the South America, and Europe (Zeller et al. 2016).     

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

In conclusion, the novel visual RT-LAMP assay is a simple, rapid, and sensitive approach for detection of SARS-CoV-2, and it is ready for application in primary care and community hospitals or health care centers, and even patients' own houses in response to the current SARS-CoV-2 epidemic because the assay does not require sophisticated equipment and skilled personnel. Furthermore, it is also ready to be used in fields for screening samples from wild animals and environments to facilitate the identification of potential intermediate hosts that mediate the cross-species transmission of SARS-CoV-2 from bats to humans.