分析超速离心技术研究膜蛋白TmrAB

去垢剂在膜蛋白的提取纯化过程中起到必要的作用,对膜蛋白的聚合状态、结晶条件以及理化性质等方面都有较大影响.分析超速离心技术(analytical ultracentrifuge,AUC)通过测定溶液中膜蛋白-去垢剂复合物在离心场中的沉降运动轨迹,可以分析获得其沉降系数、摩尔质量、流体力学半径、结合常数等水力学和热力学性质,进而判断膜蛋白-去垢剂复合物的均一性及聚合状态.本文以嗜热菌来源的ATP结合转运蛋白(ABC transporter)TmrAB作为研究对象,利用分析超速离心技术结合分子排阻层析和冷冻电镜负染技术,研究其均一性、聚合状态以及去垢剂与膜蛋白的摩尔比.结果显示,在8倍临界胶束浓度(critical micelle concentration,CMC)的DDM条件下,TmrAB性质均一,并以异二聚体的单体形式存在,DDM与Tmr AB的摩尔比为116∶1.本研究表明,分析超速离心技术是一种测定膜蛋白分子质量、研究膜蛋白聚合状态的可靠手段.     

青稞HbSnRK2.4的克隆及其序列特征与表达特性分析

干旱胁迫是制约农作物生产的重要限制因素之一,研究并增强作物的抗旱性具有重要意义。Sn RK2(Sucrose nonfermenting 1-related protein kinase 2)基因编码一类蔗糖非酵解型蛋白激酶,该酶在ABA信号转录途径和抗渗透胁迫中起着重要作用。以青稞(Hordeum vulgare subsp.vulgare)抗旱品种喜马拉雅10号为材料,利用RT-PCR技术克隆获得了Sn RK2基因全长c DNA序列,命名为Hb Sn RK2.4(登录号:KJ699389)。生物信息学分析表明,该基因全长1 310 bp,编码362个氨基酸序列,蛋白分子量为41.94 k D,等电点(p I)为5.96。Prosite Scan分析结果表明,Hb Sn RK2.4含有多个干旱胁迫响应蛋白的作用位点,如酪蛋白激酶Ⅱ磷酸化位点、酪氨酸激酶磷酸化位点、蛋白激酶C磷酸化位点及N-豆蔻酰化位点等。利用实时定量PCR方法研究了Hb Sn RK2.4在干旱胁迫条件下及复水后不同时间点的表达情况,发现Hb Sn RK2.4在土壤绝对含水量为33.4%时表达量最高,随着土壤绝对含水量的下降而下调表达;当达到作物正常生长所需的土壤绝对含水量时又开始上调表达;进行干旱胁迫后(15.5%)基因表达量下降;复水后8 h时恢复正常表达水平。     

草莓蔗糖非发酵-1-相关蛋白激酶1(SnRK1)α亚基编码基因的克隆及表达分析

植物的蔗糖非发酵-1-相关蛋白激酶1(Sn RK1)与酵母蔗糖非发酵蛋白激酶1(SNF1)以及哺乳动物AMP激活的蛋白激酶(AMPK)同源,都是异源三聚复合体结构,含有α催化亚基和β、γ两个调节亚基来维持蛋白结构的稳定和激酶活性。本试验以‘妙香7号’草莓(Fragaria×ananassa)为材料,通过反转录PCR克隆得到一个Sn RK1的α催化亚基编码基因,命名为Fa Sn RK1α。序列分析显示该基因全长1 557 bp,共编码518个氨基酸,预测Fa SnRK1α蛋白分子质量为59.159 k Da,理论等电点为8.54,定位于细胞质和细胞核。生物信息学分析发现Fa SnRK1α的氨基酸序列与其他植物Sn RK1α蛋白具有较高同源性,含有KD(kinase domain)、UBA(ubiquitin associated domain)和β-SID(β-submit interaction domain)三个保守结构域。组织特异性分析表明Fa Sn RK1α在草莓根、茎、叶、花和果实中均有表达,在果实发育进程中Fa Sn RK1α的表达水平呈上升趋势。荧光定量PCR分析表明,果实中Fa Sn RK1α受脱落酸(ABA)诱导,表明该基因可能与ABA诱导的果实发育和成熟有关。     

天蚕卵黄原蛋白cDNA的克隆及序列分析

天蚕卵黄原蛋白cDNA的全碱基序列由5 720个碱基构成,一个开读框有5 334个碱基序列,编码了包括由15个氨基酸组成的信号肽在内的共1 778个氨基酸的蛋白质前体。天蚕和柞蚕卵黄原蛋白的同源性非常高,氨基酸序列达到92.6%,碱基序列达到94%。天蚕、柞蚕和家蚕的卵黄原蛋白的糖链附加位点(N-linkedg-lycosylationsite):柞蚕有4个,家蚕有5个,天蚕和家蚕相同保存着5个。在N-末端区域,天蚕和家蚕有多聚丝氨酸区域和可被胰蛋白酶识别的RSRR部位;柞蚕虽然也具有多聚丝氨酸区域,但没有可被胰蛋白酶识别的RSRR部位。在C-末端区域里,大多数昆虫从G ICG功能部位至C-末端结尾具有7个~10个半胱氨酸(Cys)。鳞翅目的4种昆虫柞蚕、天蚕、家蚕和舞毒蛾(A.pernyi,A.yam am ai,B.m ori,L.dispar)都是7个半胱氨酸。     

分子内分子伴侣机制的研究进展

在原核生物、真核生物及病毒中,一些蛋白质的折叠不符合Anfinsen原则,即依靠自身的氨基酸序列是不够的,还需一段被称为分子内分子伴侣(IMC)的肽段来协助折叠．根据机制不同,IMC可分为两类：第一类IMC引导成熟肽折叠为具有空间结构的蛋白质;第二类IMC协助成熟肽的多聚化而使其获得生物学功能．IMC能提供比分子伴侣更契合的结构,更有效地引导成熟肽折叠,是一种更优的折叠策略．研究IMC分子机制,不仅能够确定IMC上哪些残基的协同作用引导成熟肽折叠,而且可通过改变或修饰其侧链来改造成熟肽,拓展传统的蛋白质工程．     

十字花科植物CYP86MF基因同源序列的克隆与进化分析

为了从分子水平阐明十字花科植物间的亲缘进化关系,给植物种质资源的创建提供理论依据,试验根据课题组已报道的CYP86MF基因编码的氨基酸保守区域设计特异引物,运用PCR技术分别从十字花科6个属11个物种中分离克隆到了CYP86MF基因的同源序列,经比较分析,结果表明：这些同源序列的相似性达80%以上,所推导的氨基酸序列相似性达70%以上,且两者种间差异分别为1.0% ~ 5.7%和2.6% ~ 7.3%,属间差异分别是5.6% ~ 22.5%和7.3% ~ 31.2%;由氨基酸序列构建的分子系统树可知,在亲缘进化关系上芸薹属与萝卜属较近,其他依次为蔊菜属、拟南芥属、荠菜属,而与诸葛菜属最远。因此,CYP86MF基因的核苷酸及其可能编码的氨基酸序列差异属间较种间大,它可用于属间的分类等级研究,而难用于属以下的分类等级研究。     

甜瓜乙烯信号转导途径关键因子基因CTR1的克隆及表达特性分析

在拟南芥中,CTR1在乙烯信号转导途径中发挥重要的负调控作用.目前的研究表明,在拟南芥中只存在一个CTR1基因.利用RT-PCR技术从甜瓜果实中克隆得到CTR1基因cDNA(Cm-CTR1).Cm-CTR1cDNA全长2 613 bp,编码870个氨基酸.氨基酸序列比对和同源性分析表明,所克隆的甜瓜CTR1基因与拟南芥CTR1基因相似度为53.69％.Cm-CTR1的C-末端具有明显的类似于哺乳动物和果蝇中的Raf( retro-viral protein,V-raf)蛋白激酶家族的丝/苏氨酸蛋白激酶(Serine/threonine protein kinase)的特征.该结构具有所有已知蛋白激酶所共有的11个亚结构域,其中包括ATP结合位点和丝/苏氨酸蛋白激酶位点结构域,说明CmCTR1与其他植物CTR1相似.实时荧光定量PCR分析结果表明,从授粉后第15天到第20天,甜瓜果实Cm-CTR1基因表达量显著增加,第20天的表达量是第15天的2.5倍,之后表达水平趋于稳定,第40天到第45天表达量有所下降.     

模式匹配分析方法检索免疫球蛋白样功能区

运用计算机进行核酸和蛋白质的序列分析是分子生物学研究的一个较新发展,这项技术已越来越多地用于研究大量积累的序列数据。蛋白质功能区是蛋白质分子中能独立折叠成具有一定结构并执行特定功能的结构域,所有具有同一类功能区的分子统称为一个蛋白质的超族(protein superfamily)。本文通过对免疫球蛋白(Ig)超族及其功能区序列所进行的分析,建立了一种根据功能区之保守片段残基组成的模式匹配分析检索蛋白质功能区的方法,它先根据多序列的对准比较确定某一类功能区之保守片段,再对已知的保守片段各位置上氨基酸残基组成进行统计分析,然后根据与统计数值相匹配的方法,计算待检序列残基组成的统计学意义,由此确定功能区的存在。该方法的优点在于它不仅可以检出已知的具有某一类功能区的分子,而且还可能发现新的具有该功能区的分子,从而推测后者的功能。     

菜心苯丙氨酸解氨酶基因克隆与序列分析

通过同源克隆和RACE相结合的方法,首次从菜心中克隆了苯丙氨酸解氨酶(PAL)基因的全长cDNA,命名为BcPAL1(GenBank登录号为GU245694).BcPAL1全长2 476 bp,开放阅读框2 166 bp,编码721个氨基酸.经氨基酸序列多重比较发现,BcPAL1编码的氨基酸序列与拟南芥、烟草、甘蓝型油菜等植物的PAL氨基酸序列同源性大于90%,BcPAL1编码的氨基酸序列包含与拟南芥、烟草PAL蛋白质相同的脱氨基位点和催化活性位点.基于氨基酸序列的PAL系统进化树分析结果显示,BcPAL1编码的氨基酸序列与十字花科类植物(如拟南芥)的PAL聚为一支,说明两者的亲缘关系较近.     

玉米精氨酸甲基转移酶蛋白家族生物信息学分析

蛋白质精氨酸甲基转移酶(protein arginine methyltransferases,PRMTs)是真核生物基因组中重要的表观遗传调控因子之一,主要参与组蛋白精氨酸位点甲基化修饰,改变真核基因组的染色质结构,对基因的表达进行调控。本研究鉴定了8个玉米的PRMT蛋白序列,通过与两种模式植物(拟南芥和水稻)的全部PRMT蛋白序列的同源比对和系统发生关系分析,确定玉米PRMT蛋白主要分布在3个不同的亚家族中。运用生物信息学的方法和软件预测和分析了全部玉米PRMT蛋白氨基酸序列的理化性质、信号肽、跨膜结构域、疏水性和亲水性,以及蛋白质二级及三级结构等重要参数。这些数据对后续鉴定玉米PRMT蛋白的功能具有重要的意义。     

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).