人乙醛脱氢酶2的原核表达及其条件优化

为实现人乙醛脱氢酶2(ALDH2)基因在原核生物中高效表达,将含有6×His标签和SUMO融合蛋白标签的人乙醛脱氢酶2基因的表达载体转化至宿主菌BL21(DE3)中。在异丙基硫代-β-D-半乳糖苷(IPTG)诱导下,目的基因在大肠杆菌内高效表达。通过对表达条件的优化,37℃使用终浓度0.3mmol/L的IPTG诱导3h,重组大肠杆菌的表达量可占全菌蛋白的16%。SUMO融合蛋白标签的加入以及较低的诱导温度(16℃)有利于提高人乙醛脱氢酶2基因在大肠杆菌内的可溶性表达。     

通粳1号水稻乙醛脱氢酶基因的克隆及原核表达

目的：克隆通粳1号水稻乙醛脱氢酶（aldehyde dehydrogenase,ALDH）基因,并在大肠杆菌中进行体外表达。方法：提取通粳1号水稻幼根总RNA,RT-PCR法扩增ALDH基因开放阅读框架片段,双酶切后连接至pET5a表达质粒中,转化BL21（DE3）宿主菌,平板培养筛选,挑取阳性菌落并提取质粒,酶切、电泳鉴定插入片段并测定其序列。含重组质粒的BL21（DE3）进行常规LB扩大培养,用不同浓度的IPTG作用不同时间进行诱导表达,SDS-PAGE检测表达产物。结果：质粒中插入的ALDH片段为1668bp,含有完整的开放阅读框架,序列与GenBank比较无差异,插入方向正确无误,表达蛋白分子量为61.8kDa左右,符合预期值,IPTG最佳诱导时间为3h,0.1mmol/L～0.8mmol/L浓度的IPTG作用效果无显著差异。结论：该基因的成功克隆和表达为进一步研究水稻中ALDH的作用和应用生物工程法大量获得ALDH奠定基础。     

植物醛脱氢酶在逆境胁迫中的研究进展

干旱、盐和病虫害等逆境胁迫已成为制约植物生长和作物产量的主要因素。植物在生长过程中进化出从形态到生理的一系列机制以缓解胁迫对自身的损害。逆境胁迫下醛类物质的富集会产生一系列的过氧化链式反应,危害细胞膜系统正常生理功能。过量的醛也会与蛋白质和核酸反应,破坏蛋白质和核酸的正常结构和功能,甚至直接导致植物死亡。植物体内醛脱氢酶基因(Aldehyde dehydrogenase,ALDH)在胁迫诱导条件下表达水平增加,大量累积的醛脱氢酶蛋白(ALDHs)将醛物质氧化成相应的羧酸,减少脂类物质的过氧化,参与到植物对生物及非生物的胁迫以及植物的发育调节。从ALDH的分类、功能及作用途径展开详细论述。     

醛脱氢酶基因敲除的K.pneumoniae重组菌的构建

在利用KlebsiellapneumoniaeM5aL厌氧发酵甘油生产1,3丙二醇的过程中,一部分甘油通过氧化代谢途径经醛脱氢酶(ALDH)催化合成大量副产物乙醇,降低了1,3丙二醇的产量和得率。首次以醛脱氢酶ALDH为改造目标,利用同源重组技术获得了ALDH基因敲除的K.pneumoniae重组菌。首先,采用PCR的方法分别从K.pneumoniaeM5aL基因组DNA及质粒pBR322上扩增得到ALDH基因和四环素抗性基因(Tcr);然后将ALDH基因定向插入到质粒pUC18的多克隆位点得到中间载体pUC18ALDH,该载体与Tcr基因分别用AvaI和BsaAI双酶切后进行连接,得到ALDH基因敲除的重组载体pUCAT;经DNA测序及限制性酶切电泳分析,构建的载体由5′ALDHTcr3′ALDHpUC18组成,与设计结果相符;最后,利用该载体通过同源重组技术得到两株K.pneumoniae重组菌06231hb及06231hc,经菌落PCR及酶活鉴定,两株菌的ALDH基因均已缺失。与出发菌株K.pneumonaieM5aL相比,重组菌的乙醇合成浓度降低了43%～53%,1,3丙二醇合成浓度提高了27%～42%。     

大豆醛脱氢酶基因GmALDH3-1的克隆及在生殖器官中的表达

通过基因芯片技术,从大豆中鉴定了一个花优势表达基因,其在大豆花中的表达量为叶片中的85倍。通过生物信息学方法,拼接了该基因的全长序列,并通过RT-PCR克隆了该基因。BLAST检索分析表明该基因编码醛脱氢酶,命名为GmALDH3-1。GmALDH3-1包含一个1485 bp的开放阅读框,编码494个氨基酸残基。GmADLH3-1与白杨的醛脱氢酶PtALDH3相似性最高（氨基酸相似率83%,一致率为68%）,而与来自于人的ALDH3B的氨基酸一致率和相似率分别为39%和59%。系统发生分析表明GmALDH3-1与其它植物ALDH3亚家族成员位于一个分支,且与白杨PtALDH3和拟南芥AtALDH3F1亲缘关系最近。采用实时定量RT-PCR检测了GmALDH3-1基因在大豆叶、根和花中的表达,结果表明GmALDH3-1基因在花中高丰度表达,在根和叶中未检测到表达。运用基因芯片信息分析了GmALDH3-1在种子发育过程中的表达情况,结果表明GmALDH3-1在种子发育过程中的外表皮、内表皮、外胚珠和种脐中表达量较高。文章讨论了GmALDH3-1基因在大豆生殖器官发育中可能发挥的作用。     

小麦TaMBD2原核表达载体的构建和诱导表达

构建了小麦甲基结合域蛋白基因TaMBD2的原核表达载体pGEX-TaMBD2,并在大肠杆菌BL21(DE3)工程菌株中优化了融合蛋白GST-TaMBD2的诱导表达条件.结果表明,用0.3、0.5和1.0 mm01.L-1的IPTG诱导后,融合蛋白GST-TaMBD2均能有效表达,以1.0mmol·L-1IPTG诱导的效果最好;从诱导表达的时间来看,3种浓度IPTG诱导1 h后融合蛋白均开始表达,且表达量随着诱导时间的延长而逐渐增加,但在诱导6 h后的表达量增加幅度不大,因此确定诱导融合蛋白GST-TaMBD2表达的最佳IPTG浓度为1.0 mm01.L-1诱导时间为6 h.     

pGEX载体表达马立克氏病病毒囊膜糖蛋白gI基因的最佳条件

通过聚合酶链式反应 (PCR) ,扩增出马立克氏病病毒特超强毒 (vv +MDV) 648株囊膜糖蛋白gI基因 ,并将该基因按正确的阅读框 (ORF)克隆到表达性载体质粒pGEX 6P 1中谷胱甘肽转移酶 (GST)基因的下游。重组质粒 (pGEX gI)经氯化钙转化宿主菌BL2 1 ;通过建立重组菌生长时间与OD60 0 值间的关系曲线 ,以及对诱导时间、诱导温度、IPTG浓度等条件的摸索 ,根据聚丙烯酰胺凝胶电泳 (SDS PAGE)判定GST gI融合蛋白的最佳表达条件 ,并经蛋白质印迹试验 (WesternBlotting)对表达产物进行了验证。将表达产物免疫小鼠 ,所得抗血清能与MDV感染的鸡胚成纤维细胞 (CEF)在间接免疫荧光试验 (IFA)中 ,呈较强的细胞膜荧光着色。实验结果表明 :IPTG的最佳浓度为 0 2～ 0 5mmol L ;最适的诱导时期为重组菌生长对数中期 ;温度对表达几乎没有影响。pGEX载体表达的融合蛋白至少保留了天然蛋白的部分抗原性     

Pseudomonas putida S1海藻糖合成酶表达质粒的构建及诱导条件优化

采用PCR方法从Pseudomonas putida S1中克隆出编码海藻糖合成酶的基因treS,并与质粒pQE30T相连,构建了表达质粒pQE—TS2。将此重组质粒转化宿主菌E．coliM15进行诱导表达。十二烷基磺酸钠-聚丙烯酰胺凝胶SDS—PAGE电泳结果表明,treS基因在大肠杆菌中获得了高效表达。通过对诱导温度、诱导剂浓度、加诱导剂时间和诱导时间的优化研究,在菌液生长至OD600值为0．6时,加入诱导剂IPTG至终浓度0．01mmol／L,20℃诱导20h,蛋白的表达量达到每克干细胞89mg的蛋白,粗酶液酶活达到19U／mL。     

鱼腥藻PCC7120中琥珀酸半醛脱氢酶的初步表征及结构分析

蓝藻琥珀酸半醛脱氢酶催化琥珀酸半醛到琥珀酸的转化,使得蓝藻的三羧酸循环变得完整。BLAST序列分析预测鱼腥藻PCC7120中all3556基因编码琥珀酸半醛脱氢酶。为了证实all3556基因编码蛋白的催化功能,构建了p ET28a-all3556表达质粒,并在大肠杆菌BL21(DE3)菌体中进行重组蛋白诱导表达;利用镍亲和层析方法对all3556蛋白进行了分离纯化。酶动力学测试表明all3556蛋白是一个NADP+-依赖型的琥珀酸半醛脱氢酶。生物信息学分析发现all3556蛋白和其他来源的琥珀酸半醛脱氢酶的氨基酸序列具有一定的同源性,在催化中心的氨基酸残基高度保守。     

龟裂链霉菌RsigB基因提高大肠杆菌对环境胁迫的耐受性

龟裂链霉菌RsigB是与天蓝色链霉菌与枯草芽孢杆菌中的sigmaB基因同源的一个σ因子,其也可能为一种全局压力调控蛋白,在菌体遇到生长环境改变时起着重要的调控作用。本文以龟裂链霉菌基因组为模版,PCR扩增出RsigB基因,以pET28a为载体,构建重组质粒pET28aRsigB,并且转化至Escherichia coli BL21(DE3)中。将对照菌与重组子置于不同环境胁迫条件下培养,并用IPTG诱导蛋白表达,测其生长曲线,研究RsigB对于大肠杆菌对环境胁迫的耐受性的作用。结果表明,RsigB在大肠杆菌中的表达能够提高其对温度,高渗透压以及氧化还原压力的耐受性。RsigB的成功表达以及对于逆境的耐受性能为龟裂链霉菌中RsigB因子的功能以及机理研究提供实验基础。     

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