小麦Na+/H+反转运蛋白基因的克隆和特性

以水稻(Oryza sativa L.) Na+/H+反转运蛋白cDNA片段为探针,从小麦盐胁迫cDNA文库中筛选和克隆了2个小麦Na+/H+反转运蛋白基因,分别命名为TaNHX1 和 TaNHX2.序列分析表明TaNHX1为2 029 bp,包含一个完整的1 638 bp的ORF,编码546个氨基酸,其中含有DIFFIYLLPPI跨膜区.TaNHX2为1 693 bp,包含部分ORF及808 bp的3′-UTR.这2个基因与已知的水稻、拟南芥(Arabidopsis thialiana)和滨藜(Atriplex gmelini)中的同类基因NHX的相似性约为70%.RT-PCR分析表明小麦苗经400 mmol/L NaCl处理1 h后,TaNHX1的转录水平有所提高.     

中间偃麦草Na+/H+逆向转运蛋白的分子克隆及生物信息学分析

根据小麦液泡膜Na /H 逆转运蛋白基因TaNHX1的全长序列设计引物,通过RT-PCR直接扩增的方法从中间偃麦草(Elytrigia intermedia)中克隆到了TaNHX1的同源基因,命名为TiNHX1(Acession Numeber:EF409418).TiNHX1最大开放阅读框为1 641 bp,编码含有546个氨基酸残基、分子量为59.8 kDa的蛋白,预测等电点8.0.TiNHX1含有38个碱性氨基酸,36个酸性氨基酸,256个疏水氨基酸及129个极性氨基酸.二级结构预测表明该蛋白含约44%的a-螺旋、21%的p-折叠、4%的p-转角和29%的不规则卷曲.亲疏水性分析显示,TiNHX1含有12个连续的疏水片断,其中10个可能构成穿膜螺旋.序列分析显示,TiNHX1与小麦(Triticum aestivum)、长穗偃麦草(Elytrigia elongate)、水稻(Oryza sativa)、小盐芥(Thellungiella halophila)、拟南芥(Arabidopsis thaliana)等植物的液泡膜Na /H 逆向转运蛋白高度同源,序列相似性分别为97%、96%、85%、68%、67%.序列比对结果以及进化树分析均表明TiNHX1应为定位于中间偃麦草液胞膜上的Na /H 逆向转运蛋白.     

RLM-RACE法扩增獐茅液泡膜Na^＋/H^＋逆向转运蛋白基因5′-cDNA末端序列

根据已获得的盐生植物獐茅（Aeluropus littoralisvar.sinensisDebeaux）液泡膜Na^＋/H^＋逆向转运蛋白（Na＋/H＋antiporter）基因部分cDNA片段,设计2条特异引物（GSP1、GSP2）,采用RLM-RACE（RNA ligase-medi-ated rapid amplification of 5′and 3′cDNA ends）法获得了其5-′cDNA末端序列,并找出了转录起始位点。该片段长816 bp,与芦苇液泡膜Na^＋/H^＋逆向转运蛋白基因序列同源性最高达91%,与拟南芥、盐角草、水稻、大麦、小麦的同源性分别为81%、82%、86%、87%和81%,为该基因全长的克隆及启动子的研究奠定了基础。与其它测定基因转录起始位点的方法相比,RLM-RACE方法更快捷、简便、准确。     

新疆无苞芥Na~+/H~+逆向转运蛋白基因OpNHX1的克隆、表达分析与功能验证

从耐盐植物无苞芥中克隆获得了1个Na+/H+逆向转运蛋白基因NHX1,命名为OpNHX1(GenBank登录号:KC200248)。OpNHX1基因cDNA全长2 153 bp,包含一个1 605 bp的开放阅读框,编码534个氨基酸。系统进化树分析表明,OpNHX1编码产物与拟南芥、小盐芥亲缘关系较近,属于同一进化分支。实时荧光定量PCR分析表明,该蛋白基因在无苞芥根、茎、叶、花和荚果中均有表达,其中茎中表达量最高。半定量RT-PCR分析表明,该基因受高盐、干旱、低温及ABA的诱导上调表达。进一步将该基因在拟南芥中过量表达,显著提高了转基因植株在盐胁迫下的存活率,说明OpNHX1基因参与了植物的耐盐性。酵母功能互补试验结果显示,该基因转化酵母Δnhx1后可以补充NHX1的缺失,表明OpNHX1参与Na+/H+的转运。     

小麦TaMAPK2基因的克隆及表达分析

该研究从小麦中克隆了1个MAPK基因TaMAPK2。序列分析表明,TaMAPK2基因的ORF为1 110bp,编码369个氨基酸。序列比对分析表明,该基因所编码的蛋白与粗山羊草、水稻、谷子等MAPK蛋白具有较高的一致性,分别为99%、94%、94%。进化树分析表明,TaMAPK2与水稻OsMAPK2的亲缘关系最近。实时荧光定量PCR分析表明,该基因的表达显著受渗透胁迫、低温胁迫、高盐胁迫、乙烯和双氧水诱导,受ABA抑制。研究表明,TaMAPK2可能参与非生物逆境胁迫及相关信号分子应答。     

小麦尿卟啉原Ⅲ合成酶基因克隆及序列分析

根据水稻已公布的尿卟啉原Ⅲ合成酶(UROS)基因和小麦EST的保守序列,设计特异性引物对小麦尿卟啉原Ⅲ合成酶基因的部分片段进行克隆,得到了364 bp的cDNA(命名为UROS1)。以UROS1作为种子进行电子克隆,得到一段长为1210 bp的cDNA序列,并设计特异性引物克隆到1个1077 bp cDNA序列。对该片段分析结果表明,克隆得到的小麦UROS基因包含了信号肽区和全长的成熟肽区。小麦UROS基因与水稻UROS基因的同源性为86%左右,其推导氨基酸序列与水稻和拟南芥蛋白序列同源性分别约91%和79%。动物、植物以及微生物间核酸序列的保守性较低,氨基酸序列保守性也不高,但都存在UROS保守结构域(Hem D)。进化分析显示,该酶在不同物种间的进化速度差异较大。     

小麦锌指蛋白基因TaLOL2的克隆及特征分析

通过电子克隆与RT-PCR相结合的方法,在条锈菌诱导的小麦叶片中克隆获得1个新的LSD1型锌指蛋白基因TaLOL2,并用qRT-PCR技术分析了其转录表达特征。结果显示:(1)小麦锌指蛋白基因TaLOL2的cDNA全长1 095bp,编码179个氨基酸。(2)TaLOL2含有3个典型的zf-LSD1型(CxxCxRxxLMYxxGASxVxCxxC)保守结构域,与水稻、拟南芥、大麦等植物LSD1型锌指蛋白序列具有高度相似性,其中与水稻OsLOL2相似度达86.0%。(3)进化树分析表明,TaLOL2与水稻、拟南芥和大麦中部分含有3个保守zf-LSD1锌指结构的基因亲缘关系较近,而与其它包含不同数目的zf-LSD1锌指结构的基因亲缘关系较远。(4)qRT-PCR定量分析表明,TaLOL2在条锈菌侵染前期呈上调表达,在亲和及非亲和反应中差异表达。研究表明,TaLOL2参与了条锈菌诱导的小麦抗病防卫反应,很可能作为正调控因子参与了小麦-条锈菌非亲和互作中对条锈菌的抗性信号途径。     

菊芋Na+/H+逆向转运蛋白基因的克隆与表达分析

根据同源序列设计简并引物,通过RT-PCR及RACE的方法从菊芋中克隆了Na /H 逆向转运蛋白基因。序列分析表明,该基因全长2148 bp,开放读码框为1647 bp,可编码长549个氨基酸的多肽,与其它植物已克隆的Na /H 逆向转运蛋白具有很高的同源性。系统发育分析表明该蛋白(HtNHX1)与液泡型Na /H 逆向转运蛋白的亲缘关系较近,与质膜型Na /H 逆向转运蛋白亲缘关系较远。NaCl胁迫条件下RT-PCR检测结果表明,HtNHX1随NaCl浓度增加和处理时间延长表达持续增强,但到了第3天表达量开始下降。HtNHX1逆向转运蛋白基因的转录调控可能是决定菊芋耐盐能力的一个重要因素。     

内蒙古白绒山羊erk2基因的克隆及表达模式分析

旨在克隆内蒙古白绒山羊erk2基因cDNA并分析其基本表达模式。采用RT-PCR方法克隆白绒山羊erk2基因cDNA。通过在线软件Blast进行核酸序列分析,用SMART与Psite进行氨基酸序列分析。定量RT-PCR检测erk2基因在绒山羊组织中的表达特异性。免疫组化法检测绒山羊睾丸中erk2表达。克隆到的内蒙古白绒山羊erk2基因cDNA片段 (GenBank Accession No.JX569765) 长1 083 bp,包含了编码360个氨基酸残基的全长ORF,氨基酸序列与牛的ERK2 (Bos Taurus BC133588.1) 同源性为100％。SMART分析表明,ORF编码的蛋白包含了活化位点“TEY”及具有丝氨酸/苏氨酸激酶催化活性的S-TKc结构域。Psite分析表明,含2个N-糖基化位点、1个依赖于cAMP/cGMP的蛋白激酶磷酸化位点、3个蛋白激酶c磷酸化位点、5个酪蛋白激酶Ⅱ磷酸化位点、2个N-豆蔻酰化位点、2个异戊二烯基结合区 (CAAX box)、7个微体羧基端靶向信号、2个蛋白激酶ATP结合区标记及一个丝/苏氨酸蛋白激酶活性区域标记。PSORT (k-NN prediction) 程序预测其定位于细胞质中。定量RT-PCR分析显示erk2基因mRNA丰度在心脏、皮肤以及乳腺组织中mRNA丰度较高,脾、肾中的表达相对较低。在睾丸中检测到ERK2蛋白表达。     

RLM-RACE法扩增獐茅液泡膜Na+/H+逆向转运蛋白基因5'-cDNA末端序列

根据已获得的盐生植物獐茅(Aeluropus littoralis var.sinensis Debeaux)液泡膜Na+/H+逆向转运蛋白(Na+/H+antiporter)基因部分cDNA片段,设计2条特异引物(GSP1、GSP2),采用RLM-RACE(RNA ligase-mediated rapid amplification of 5'and 3'cDNA ends)法获得了其5'-cDNA末端序列,并找出了转录起始位点.该片段长816 bp,与芦苇液泡膜Na+/H+逆向转运蛋白基因序列同源性最高达91%,与拟南芥、盐角草、水稻、大麦、小麦的同源性分别为81%、82%、86%、87%和81%,为该基因全长的克隆及启动子的研究奠定了基础.与其它测定基因转录起始位点的方法相比,RLM-RACE方法更快捷、简便、准确.     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

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.     

Synthesis and antimicrobial studies of hydrazone derivatives of 4-[3-(2,4-difluorophenyl)-4-formyl-1H-pyrazol-1-yl]benzoic acid and 4-[3-(3,4-difluorophenyl)-4-formyl-1H-pyrazol-1-yl]benzoic acid

Microbial resistance to antibiotics is an unresolved global concern, which needs urgent and coordinated action. One of the guidelines of the Centers for Disease Control and Preventions (CDC) to combat antibiotic resistance is the development of new antibiotics to treat drug-resistant bacteria. In our effort to find new antibiotics, we report the synthesis and antimicrobial studies of 30 new pyrazole derivatives. These novel molecules have been synthesized by using readily available starting materials and benign reaction conditions. Some of these molecules have shown activity with MIC values as low as 0.78?µg/mL against four bacterial strains; Staphylococcus aureus, methicillin-resistant S. aureus, Bacillus subtilis, and Acinetobacter baumannii. Furthermore, active molecules are non-toxic to mammalian cell line.

     

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.