NaHCO3胁迫下紫杆柽柳一些基因的表达

应用差异显示技术研究了NaHCO3胁迫下紫杆柽柳(Tamarix androssowii)基因的表达.经Northern检测共获得了17个基因片段,BLASTX分析表明,有2个基因与编码F-box类蛋白家族成员的基因同源性较高(F-box蛋白的功能为调节细胞周期转变、转录调控和信号转换,在植物抗逆防御系统中起重要作用);1个基因与翻译起始因子eIF成员有高的同源性(eIF在植物和酵母的抗盐胁迫中起重要作用);2个与分泌型过氧化物酶和过氧化物酶基因同源性高的基因片段;5个与盐碱胁迫密切相关的新基因,它们在胁迫前后差异表达明显.另外7个与已知基因同源性较高或有一定相似性,它们在抗盐碱胁迫中的作用尚待研究.     

雪里蕻非特异脂质转运蛋白基因的克隆与分析

非特异脂质转运蛋白是植物生命活动中一类重要的活性蛋白,这类蛋白在植物的抗性和防御中行使着重要的功能。近年来研究发现这类蛋白还与植物的有性生殖密切相关。通过已得到的普通白菜的脂质转运蛋白基因BcMF15的核苷酸序列,在其基因全长两侧设计引物,从雪里蕻中克隆得到该类活性蛋白基因,命名为BjLTP (登陆号: EU082009)。该基因全长650bp,不含内含子。不同组织的表达特征分析发现,BjLTP在雪里蕻的花蕾、开放的花中特异表达,推测BjLTP可能与花粉的发育有关。蛋白质特征预测及蛋白序列结构分析发现BjLTP是一个跨膜蛋白,具有显著的疏水区。序列同源比对表明该基因与白花芥蓝、拟南芥等的脂质转运蛋白基因有很高的相似性,证明BjLTP是LTP家族的成员之一。     

植物脂质转运蛋白的研究进展

高等植物脂质转运蛋白(lipid-transfer proteins,LTP)是一类小分子(约9 ku)的碱性蛋白质,已从多种植物中纯化出了LTP,且编码LTP的cDNA及基因也从不同植物中克隆.LTP能够在生物膜之间转运磷脂,因而认为LTP参与了细胞内生物膜形成.而近期的研究又发现LTP具信号肽,可从细胞内分泌到细胞外,位于细胞壁上,因而又对其在细胞内的转运脂质能力产生疑问.而有证据表明LTP参与了角质与腊质的形成、植物的抗病反应和植物对环境变化（温度、盐、干旱协迫）的适应.     

柽柳金属硫蛋白基因的克隆及序列分析

用木麻黄(Casuarina glauca)的金属硫蛋白基因(metallothionein 1)氨基酸序列对柽柳ESTs序列本地数据库进行tBlastn检索,获得了柽柳金属硫蛋白基因全长cDNA序列,去除polyA后该基因全长366 bp,其中5′非翻译区97 bp,3′非翻译区59 bp,开放读码框(ORF)长210 bp,编码70 个氨基酸组成的多肽,蛋白分子量为6.793 kD,理论等电点为4.99,含10个Cys,集中分布在肽链的N端和C端。BlastP同源性分析表明该基因与花生同源性最高,与小豆同源性最低。该基因的EST序列在GenBank登录(登录号：CV792539)。     

紫杆柽柳MnSOD基因在非生物胁迫中的作用

构建了酵母表达载体pYES2-MnSOD,并将其转入酿酒酵母INVSc1,以转空载体pYES2的酵母INVSc1为对照,检测其抗NaCl、Na2CO3、NaHCO3和紫外线胁迫能力。结果表明,转MnSOD基因酵母在以上逆境因素胁迫下的存活率有明显提高,说明MnSOD基因具有较强的抗NaCl、Na2CO3、NaHCO3和紫外线胁迫能力。     

植物非特异脂质转运蛋白研究现状与展望

植物非特异脂质转运蛋白(nsLTP)是一类含量丰富的小分子碱性蛋白, 能够在体外与多种疏水分子可逆地结合。目前已从多种植物中分离到9种类型的nsLTP基因。所有nsLTP蛋白质都具有8个半胱氨酸残基模体的保守结构, 它们的三维结构内部有一个具有脂质结合位点的疏水腔。根据基因结构、表达、调控和体外活性等研究, nsLTP被认为可能与蜡质合成与运输、抗逆、抗病以及生殖发育等重要生理过程有关。文章全面介绍nsLTP基因及其蛋白质研究的最新进展, 内容包括基本特征、分类、基因表达、基因克隆与功能研究等, 最后对今后的研究方向进行了讨论和展望。     

红花脂质转运蛋白基因的克隆及表达分析北大核心CSCD

克隆红花脂质转运蛋白基因(Lipid transfer protein,LTP),并进行生物信息学及表达分析,旨为研究LTP在红花抵抗逆境胁迫中的作用提供依据。通过RT-PCR方法从红花种子中克隆LTP基因序列,通过生物信息学对该基因蛋白的特征进行分析,构建LTP与相关物种LTP的系统进化树,利用Real-time PCR方法分析在红花不同号组织中LTP基因的表达量。结果显示,LTP基因ORF全长294 bp,编码97个氨基酸,相对分子量为7.46 kD,等电点为8.91。红花LTP蛋白包含一个长为29个氨基酸残基的信号肽序列;该蛋白含有一个丝氨酸磷酸化位点;三级结构预测表明该蛋白是由3个α-螺旋和一个β-转角简单的缠绕在无规则卷曲上的简单结构。分子进化表明,红花LTP基因与十字花科的甘蓝型油菜进化关系最近。通过荧光定量PCR对红花LTP基因的组织表达特异性进行分析,结果表明Ct LTP在不同组织的表达水平具有显著差异,在种子和花中呈现高表达,而在其他组织中低表达。     

茶树硝酸盐转运蛋白基因的克隆和表达分析

硝酸盐转运蛋白（NRT）是植物吸收和利用硝态氮的一种关键蛋白。运用RACE技术从茶树中扩增出NRT基因的cDNA,并利用实时荧光定量PCR检测了CsNRT基因在不同茶树器官与品种之间的差异表达。结果表明：CsNRT基因的cDNA全长2 061 bp,开放阅读框为1 818 bp,编码含由605个氨基酸组成的蛋白质,GenBank登录号为KJ160503,属于NRT2基因家族。CsNRT为组成型基因,对不同处理的水培茶苗进行定量表达分析显示,该基因在根、茎、叶中都有表达,其中在根部的表达水平最高,1.0 mmol·L-1的NO3-可诱导其表达量上升7.53倍。不同茶树品种中CsNRT基因的表达也有较大差异,‘龙井长叶’和‘凫早2号’的表达量较高,前者强烈响应0.5和1.0 mmol·L-1 NO3-的诱导,后者的响应浓度为1.0和2.0mmol·L-1,而‘舒茶早’在各浓度下的表达差异不明显。     

白花柽柳质膜水孔蛋白基因克隆及序列分析

植物水孔蛋白在植物体内形成水选择性运输通道,在植物种子萌发、细胞伸长、气孔运动、受精等过程中调节水分的快速跨膜运输。有的水孔蛋白还在干旱胁迫应答中起重要作用。本文根据白花柽柳的PEG6000胁迫处理构建的SSH消减文库的水孔蛋白基因表达序列标签(EST),设计基因特异性引物进行5′RACE,克隆出一个1 043 bp的核苷酸序列。应用生物信息学软件进行分析,预测该序列编码287个氨基酸,具有6个跨膜区,有MIP家族信号序列SGXHXNPAVT,高等植物PIP高度保守序列GGGANXXXXGY和TGI/TNPARSL/FGAAI/VI/VF/YN,这是质膜水孔蛋白基因的典型的结构特征。经NCBI比对,与Arabidopsis thaliana (MIP-C),同源性达到95%,预测该蛋白的相对分子量是30.9KD,理论等电点是8.84。     

柽柳翻译起始因子(eIF-5A)基因的克隆及原核表达

根据柽柳cDNA文库中获得的eIF-5A基因片段,用RACE技术克隆出其全长cDNA序列.cDNA长度为799 bp,编码159个氨基酸.将该cDNA序列克隆到原核表达载体pET28a中,获得重组质粒pET28a-eIF5A.不同浓度NaCl胁迫下大肠杆菌(Escherichia coli)BL21(pET28a-eIF5A)比E.coli BL21(pET28a)有明显的抗盐性,前者菌株存活率在1.0 mo1·L-1NaCl盐胁迫下是后者的9.3倍,据此认为E.coliBL21(pET28a-eIF5A)的耐盐性可能与eIF-5A基因的表达相关.该基因的GenBank登录号为AY587771(基因)、AAT01416(蛋白).     

An Antifungal Protein Purified from Pearl Millet Seeds Shows Sequence Homology to Lipid Transfer Proteins

In the course of a search for antifungal proteins from plant seeds, we observed inhibition of mycelial growth of Trichoderma viride with extracts of pearl millet. We have identified several proteins with antifungal properties in the seeds of pearl millet. One of these proteins has been purified to homogeneity and characterized. The purified protein has a molecular mass of 25 kDa. The N-terminal sequence of the protein (25 residues) shows homology to non-specific lipid transfer proteins (LTPs) of cotton, wheat and barley. The purified LTP inhibited mycelial growth of T. viride and the rice sheath blight fungus, Rhizoctonia solani in vitro.     

柽柳(Tamarix androssowii)Tadir基因的克隆及分析

在柽柳cDNA文库测序中获得了Tadir基因的全长cDNA序列,去除PolyA后,该基因全长724bp。其中5′非翻译区26bp,3′非翻译区143bp,开放阅读框(ORF)长555bp,编码184个氨基酸。基因编码蛋白的分子量为19.69kD,理论等电点为6.96。疏水性分析表明,蛋白的前41个氨基酸为亲水性的。该基因的Genbank登录号为DQ462418(基因),ABE73781(蛋白)。实时荧光定量PCR分析结果表明,0.4mol·L-1NaCl和NaHCO3胁迫后该基因表达量发生变化,其可能与柽柳的耐盐性有关。     

Distinct Unfolding and Refolding Pathways of Lipid Transfer Proteins LTP1 and LTP2

Plant non-specific lipid transfer proteins (ns-LTPs) comprise two families, LTP1s and LTP2s, all structurally stabilized by four native disulfide bonds. Solution and crystal structures of both LTP1s and LTP2s from various plants have been determined. Despite the similarities of their biological function and backbone folds, the biophysical properties of LTP1s and LTP2s differ significantly. In this report, the mechanisms of unfolding and refolding of rice LTP1 and LTP2 have been investigated using the technique of disulfide bonds scrambling. LTP1 is shown to unfold and refold via predominant species of partially structured intermediates. Four isomers of partly unfolded and extensively unfolded LTP1 were identified, isolated and their disulfide structures were determined. By contrast, unfolding and refolding of LTP2 adopt a (close to) two-state mechanism, and undergo a reversible conversion between the native and a single extensively unfolded isomer without accumulation of any significant intermediate.     

巴西橡胶树磷脂酰肌醇转移蛋白cDNA的克隆及其序列分析

本文从巴西橡胶树(Hevea brasiliensis)差减cDNA文库中筛选到一个与磷脂酰肌醇转移蛋白(phos-phatidylinositol transfer protein)同源性较高的基因片段,并根据该基因片段序列信息,设计特异性引物,采用cDNA末端快速扩增技术RACE(rapid amplification of cDNA ends)进行差异片段的5'和3'端的扩增,并获得长度为1081bp的全长cDNA克隆R291(GenBank登陆号:AY589690)。序列分析表明,该基因包含702bp的开放阅读框,编码234个氨基酸,推测其蛋白质的分子量为26.8kD,等电点为6.51,有一个的跨膜螺旋区(氨基酸位点为83～103)。R291基因含有一个脂质结合保守区(Sec14p-like lipid-binding domain),具有CRAL-TRIO脂质结合结构域,推测该基因是一个磷脂酰肌醇转移蛋白基因。该基因的克隆将为橡胶树磷脂酰肌醇代谢的研究奠定了基础,将有助于进一步了解磷脂酰肌醇代谢与胶乳再生之间的关系。     

粗糙脉孢菌基因组分泌蛋白的初步分析

文章报道利用信号肽预测软件SignalP v3.0和PSORT,跨膜螺旋结构预测软件TMHMMv2.0和THUMBUP,GPI-锚定位点预测软件big-PI Predictor和亚细胞器中蛋白定位分布预测软件TargetP v1.01对粗糙脉孢菌全基因组数据库中已公布的10 082个氨基酸序列进行预测分析。结果表明在粗糙脉孢菌中有437个蛋白为分泌蛋白,编码这些蛋白最小的可读框（open reading frame,ORF）为252 bp,最大为6 604 bp,平均1 433 bp,分泌蛋白信号肽长度介于15~59个氨基酸之间。在437个分泌蛋白中,205个具有功能描述,主要包括各种酶类、细胞能量生成、运转以及自身修复、防卫等多种功能。这些蛋白所参与的生化过程可能发生在膜外的周质空间或是菌体外的场所,为该物种营养的摄取,以及对环境做出响应服务。      

Similar Organization of the Lipopolysaccharide-Binding Protein (LBP) and Phospholipid Transfer Protein (PLTP) Genes Suggests a Common Gene Family of Lipid-Binding Proteins

The transfer of lipids in aqueous environments such as serum has been attributed to a recently characterized class of proteins. Abnormal regulation of serum lipids by these proteins is thought to be a key event in the pathophysiology of cardiovascular diseases. Lipopolysaccharide (endotoxin) binding protein (LBP) was identified by virtue of its ability to bind bacterial lipid A. We have analyzed the exon–intron organization of the LBP gene and the nucleotide sequence of its approximately 20 kb spanning 5′- and 3′-untranslated regions. When comparing the genomic organization of LBP with that of two other genes coding for lipid transfer proteins, significant homologies were found. The LBP gene includes 15 exons, and the 2-kb promoter contains recognition elements of acute phase-typical reactants and a repetitive 12-mer motif with an as yet unknown protein-binding property. Detailed sequence comparison revealed a closer relatedness of LBP with PLTP than with CETP as demonstrated by an almost identical intron positioning. This high degree of similarity supports functional studies by others suggesting that like LBP, PLTP may also be able to bind and transport bacterial lipopolysaccharide.     

Modulation of Src Homology 3 Proteins by the Proline-Rich Adaptor Protein Shb

In the present study we have investigated a possible role for the proline-rich SH2 domain protein Shb as a regulator of expression or activity of certain SH3 domain proteins and MAP kinase. The expression of the Shb binding proteins Eps8, Src, and p85 PI3-kinase, PI3-kinase activity, and MAP kinase activation were assessed in wild-type NIH3T3 cells and in NIH3T3 cells overexpressing the Shb cDNA. In addition, the expression of the SH3 domain STAT1 proteins was assessed in wild-type and Shb overexpressing cells. The Eps8 protein content and Eps8 mRNA steady-state levels were downregulated, whereas the protein contents of Src and p85 PI3-kinase were unaffected by Shb overexpression. There was, however, an increased basal PI3-kinase activity in Shb transfected cells after a 3-h serum starvation. Increased steady-state levels of STAT1 mRNA were accompanied by an increased STAT1 protein content in Shb overexpressing cells. Shb overexpression was not associated with an altered activation of p44 or p42 MAP kinases in response to PDGF stimulation. The data presented in this study suggest novel functions for the adaptor protein Shb regulating the expression of certain signal-transducing SH3 domain proteins and modulating PI3-kinase activity.     

Direct Molecular Fishing of Protein Partners for Proteins Encoded by Genes of Human Chromosome 18 in HepG2 Cell Lysate

Russian Journal of Bioorganic Chemistry - The aim of this work was to identify the spectrum of possible protein-protein interactions (PPI) for six target proteins (CYB5A, RAB27B, SMAD4, CXXC1,...