棉花Gh4CL1基因克隆及表达

根据棉花纤维特异表达cDNA文库分析得到的4-香豆酸辅酶A连接酶基因EST序列设计引物,采用RT-PCR技术从棉花中克隆了1个4CL基因,命名为Gh4CL1(GenBank登录号为FJ479707).结果表明:Gh4CL1基因cDNA全长2 331 bp,具有1个1 722 bp的开放阅读框,5′非编码区为64 bp,3′非编码区为445 bp,编码573个氨基酸,预测分子量约为61.951 kD,等电点为5.70.氨基酸同源性分析发现,Gh4CL1与来自白杨、大豆和紫草的4CL同源性较高.半定量RT-PCR检测表明,Gh4CL1基因在不同发育阶段的棉纤维中均有表达,在开花后20 d的棉纤维中表达量最大,说明该基因可能参与调控棉纤维细胞的伸长和次生壁的增厚.Gh4CL1基因在棉花花瓣中表达量最高,在其他组织中低水平表达或不表达.     

柞蚕14-3-3基因的鉴定及表达分析

14-3-3蛋白是一种可以改变其结合蛋白构象的酸性蛋白质.柞蚕14-3-3 cDNA序列全长1 220 bp,包括一个126 bp的5'非编码区和一个350 bp的3'非编码区.该基因的开放读码框长度为744 bp,编码247个氨基酸.序列比对结果表明,柞蚕14-3-3蛋白与家蚕的14-3-3蛋白具有高度同源性.此外对柞蚕14-3-3基因进行了原核表达和重组蛋白纯化.SDS-PAGE和免疫印迹结果表明,分子量大小约32 kD的重组蛋白在大肠杆菌中得到了成功表达.     

14-3-3蛋白研究进展

14-3-3蛋白是高度保守的、所有真核生物细胞中都普遍存在的、在大多数生物物种中由一个基因家族编码的一类蛋白调控家族。它几乎参与生命体所有的生理反应过程,人们在各种组织细胞中发现了各种不同的14-3-3蛋白。作为与磷酸丝氨酸／苏氨酸结合的第一信号分子,14-3-3蛋白在细胞的信号转导中起着至关重要的作用,尤其是它直接参与调节蛋白激酶和蛋白磷酸化酶的活性,被称为蛋白质与蛋白质相互作用的”桥梁蛋白”;它可以与转录因子结合形成复合体,调节相关基因的表达。一些研究表明,14-3-3蛋白调控机制的紊乱可以直接导致疾病的发生,在临床上14-3-3蛋白常常可以作为诊断的标志物。     

菠菜14-3-3蛋白基因的克隆及硝酸盐胁迫下的表达分析

利用RT-PCR和RACE技术,从菠菜中首次获得了14-3-3蛋白基因的全长cDNA序列(GenBank登录号JX952165),命名为So14-3-3.该基因全长1 166 bp,开放阅读框801 bp,编码266个氨基酸.序列比对发现So143 3蛋白与其他植物14-3-3蛋白氨基酸序列一致性高达77.6％～84.7％.半定量RT-PCR表明,随NO3-胁迫处理时间的延长和浓度的增加,菠菜根和叶中So14-3-3基因的表达增强.实验构建了pGEX4T-So14-3-3原核表达载体,并通过IPTG诱导后获得分子量约为56 kD的蛋白.进一步的蛋白质印迹检测结果表明,随着NO3处理时间的延长和浓度的增加,So14-3-3蛋白表达也增加.该实验结果为进一步研究So14 3-3蛋白功能提供了基本的实验基础.     

14-3-3蛋白参与植物应答非生物胁迫的研究进展

14-3-3蛋白是一种在真核生物细胞中普遍存在且高度保守的蛋白。该蛋白在大多数物种中由一个基因家族编码,并以同源或异源二聚体的形式存在。不同的14-3-3蛋白同工型具有不同的细胞特异性,可通过识别特异的磷酸化或非磷酸化序列与靶蛋白相互作用。14-3-3蛋白在植物生长和发育的各个方面都起重要作用。本文主要围绕植物14-3-3蛋白的种类、结构、磷酸化或非磷酸化识别序列及其响应干旱、冷冻、盐碱、营养和机械胁迫等的分子机制研究进展进行综述。     

粘虫甘油醛-3-磷酸脱氢酶基因cDNA的克隆、序列分析及表达量检测

运用RT-PCR和RACE技术,以粘虫(Mythimna separata(Walker))cDNA为模板,对甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)基因进行克隆获得全长cDNA序列,并利用生物信息学方法,对GAPDH全长cDNA序列及推测得到的GAPDH蛋白序列进行分析.结果表明,获得的粘虫GAPDH基因cDNA序列长度为1 317 bp,其中包括80 bp的5′非编码区、238 bp的3′非编码区和999 bp的开放阅读框(Open Reading Frame,ORF),编码一个332个氨基酸蛋白,具有GAPDH蛋白家族的两个功能结构域.该GAPDH蛋白理论相对分子质量为35.498 6 kDa,等电点为7.63,富含6种类型的特定功能位点.该蛋白序列与其他动物GAPDH蛋白序列具有77.4%～92.9%高度同源性.GAPDH基因表达量检测结果显示GAPDH在粘虫6种不同组织间表达量无显著差异(P0.05),表明GAPDH可作为研究粘虫功能基因表达量分析的可靠内参基因.该基因的cDNA序列已经递交GenBank并获得登录号为HM055756.     

4个棉花ADF基因的分子鉴定及其差异表达

肌动蛋白解聚合因子(actin-depolymerizing factor, ADF)是一种在真核生物中广泛存在的低分子量的肌动蛋白结合蛋白,它在调控细胞内肌动蛋白纤丝的解聚合和再聚合中起着关键作用。我们在棉纤维cDNA文库中分离克隆了4个ADF基因(cDNAs),分别命名为GhADF2,GhADF3,GhADF4,GhADF5。GhADF2 cDNA 长度为705 bp,编码139个氨基酸;GhADF3 cDNA长度为819 bp,编码139个氨基酸;GhADF4 cDNA长度为804 bp,编码143个氨基酸;GhADF5 cDNA长度为644 bp,编码141个氨基酸。分析表明,GhADF2与GhADF3的氨基酸序列同源性为99%。而且,GhADF2/3与矮牵牛PeADF2之间的氨基酸序列同源性也高达89%。GhADF4与拟南芥AtADF6的亲缘关系较近,二者的氨基酸序列同源性为78%。GhADF5与拟南芥AtADF5的亲缘关系较近,氨基酸序列的同源性为83%。上述结果表明植物ADF基因在进化中具有高度保守性。RT-PCR分析表明,GhADF2在纤维中优势表达,而GhADF5基因则在子叶中表达量最高。另一方面,GhADF3和GhADF4似乎不具有组织特异性或偏爱性表达。同一组织中不同GhADF基因表达量有较大的差异,表明它们可能涉及棉花不同组织生长发育过程的调节。而且,在进化过程中,各ADF同分异构体之间可能发展形成某种功能上的差异性。     

陆地棉XTH基因家族全基因组鉴定及在纤维发育过程的表达分析

本研究从陆地棉TM-1基因组中鉴定出72个XTH家族基因,编码木葡聚糖内转糖苷酶/水解酶(XTH,xyloglucan endotransglycosylase/hydrolase),分别命名为Gh XTH01~Gh XTH72,分析了其基因结构、保守基序、系统进化、理化性质、亚细胞定位,并探究其在棉纤维发育不同时期的表达规律。结果表明,XTH家族基因分布在除At 07、Dt 07以外的24条棉花染色体上,根据系统发育树,将XTH家族基因分为3个亚组;XTH氨基酸序列有3个保守基序,保守性较强;多数XTH蛋白定位在细胞外。根据XTHs在纤维发育不同时期的表达量变化,将其分为4类。通过构建陆地棉与拟南芥XTH氨基酸序列进化树,推测Gh XTH15、Gh XTH28、Gh XTH36、Gh XTH49、Gh XTH59、Gh XTH62、Gh XTH63等基因在棉纤维发育过程中发挥重要作用。通过比较XTH家族基因在不同纤维品质陆地棉品种中的表达差异,推测在优质棉花品种中优势表达基因Gh XTH03、Gh XTH12、Gh XTH17、Gh XTH22、Gh XTH23、Gh XTH28、Gh XTH33、Gh XTH44、Gh XTH46、Gh XTH59等在纤维发育伸长过程中可能发挥着重要作用。上述结果为研究陆地棉XTH基因家族在棉纤维发育中的功能提供了参考依据。     

棉花4-香豆酸辅酶A连接酶基因克隆及原核表达

本研究从棉花中克隆了一个4CL基因,命名为Gh4CL2(GenBank登录号为FJ848870)。研究结果表明:Gh4CL2基因cDNA全长2332bp,具有一个1725bp的开放阅读框,5′非编码区为64bp,3′非编码区为543bp,编码574个氨基酸,预测分子量约为62.106kD,等电点为5.94。氨基酸同源性分析发现,Gh4CL2与来自白杨、大豆和紫草的4CL一致性较高。进一步研究Gh4CL2基因的功能,构建了该基因的原核表达载体pET-28a-4CL2,经酶切鉴定后转化到大肠杆菌BL21(DE3)中。SDS-PAGE分析表明,最佳诱导表达条件为0.5mmol/LIPTG在37℃下诱导4h,重组蛋白主要以包涵体形式出现。     

马氏珠母贝Caspase-3基因克隆和组织表达分析

半胱氨酸蛋白酶3 (Caspase-3)作为细胞凋亡通路中重要的效应蛋白,在细胞凋亡过程中发挥着重要作用.为初步探究马氏珠母贝Caspase-3(PmCaspase-3)的生物学功能,本研究利用cDNA末端快速扩增(RACE)技术克隆获得PmCaspase-3基因cDNA的全长序列并对其序列特征进行分析;同时利用实时荧光定量PCR (RT-qPCR)方法分析了PmCaspase-3基因mRNA在马氏珠母贝不同组织和不同发育时期的表达差异.结果 显示,PmCaspase-3基因cDNA全长为2233 bp,其中5'端非编码区长度为80 bp,3'端非编码长度为31 bp,开放阅读框长度为2088 bp,共编码695个氨基酸;生物信息学分析显示,PmCaspase-3含有Caspase家族特有的CASc结构域和半胱氨酸蛋白酶家族p20、p10活性位点以及多种磷酸化位点,经进化分析以及多序列比对可知与其他物种Caspase-3蛋白同源性较高.RT-qPCR结果表明,PmCaspase-3在肝胰腺中的表达量最高,在闭壳肌中的表达量最低;在发育过程中D型幼虫期和眼点期的表达量较高.     

Cloning and characterization of the 14-3-3 protein gene from the halotolerant alga Dunaliella salina

Previous studies have demonstrated that 14-3-3 proteins exist in all the eukaryotic organisms studied; however, studies on the 14-3-3 proteins have not been involved in the halotolerant, unicellular green alga Dunaliella salina so far. In the present study, a cDNA encoding 14-3-3 protein of D. salina was cloned and sequenced by PCR and rapid amplification of cDNA end (RACE) technique based on homologous sequences of the 14-3-3 proteins found in other organisms. The cloned cDNA of 1485 bp in length had a 29.2 kDa of molecular weight and contained a 774 bp of open reading frame encoding a polypeptide of 258 amino acids. Like the other 14-3-3 proteins, the deduced amino acid sequences of the D. salina 14-3-3 protein also contained two putative phosphorylation sites within the N-terminal region (positions 62 and 67). Furthermore, an EF hand motif characteristic for Ca²⁺-binding sites was located within the C-terminal part of this polypeptide (positions 208–219). Analysis of bioinformatics revealed that the 14-3-3 protein of D. salina shared homology with that of other organisms. Real-time quantitative PCR demonstrated that expression of the 14-3-3 protein gene is cell cycle-dependent.     

A Cdc2-related protein kinase hPFTAIRE1 from human brain interacting with 14-3-3 proteins

hPFTAIRE1 （PFTK1）, a Cdc2-related protein kinase, is highly expressed in human brain. It exhibits cytoplasmic distribution in Hela cells, although it contains two nuclear localization signals （NLSs） in its N-terminus. To search for its substrates and regulatory components, we screened a two-hybrid library by using the full-length hPFTAIRE1 as a bait. Four 14-3-3 isoforms （β,ε,η,τ） were identified interacting with the hPFTAIRE1. We found a putative 14-3-3 binding consensus motif（RHSSPSS） in the hPFTAIRE 1, which overlapped with its second NLS. Deletion of the RHSSPSS motif or substitution of Ser^119 gwithAla in the conserved binding motif abolished the specific interaction between the hPFTAIRE 1 and the 14-3 -3 proteins. The mutant S 120A hPFTAIRE1 also showed a weak interaction to the 14-3-3 proteins. The results suggested that the Ser^119 is crucial for the interaction between hPFTAIREI and the 14-3-3 proteins. All the hPFTAIRE1 mutants distributed in cytoplasm of Hela cells and human neuroblastoma cells （SH-SY5Y） when fused to the C-terminus of a green fluorescent protein （GFP）, indicating that binding with the 14-3-3 proteins does not contribute to the subcellular localization of the hPFTAIRE1, although the binding may be involved in its signaling regulation.     

Molecular characterization of a 14-3-3 zeta gene from Plodia interpunctella: A potential marker for phylogenetic inference

The 14-3-3 proteins are a large family of approximately 30 kDa acidic proteins and acting in the regulation of many biological processes. In this study, a 14-3-3 zeta (Pi14-3-3z) gene from the Indian meal moth, Plodia interpunctella (Lepidoptera, Pyralidae) was isolated and characterized. The full-length cDNA of Pi14-3-3z is 1382 bp, including a 5'-untranslated region (UTR) of 141 bp, 3′-UTR of 497 bp and an open reading frame (ORF) of 744 bp encoding a polypeptide of 247 amino acids which contains a 14-3-3 homologues domain (PF00244). The deduced Pi14-3-3z protein sequence has 81%–100% identity with the homologues in comparison to with other individuals. qPCR analysis revealed that Pi14-3-3z was expressed at the four developmental stages and in all tissues tested. Based on the amino acid of 14-3-3z, phylogenetic analysis demonstrated a similar topology with the traditional classification, suggesting 14-3-3z protein has the potential value in phylogenetic inference.     

两个小麦14-3-3基因的特征、亚细胞定位及其对非生物胁迫的响应(英文)

为了探讨14-3-3基因在小麦逆境胁迫应答中的调控作用,利用RACE技术克隆了两个包含完整编码框的14-3-3基因(命名为Ta14R1和Ta14R2),其中Ta14R1 cDNA长999 bp,编码262个氨基酸,而Ta14R2 cDNA长897 bp,编码261个氨基酸。Ta14R1/Ta14R2-GFP融合载体瞬时表达结果显示,Ta14R1和Ta14R2蛋白均定位于细胞质和细胞膜,但不在叶绿体中。荧光定量PCR分析表明,Ta14R1和Ta14R2均在萌发1 d的胚芽鞘中表达量最高;在高温、低温、模拟干旱和ABA处理下,两个基因在小麦的根和叶中都受胁迫诱导而且显著上调表达,推测这两个14-3-3基因通过依赖ABA的非生物胁迫响应途径发挥作用,可能参与了小麦中高温、低温和干旱胁迫的耐受调节过程。     

Cotton (Gossypium hirsutum) 14‐3‐3 proteins participate in regulation of fibre initiation and elongation by modulating brassinosteroid signalling

Cotton (Gossypium hirsutum) fibre is an important natural raw material for textile industry in the world. Understanding the molecular mechanism of fibre development is important for the development of future cotton varieties with superior fibre quality. In this study, overexpression of Gh14‐3‐3L in cotton promoted fibre elongation, leading to an increase in mature fibre length. In contrast, suppression of expression of Gh14‐3‐3L, Gh14‐3‐3e and Gh14‐3‐3h in cotton slowed down fibre initiation and elongation. As a result, the mature fibres of the Gh14‐3‐3 RNAi transgenic plants were significantly shorter than those of wild type. This ‘short fibre’ phenotype of the 14‐3‐3 RNAi cotton could be partially rescued by application of 2,4‐epibrassinolide (BL). Expression levels of the BR‐related and fibre‐related genes were altered in the Gh14‐3‐3 transgenic fibres. Furthermore, we identified Gh14‐3‐3 interacting proteins (including GhBZR1) in cotton. Site mutation assay revealed that Ser163 in GhBZR1 and Lys51/56/53 in Gh14‐3‐3L/e/h were required for Gh14‐3‐3‐GhBZR1 interaction. Nuclear localization of GhBZR1 protein was induced by BR, and phosphorylation of GhBZR1 by GhBIN2 kinase was helpful for its binding to Gh14‐3‐3 proteins. Additionally, 14‐3‐3‐regulated GhBZR1 protein may directly bind to GhXTH1 and GhEXP promoters to regulate gene expression for responding rapid fibre elongation. These results suggested that Gh14‐3‐3 proteins may be involved in regulating fibre initiation and elongation through their interacting with GhBZR1 to modulate BR signalling. Thus, our study provides the candidate intrinsic genes for improving fibre yield and quality by genetic manipulation.     

Subcellular localization of 14-3-3 proteins in Toxoplasma gondii tachyzoites and evidence for a lipid raft-associated form

A polyclonal antibody was raised against a Toxoplasma gondii 14-3-3-gluthatione S-transferase fusion protein obtained by cloning a 14-3-3 cDNA sequence determined from the T. gondii database. This antibody specifically recognized T. gondii 14-3-3 without any cross-reaction with mammalian proteins. Immunofluorescence microscopy studies of the tachyzoites or the T. gondii-infected cells suggested cytosolic and membranous localizations of 14-3-3 protein. Different subcellular fractions were prepared for electrophoresis analysis and immunodetection. 14-3-3 proteins were found in the cytosol, the membrane fraction and Triton X-100-resistant membranes. Two 14-3-3 isoforms were detected. The major one was mainly cytoplasmic and to a lesser extent membrane-associated, whereas the minor isoform was associated with the detergent-resistant lipid rafts.     

Molecular characterization and expression analysis of nine cotton GhEF1A genes encoding translation elongation factor 1A

The translation elongation factor 1A, eEF1A, plays an important role in protein synthesis, catalyzing the binding of aminoacyl-tRNA to the A-site of the ribosome by a GTP-dependent mechanism. To investigate the role of eEF1A for protein synthesis in cotton fiber development, nine different cDNA clones encoding eukaryotic translation elongation factor 1A were isolated from cotton (Gossypium hirsutum) fiber cDNA libraries. The isolated genes (cDNAs) were designated cotton elongation factor 1A gene GhEF1A1, GhEF1A2, GhEF1A3, GhEF1A4, GhEF1A5, GhEF1A6, GhEF1A7, GhEF1A8, GhEF1A9, respectively. They share high sequence homology at nucleotide level (71-99% identity) in the coding region and at amino acid level (96-99% identity) among each other. Phylogenetic analysis demonstrated that the nine GhEF1A genes can be divided into 5-6 subfamilies, indicating the divergence occurred in structures of the genes as well as the deduced proteins during evolution. Real-time quantitative RT-PCR analysis revealed that GhEF1A genes are differentially expressed in different tissues/organs. Of the nine GhEF1A genes, five are expressed at relatively high levels in young fibers. Further analysis indicated that expressions of the GhEF1As in fiber are highly developmental-regulated, suggesting that protein biosynthesis is very active at the early fiber elongation.     

Molecular dynamics and in vitro analysis of Connexin43: A new 14-3-3 mode-1 interacting protein

The interaction of cellular proteins with the gap junction protein Connexin43 (Cx43) is thought to form a dynamic scaffolding complex that functions as a platform for the assembly of signaling, structural, and cytoskeletal proteins. A high stringency Scansite search of rat Cx43 identified the motif containing Ser373 (S373) as a 14-3-3 binding site. The S373 motif and the second best mode-1 motif, containing Ser244 (S244), are conserved in rat, mouse, human, chicken, and bovine, but not in Xenopus or zebrafish Cx43. Docking studies of a mouse/rat 14-3-3 homology model with the modeled phosphorylated S373 or S244 peptide ligands or their serine-to-alanine mutants, S373A or S244A, revealed that the pS373 motif facilitated a greater number of intermolecular contacts than the pS244 motif, thus supporting a stronger 14-3-3 binding interaction with the pS373 motif. The alanine substitution also reduced more than half the number of intermolecular contacts between 14-3-3 and the S373 motif, emphasizing the phosphorylation dependence of this interaction. Furthermore, the ability of the wild-type or the S244A GST-Cx43 C-terminal fusion protein, but not the S373A fusion protein, to interact with either 14-3-3 or 14-3-3zeta in GST pull-down experiments clearly demonstrated that the S373 motif mediates the direct interaction between Cx43 and 14-3-3 proteins. Blocking growth factor-induced Akt activation and presumably any Akt-mediated phosphorylation of the S373 motif in ROSE 199 cells did not prevent the down-regulation of Cx43-mediated cell-cell communication, suggesting that an Akt-mediated interaction with 14-3-3 was not involved in the disruption of Cx43 function.