蕨类植物问荆DEAD box RNA解旋酶基因的克隆与分析

DEAD box RNA解旋酶参与RNA多方面的代谢,在植物生长发育和逆境反应中起重要作用。本研究从蕨类植物问荆(Equisetum arvense)中克隆到一条DEAD box RNA解旋酶cDNA全长序列,命名为EaRH1,并在GenBank注册登记（KJ734026）。序列分析显示：该cDNA全长3230bp,包含一个从487bp到2799bp编码770个氨基酸的开放读码框,其对应的蛋白序列包含9个保守模块结构。EaRH1与其它物种DEAD box RNA 解旋酶蛋白序列比对结果显示：模块Ⅰa、Ⅱ和Ⅲ序列几乎完全相同,模块Q、Ⅰ和 Ⅳ序列存在一些差异。EaRH1与江南卷柏(Selaginella moellendorffii)基因组一条假定序列相似度高达69%,其中相似度最高的区域集中在包含9个保守模块的结构域。系统进化树分析显示：EaRH1与拟南芥(Arabidopsis thaliana)DEAD box RNA解旋酶At3g22320在氨基酸序列上有相对较高的同源性。序列结构比较和进化分析可推测出EaRH1可能参与植物体生长发育、miRNA生物合成、与RNA结合蛋白的相互作用和非生物胁迫应答。本文的研究为探索问荆DEAD box RNA解旋酶的进一步功能提供参考。     

DEAD-box基因家族在拟南芥生长发育中的作用

在RNA代谢过程中,需要许多蛋白和核酸的参与,其中一类蛋白就是RNA解旋酶。RNA解旋酶通过水解ATP获得能量来参与RNA代谢的多个方面,包括核内转录、pre-mRNA的剪切、核糖体发生、核质运输、蛋白质翻译、RNA降解、细胞器内基因的表达。DEAD-box蛋白家族是RNA解旋酶中最大的亚家族,它具有9个保守结构域,因motifyⅡ的保守氨基酸序列Asp-Glu-Ala-Asp(DEAD)而命名。该家族在酵母、拟南芥(Arabidopsis thaliana Heynh.)和人类基因组中都有较多的家庭成员。近年来,研究者对拟南芥DEAD-box蛋白家族的结构和功能进行了一些研究,本文着重总结DEAD-box基因家族对拟南芥生长发育的影响。     

玉米DEAD-box RNA解旋酶基因的克隆及分析

DEAD-box RNA解旋酶参与RNA转录、前体mRNA剪切、核糖体发生、核质运输、蛋白质翻译、RNA降解等重要的生命活动.根据本室在S-Mo17Rf3Rf3cDNA芯片研究中,检测到花粉发育后期RNA解旋酶上调表达的结果,应用RACE技术从S-Mo17Rf3Rf3花粉中克隆得到该RNA解旋酶基因全长cDNA,命名为ZmRH2并在GenBank注册登记 (DQ327709).序列分析表明：该cDNA全长1 652bp,从第163 bp开始到1 386bp含有一个开放阅读框,编码407个氨基酸.其编码的蛋白质具有DEAD-box RNA解旋酶特有的9个保守模体,与水稻、拟南芥和豌豆中的DEAD-box RNA解旋酶的氨基酸序列存在着很高的同源性.RT-PCR分析表明,该基因在近等基因系S-Mo17Rf3Rf3和S-Mo17rf3rf3的叶、根、和雌穗中的表达没有差异,但在花丝和花粉中有明显差异.     

马氏珠母贝Pm-vasa基因的克隆与表达分析

vasa蛋白是DEAD-box家族蛋白的一员,在真核生物原生殖细胞形成过程中起关键作用。本实验利用RACE技术克隆获得了马氏珠母贝vasa基因(Pm-vasa),并对其结构和组织表达模式进行了分析。结果表明:Pm-vasa c DNA序列全长为1 709 bp,其中开放式阅读框1 431 bp、5'UTR 148 bp、3'UTR 131 bp,共编码476个氨基酸,分子量为52.139 k D,理论等电点为6.24。SMART软件分析显示Pm-vasa蛋白具有典型的DEAD-box结构域,且具DEAD-box家族蛋白9个典型保守基序。多序列比对结果表明Pm-vasa与紫贻贝vasa同源性最高,为74%;系统进化分析发现,Pm-vasa与紫贻贝等软体动物聚为一支。组织表达定量分析发现Pm-vasa基因m RNA在性腺中显著高表达。我们的研究结果表明Pm-vasa可能参与马氏珠母贝的性腺发育。     

DEAD-box解旋酶在植物非生物胁迫响应中的功能研究进展

解旋酶广泛存在于从病毒到人类几乎所有已知的原核和真核生物体中,它们能利用NTP水解获得能量促进双链DNA/RNA解链。DEAD-box解旋酶是最大的解旋酶家族,在DNA复制、修复、重组、转录、核糖体发生和翻译起始等几乎所有涉及DNA/RNA代谢的细胞过程中均发挥重要作用。近年研究表明,DEAD-box解旋酶除具有持家基因功能外,还广泛参与植物生长发育及胁迫响应过程。目前,由解旋酶介导的胁迫应答具体机制依然不清晰。现重点介绍近年来高等植物DEAD-box解旋酶在非生物胁迫响应中的功能研究进展,同时探讨解旋酶参与胁迫应答的可能机制。     

番茄DEAD-box RNA解旋酶基因SlDEAH1的克隆及表达分析

DEAD-box RNA解旋酶是一种特殊的RNA分子伴侣,参与了RNA代谢,包括前体RNA剪接、核糖体合成、RNA降解以及基因表达,并对植物的发育和抗性等也具有重要作用。根据已报道的拟南芥DEAD-box蛋白,通过同源比对,在NCBI据库中筛选得到一个DEAD-box RNA解旋酶同源蛋白,命名为SlDEAH1,并根据其基因序列设计特异引物,应用RT-PCR方法从野生型番茄(Solanum lycopersicum)AC++中克隆得到了该基因的全长编码区序列。利用生物学网站、软件及实时荧光定量PCR方法,对其进行生物信息学、表达模式、胁迫及激素处理分析。结果表明:SlDEAH1包括2 073 bp的开放阅读框,编码690个氨基酸残基,其编码蛋白有9个保守结构基序,其所涉及到的ATP结合、ATP水解及RNA结合等功能对于解旋酶活性是至关重要的;表达模式分析表明SlDEAH1基因可能在野生型番茄萼片、叶片发育及果实成熟方面起到重要作用;高温、低温、脱水、伤害、盐胁迫不同程度的诱导了SlDEAH1的表达,但在根中该基因的表达受盐胁迫抑制;ABA、ACC、IAA、GA3、MeJA和ZT均不同程度诱导了SlDEAH1的表达,其中ABA诱导效应最为明显。这些结果为进一步研究SlDEAH1在番茄发育和胁迫响应中的功能奠定了基础。     

马氏珠母贝生长激素诱导跨膜蛋白基因GHITM克隆与表达分析

生长激素诱导跨膜蛋白(growth hormone-induced transmembrane protein,GHITM)是一类在进化上高度保守的跨膜蛋白,参与机体生长发育、老化及先天免疫等过程。为探究GHITM在马氏珠母贝生长发育过程中的作用,本实验利用c DNA末端快速扩增技术(rapid amplification of c DNA ends,RACE),克隆得到马氏珠母贝GHITM(Pinctada martensii GHITM,Pm-GHITM)基因c DNA全长序列,并对其序列特征以及在组织中的表达进行分析。结果显示:Pm-GHITM基因c DNA全长为1 468 bp,其中,开放阅读框(open reading frame,ORF)为1 017 bp,编码338个氨基酸,5'UTR长62 bp,3'UTR长389 bp,分子量约为35.48 ku,等电点(p I)为9.87;多序列及系统进化树分析表明Pm-GHITM与其他物种的GHITM有较高的同源性,与太平洋牡蛎(Crassostrea gigas)的GHITM相似度最高,为67%;结构域分析结果表明Pm-GHITM序列中包含一个高度保守的BI-1(Bax inhibitor-1)结构域;荧光定量数据指出Pm-GHITM在马氏珠母贝的血淋巴、外套膜、足、鳃、性腺、闭壳肌以及肝胰腺中均有表达,在性腺的表达量最高,肝胰腺和闭壳肌次之,血淋巴中的表达量最低(p0.05)。本研究可以为进一步探究Pm-GHITM在马氏珠母贝的生长发育过程中的作用做铺垫。     

朱砂叶螨几丁质合成酶基因1全长cDNA的克隆与表达分析

【目的】克隆朱砂叶螨Tetranychus cinnabarinus几丁质合成过程中的关键酶几丁质合成酶基因,并检测该基因在朱砂叶螨生长发育不同阶段的相对表达量。【方法】本研究采用逆转录聚合酶链反应(RT-PCR)以及c DNA末端快速扩增(RACE)技术首次克隆获得朱砂叶螨几丁质合成酶基因1的全长c DNA序列(命名为Tc CHS1,Gen Bank登录号为KM242062),并使用实时荧光定量PCR技术首次检测了Tc CHS1基因在朱砂叶螨生长发育不同阶段的相对表达量。【结果】朱砂叶螨Tc CHS1基因的c DNA全长为4 881 bp,包括198 bp的5'非翻译区(5'-UTR),4 425 bp的开放阅读框(ORF),258 bp的3'非翻译区(3'-UTR),开放阅读框编码1 474个氨基酸,预测其蛋白质分子质量约为168.35 ku,理论等电点为6.26。其包含EDR和QRRRW这2个几丁质合成酶基因的标签序列。氨基酸序列同源性分析结果表明:Tc CHS1与其他昆虫该基因编码蛋白的氨基酸序列相似度在50%左右,与二斑叶螨Tetranychus urticae的氨基酸相似度最高(98%),与西方盲走螨Metaseiulus occidentalis的相似度为55%。分子系统进化的结果也表明Tc CHS1与其他昆虫的CHS1聚在一起,并且和二斑叶螨具有最近的亲缘关系。荧光定量分析表明Tc CHS1基因在朱砂叶螨生长发育的不同阶段(卵、幼螨、第1若螨、第2若螨、雌成螨和雄成螨)均有表达,在卵和雌成螨中的表达量较高,在第2若螨的表达量最低。【结论】Tc CHS1基因可能在朱砂叶螨生长发育过程中具有重要作用。     

番茄 DEAD-box RNA 解旋酶基因 SlDEAH1 的克隆及表达分析简

DEAD-box RNA解旋酶是一种特殊的RNA分子伴侣,参与了RNA代谢,包括前体RNA剪接、核糖体合成、RNA降解以及基因表达,并对植物的发育和抗性等也具有重要作用。根据已报道的拟南芥DEAD-box蛋白,通过同源比对,在NCBI据库中筛选得到一个DEAD-box RNA解旋酶同源蛋白,命名为SlDEAH1,并根据其基因序列设计特异引物,应用RT-PCR方法从野生型番茄（Solanum lycopersicum）AC⁺⁺中克隆得到了该基因的全长编码区序列。利用生物学网站、软件及实时荧光定量PCR方法,对其进行生物信息学、表达模式、胁迫及激素处理分析。结果表明：SlDEAH1包括2 073 bp的开放阅读框,编码690个氨基酸残基,其编码蛋白有9个保守结构基序,其所涉及到的ATP结合、ATP水解及RNA结合等功能对于解旋酶活性是至关重要的;表达模式分析表明SlDEAH1基因可能在野生型番茄萼片、叶片发育及果实成熟方面起到重要作用;高温、低温、脱水、伤害、盐胁迫不同程度的诱导了SlDEAH1的表达,但在根中该基因的表达受盐胁迫抑制;ABA、ACC、IAA、GA₃、MeJA和ZT均不同程度诱导了SlDEAH1的表达,其中ABA诱导效应最为明显。这些结果为进一步研究SlDEAH1在番茄发育和胁迫响应中的功能奠定了基础。     

眼镜王蛇泛素融合蛋白基因和核糖体蛋白L30基因的克隆及分析

从广西产眼镜王蛇（Ophiophagus hannah）毒腺中抽提总RNA,经mRNA纯化后构建眼镜王蛇毒腺cDNA文库。从所构建的cDNA文库中,随机筛选200个克隆测序,得到两个在进化上高度保守的基因：泛素融合蛋白基因（GenBank登录号为AF297036）和核糖体蛋白L30基因（GenBank登录号是AF297033）。前者cDNA的开放阅读框为387bp,后者为348bp。前者编码128个氨基酸残基组成的泛素融合蛋白前体;后者编码115个氨基酸残基组成的核糖体蛋白L30前体。由cDNA序列推导出的氨基酸序列分析表明,泛素融合蛋白前体包括N-末端的泛素结构域（76个氨基酸残基）和C-末端的核糖体蛋白L40结构域（52个氨基酸残基）。该蛋白为一高碱性蛋白,C末端含有一个“锌指”模式结构。与16个物种比较的结果表明,眼镜王蛇与脊椎动物的泛素融合蛋白氨基酸序列相似度较高,具有高度的保守性。     

Crystal structure of the N-terminal RecA-like domain of a DEAD-box RNA helicase, the Dugesia japonica vasa-like gene B protein

The Dugesia japonica vasa-like gene B (DjVLGB) protein is a DEAD-box RNA helicase of a planarian, which is well known for its strong regenerative capacity. DjVLGB shares sequence similarity to the Drosophila germ-line-specific DEAD-box RNA helicase Vasa, and even higher similarity to its paralogue, mouse PL10. In this study, we solved the crystal structure of the DjVLGB N-terminal RecA-like domain. The overall fold and the structures of the putative ATPase active site of the DjVLGB N-terminal RecA-like domain are similar to those of the previously reported DEAD-box RNA helicase structures. In contrast, the surface structure of the side opposite to the putative ATPase active site is different from those of the other DEAD-box RNA helicases; the characteristic hydrophobic pockets are formed with aromatic and proline residues. These pocket-forming residues are conserved in the PL10-subfamily proteins, but less conserved in the Vasa orthologues and not conserved in the DEAD-box RNA helicases. Therefore, the structural features that we found are characteristic of the PL10-subfamily proteins and might contribute to their biological roles in germ-line development.     

Entamoeba histolytica EhDEAD1 is a conserved DEAD-box RNA helicase with ATPase and ATP-dependent RNA unwinding activities

RNA helicases are widely conserved key enzymes that perform multiple functions in RNA metabolism. Here, we present the cloning, expression and functional characterization of the EhDEAD1 RNA helicase in the protozoan parasite Entamoeba histolytica. According to its primary structure, EhDEAD1 is evolutionary related to yeast DED1 and human DDX3X RNA helicases, both involved in translation and cell cycle regulation. The EhDEAD1 predicted amino acid sequence exhibits the nine conserved motifs described for the DEAD-box SFII superfamily members reported in other organisms and it is evolutionary close to protozoan homologues. Purified recombinant EhDEAD1 protein presented ATPase activity and it was able to bind and unwind RNA in an ATPase-dependent manner in vitro. RT-PCR assays showed that EhDead1 gene is overtranscribed in the cell cycle S phase. Moreover, inhibition of EhDead1 gene expression by antisense RNA seemed to facilitate transition from S to G2/M phase. Intriguingly, our results showed that EhDEAD1 was unable to rescue two yeast Ded1 RNA helicase mutants affected in translation, in spite of the high sequence homology with yeast DED1.     

A new DEAD-box helicase ATP-binding protein (OsABP) from rice is responsive to abiotic stress

The DEAD-box RNA helicase family comprise enzymes that participate in every aspect of RNA metabolism, associated with a diverse range of cellular functions including response to abiotic stress. In the present study, we report on the identification of a new DEAD-box helicase ATP-binding protein (OsABP) from rice which is upregulated in response e to multiple abiotic stress treatments  including NaCl, dehydration, ABA, blue and red light. It possesses an ORF of 2772 nt, encoding a protein of 923 aa, which contains the DEAD and helicase C-terminal domains, along with the nine conserved motifs specific to DEAD-box helicases. The in silico putative interaction with other proteins showed that OsABP interacts with proteins involved in RNA metabolism, signal transduction or stress response. These results imply that OsABP might perform important functions in the cellular response to specific abiotic stress.     

Crystallographic structure of the amino terminal domain of yeast initiation factor 4A, a representative DEAD-box RNA helicase

The eukaryotic translation initiation factor 4A (elF4A) is a representative of the DEAD-box RNA helicase protein family. We have solved the crystallographic structure of the amino-terminal domain (residues 1-223) of yeast elF4A. The domain is built around a core scaffold, a parallel alpha-beta motif with five beta strands, that is found in other RNA and DNA helicases, as well as in the RecA protein. The amino acid sequence motifs that are conserved within the helicase family are localized to the beta strand-->alpha helix junctions within the core. The core of the amino terminal domain of elF4A is amplified with additional structural elements that differ from those of other helicases. The phosphate binding loop (the Walker A motif) is in an unusual closed conformation. The crystallographic structure reveals specific interactions between amino acid residues of the phosphate binding loop, the DEAD motif, and the SAT motif, whose alteration is known to impair coupling between the ATPase cycle and the RNA unwinding activity of elF4A.     

RNA helicase-related genes of Plasmodium falciparum and Plasmodium cynomolgi

RNA helicases play many essential roles including cell development and growth. Using degenerate oligonucleotide primers designed to amplify DNA fragments flanked by the highly conserved helicase motifs VLDEAD and YIHRIG and genomic DNAs from the malarial parasites as a template, we have cloned two putative RNA helicase genes (546 and 540 bp) from P. falciparum and one gene (546 bp) from P. cynomologi. Southern blot analysis revealed that these could be multiple and single-copy genes in P. falciparum and P. cynomolgi, respectively. Several members of the RNA helicase gene family share sequence identity with malarial parasite's helicases ranging from 30 to 76%, suggesting that they are functionally related. The discovery of such a multitude of putative RNA helicase genes in malarial parasites suggested that RNA helicase activities may be involved in many essential biological processes. Further characterization of these helicases may also help in designing parasite-specific inhibitors/drugs which specifically inhibit the parasite's growth without affecting the host.     

OsBIRH1, a DEAD-box RNA helicase with functions in modulating defence responses against pathogen infection and oxidative stress

DEAD-box proteins comprise a large protein family with members from all kingdoms and play important roles in all types of processes in RNA metabolism. In this study, a rice gene OsBIRH1, which encodes a DEAD-box RNA helicase protein, was cloned and characterized. The predicted OsBIRH1 protein contains a DEAD domain and all conserved motifs that are common characteristics of DEAD-box RNA helicases. Recombinant OsBIRH1 protein purified from Escherichia coli was shown to have both RNA-dependent ATPase and ATP-dependent RNA helicase activities in vitro. Expression of OsBIRH1 was activated in rice seedling leaves after treatment with defence-related signal chemicals, for example benzothiadiazole, salicylic acid, l-aminocyclopropane-1-carboxylic acid, and jasmonic acid, and was also up-regulated in an incompatible interaction between a resistant rice genotype and the blast fungus, Magnaporthe grisea. Transgenic Arabidopsis plants that overexpress the OsBIRH1 gene were generated. Disease resistance phenotype assays revealed that the OsBIRH1-overexpressing transgenic plants showed an enhanced disease resistance against Alternaria brassicicola and Pseudomonas syringae pv. tomato DC3000. Meanwhile, defence-related genes, for example PR-1, PR-2, PR-5, and PDF1.2, showed an up-regulated expression in the transgenic plants. Moreover, the OsBIRH1 transgenic Arabidopsis plants also showed increased tolerance to oxidative stress and elevated expression levels of oxidative defence genes, AtApx1, AtApx2, and AtFSD1. The results suggest that OsBIRH1 encodes a functional DEAD-box RNA helicase and plays important roles in defence responses against biotic and abiotic stresses.     

The newly discovered Q motif of DEAD-box RNA helicases regulates RNA-binding and helicase activity

DEAD-box proteins are the most common RNA helicases, and they are associated with virtually all processes involving RNA. They have nine conserved motifs that are required for ATP and RNA binding, and for linking phosphoanhydride cleavage of ATP with helicase activity. The Q motif is the most recently identified conserved element, and it occurs approximately 17 amino acids upstream of motif I. There is a highly conserved, but isolated, aromatic group approximately 17 amino acids upstream of the Q motif. These two elements are involved in adenine recognition and in ATPase activity of DEAD-box proteins. We made extensive analyses of the Q motif and upstream aromatic residue in the yeast translation-initiation factor Ded1. We made site-specific mutations and tested them for viability in yeast. Moreover, we purified various mutant proteins and obtained the Michaelis-Menten parameters for the ATPase activities. We also measured RNA affinities and strand-displacement activities. We find that the Q motif not only regulates ATP binding and hydrolysis but also regulates the affinity of the protein for RNA substrates and ultimately the helicase activity.     

Intermolecular interactions within the abundant DEAD-box protein Dhh1 regulate its activity in vivo

Dhh1 is a highly conserved DEAD-box protein that has been implicated in many processes involved in mRNA regulation. At least some functions of Dhh1 may be carried out in cytoplasmic foci called processing bodies (P-bodies). Dhh1 was identified initially as a putative RNA helicase based solely on the presence of conserved helicase motifs found in the superfamily 2 (Sf2) of DEXD/H-box proteins. Although initial mutagenesis studies revealed that the signature DEAD-box motif is required for Dhh1 function in vivo, enzymatic (ATPase or helicase) or ATP binding activities of Dhh1 or those of any its many higher eukaryotic orthologues have not been described. Here we provide the first characterization of the biochemical activities of Dhh1. Dhh1 has weaker RNA-dependent ATPase activity than other well characterized DEAD-box helicases. We provide evidence that intermolecular interactions between the N- and C-terminal RecA-like helicase domains restrict its ATPase activity; mutation of residues mediating these interactions enhanced ATP hydrolysis. Interestingly, the interdomain interaction mutant displayed enhanced mRNA turnover, RNA binding, and recruitment into cytoplasmic foci in vivo compared with wild type Dhh1. Also, we demonstrate that the ATPase activity of Dhh1 is not required for it to be recruited into cytoplasmic foci, but it regulates its association with RNA in vivo. We hypothesize that the activity of Dhh1 is restricted by interdomain interactions, which can be regulated by cellular factors to impart stringent control over this very abundant RNA helicase.