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1.
植物中的金属蛋白酶FtsH   总被引:1,自引:0,他引:1  
FtsH是一种对ATP和Zn^2+依赖型金属蛋白酶,广泛存在于原核生物和真核生物中。具有高度保守的AAA结构域和Zn^2+结合模块,在真核生物中是多基因家族。FtsH具有ATP酶活性,蛋白水解活性和分子伴侣活性,参与蛋白质质量平衡控制,还与热激、高渗、光胁迫、低温、病害等胁迫响应有联系。文章介绍FtsH基因的发现和分布,结构、FtsH的底物识别机制以及FtsH功能的研究概况。  相似文献   

2.
FtsH(Filamentation temperature-sensitive H)是一种广泛存在于原核生物和真核生物中的ATP依赖型金属蛋白酶。同源性分析表明,在拟南芥和水稻基因组中分别有12个和9个ftsH基因。ftsH基因在染色体上的分布有明显的偏爱性,如拟南芥的1、2、5号染色体和水稻的1、5号染色体。亚细胞定位分析表明,所有FtsH蛋白均定位于叶绿体或线粒体中。系统进化分析表明,21个FtsH蛋白成员可分为8个类群,其中AtFtsH12在水稻中没有发现种间同源物。每个类群成员的蛋白序列高度保守,种内同源物显示出大于80%的相似性,而种间同源物的相似性也大于70%。类群内的同源基因并非平行进化产生的,拟南芥基因组中进化出AtftsH1/5、AtftsH2/8、AtftsH3/10和AtftsH7/9共4个同源基因对,而水稻基因组中只有OsftsH3/8和OsftsH4/5两个同源基因对。每一类群中的成员在基因外显子-内含子边界分布上表现出高度保守性,在蛋白功能结构域的可变残基上具有偏爱性,而内含子在碱基组成和序列长度上表现出广泛的变异。拟南芥和水稻ftsH基因家族的比较分析为其他物种ftsH基因的特...  相似文献   

3.
粉纹夜蛾颗粒体病毒增强蛋白锌离子结合域定点突变   总被引:2,自引:0,他引:2  
尹隽  单梁  宋大新  钟江 《昆虫学报》2007,50(11):1111-1115
粉纹夜蛾颗粒体病毒(Trichoplusia ni granulovirus, TnGV)增强蛋白(enhancin)具有增强病毒感染力的作用。该蛋白包含一个多种杆状病毒增强蛋白都具有的保守结构域HELGH,是典型的金属蛋白酶锌离子结合域,但该结构域对增强蛋白生物活性的重要性尚未得到研究。本研究通过定点突变构建了该结构域的5个氨基酸分别突变为2种不同氨基酸的共10种增强蛋白突变体基因,并用杆状病毒载体进行了重组表达。活性测定发现,10种突变型增强蛋白大部分都丧失了野生型增强蛋白所具有的降解粉纹夜蛾幼虫围食膜粘蛋白的生物学功能,只有1种(第4位G突变为A)保留该生物学活性。这一结果表明锌离子结合域对增强蛋白生物活性具有重要作用,也提示增强蛋白确是一种金属蛋白酶。  相似文献   

4.
为研究单增李斯特菌(LM)核糖核酸酶Rnase Ⅲ RncS氨基酸突变对RNA降解活性的影响。利用生物信息学软件分析单核细胞增生李斯特菌(LM)野毒株SB5中rncS基因编码的Rnase Ⅲ的结构域,并选择关键氨基酸利用基因重叠延伸PCR(SOE-PCR)技术对其进行了基因突变;然后将rncS突变基因片段D50A、E122A克隆至表达载体pET-32a(+),在大肠杆菌中利用IPTG进行诱导表达;应用SDS-PAGE和Western Blot鉴定重组蛋白的表达情况及其抗原特异性;通过体外酶活试验研究其对RNA降解活性的影响。结构域分析结果显示,LM-Rnase Ⅲ氨基酸序列含有1个双链RNA结合结构域(DSRM)和1个核酸酶结构域(RIBOc),其中结构域RIBOc含有5个活性位点。SDS-PAGE检测结果显示,表达的重组突变型Rnase Ⅲ -D50A和Rnase Ⅲ -E122 A蛋白相对分子质量均为42.5 kD,与理论值相符;Western blot分析表明重组突变型Rnase Ⅲ -D50A和Rnase Ⅲ -E122A蛋白可与LM阳性血清发生免疫学反应。体外酶活实验表明,Rnase Ⅲ发挥降解活性依赖于Mn2+或Mg2+,将其第50位天冬氨酸突变后,Rnase Ⅲ RncS的降解活性有所降低(P0.001);第122位谷氨酸突变后,Rnase Ⅲ RncS降解活性极显著下降(P0.0001),提示第122位谷氨酸是维持LM Rnase Ⅲ RncS酶活性的关键位点。  相似文献   

5.
转入甜椒热激蛋白基因CaHSP18提高番茄的耐冷性   总被引:2,自引:0,他引:2  
利用农杆菌介导法将甜椒热激蛋白基因转化番茄,Northern和Western杂交表明CaHSP18在番茄植株中表达,获得转CaHSP18的番茄植株。Northern杂交显示,CaHSP18基因受低温诱导,表达量随低温处理时间的延长而增加,6h时表达量最高。低温胁迫导致野生型和转基因番茄植株的相对电导率升高,光爱统Ⅱ(PSⅡ)最大光化学效率(Fv/Fm)和放氧速率下降,但转基因番茄植物维持较低的膜透性,较高的Fv/Fm和放氧速率。这些显示,在番茄植株中CaHSP18表达后耐冷性有提高。  相似文献   

6.
ADAMs是近年发现的一个新的具有多个结构域和多种功能的蛋白质家族。本文多方面比较了大鼠实质肝细胞和非实质肝细胞的ADAMs与再生肝的ADAMs异同。检测多种因子对ADAMs体外加工的影响发现,活性的最适pH为3.5-4的酸性蛋白水解酶在细胞ADAMs降解加工中起关键作用;活性的最适pH为7.5-8.5的偏碱性蛋白酶在基质ADAMs降解加工中起重要作用;外源的胶原酶能高效降解75kDADAMs和140kDMDC15,可见,体内胶原酶样的蛋白水解酶是ADAMs降解加工的重要酶类;Fe^3 和Zn^2 等可在体外活化ADAMs的降解,看来,体内降解加工ADAMs的酶属依赖于Fe^3 或Zn^2 的蛋白水解酶。根据研究结果推测,体内许多不同性能的蛋白水解酶,如丝氨酸蛋白水解酶,半胱氨酸蛋白水解酶,天冬氨酸蛋白水解酶和金属蛋白水解酶等参与ADAMs的降解加工。  相似文献   

7.
根据丙型肝炎病毒 (HCV)丝氨酸蛋白酶晶体结构特点 ,设计并构建了一种新的单链型丝氨酸蛋白酶分子 .该分子由辅因子NS4A的核心序列、柔性连接子GSGS和NS3丝氨酸蛋白酶结构域组成 .利用设计的 3条引物 ,通过 2轮PCR获得单链丝氨酸蛋白酶基因 ,插入原核表达载体pQE30中 ,转化大肠杆菌M15 ,获得重组克隆 .经低剂量诱导和低温培养 ,目的基因获得高水平可溶表达 .以金属螯合层析法纯化的重组蛋白纯度达 95 %以上 .间接ELISA法检测 98份血清证实 ,该蛋白具有良好的抗原性和特异性 ;以重组蛋白底物NS5ab和单链丝氨酸蛋白酶建立了简便、实用的丝氨酸蛋白酶体外活性检测系统 ;以该系统观察了PMSF和EDTA对蛋白酶活性的影响 .结果表明 ,PMSF能够抑制蛋白酶的酶切活性 ,而EDTA不能抑制酶的活性 .单链型HCV丝氨酸蛋白酶的成功表达以及体外活性检测系统的建立 ,为丝氨酸蛋白酶抑制剂的研制奠定了物质基础 .  相似文献   

8.
脱落酸-胁迫-成熟诱导蛋白(Abscisic acid-stress-ripening,ASR)在植物对非生物逆境胁迫的应答过程中发挥着重要作用。利用PCR技术从木薯中克隆了第一个ASR基因Me ASR,序列分析表明该基因开放阅读框(ORF)330 bp,编码109个氨基酸。多序列比对和进化树分析表明该基因所编码的蛋白具有ASR家族蛋白的保守结构域,与番茄ASR家族蛋白Sl ASR4具有较近的亲缘关系。亚细胞定位分析表明Me ASR定位在细胞核,实时荧光定量PCR分析表明该基因的表达显著受渗透胁迫和ABA诱导。结果表明,Me ASR可能作为转录因子参与木薯对干旱逆境胁迫应答及ABA信号调节。  相似文献   

9.
通过拟南芥芯片杂交分析发现推测的钙调素基因(GenBank accession No.Atlg76650)与低磷胁迫有关.对该基因的结构研究确认了该基因编码一个含有三个EF-hand结构域的类似钙调素的蛋白,Northern检测表明该基因在缺钾、缺磷条件下诱导表达,但在缺氮、高盐等胁迫条件下不受影响.经RT-PCR和启动子融合GUS转基因植株的组织化学染色分析,表明该基因在拟南芥中为全株表达,但各器官中表达丰度不尽相同.  相似文献   

10.
MYB转录因子参与植物细胞形态与模式建成、次级代谢的调控以及生物和非生物胁迫应答等反应。该研究采用RT-PCR方法扩增了番茄SlMYB86基因,并进行了聚类分析和保守域序列分析,构建原核表达载体和诱导纯化蛋白,利用qRT-PCR和Western blot检测SlMYB86在缺氮复氮下的表达水平,为深入探究番茄MYB86转录因子在缺氮胁迫下的功能奠定基础。结果表明:(1)番茄SlMYB86与番茄SlMYB26在进化树上属于同一分支,亲缘关系较近,且SlMYB86含有2个Myb_DNA-binding保守结构域,属于R2R3-MYB型转录因子。(2)qRT-PCR分析发现,SlMYB86基因在番茄根和叶中均有表达,缺氮胁迫下SlMYB86表达较对照显著增加。(3)成功构建pET-28a-SlMYB86原核表达载体并转化E.coli BL21(DE3),SDS-PAGE和Western blot结果表明,SlMYB86蛋白的最佳诱导条件为0.5 mmol/L的IPTG、37℃诱导8 h;目的蛋白相对分子量大约为41 kD,与预期大小一致,并获得较高纯度的SlMYB86原核蛋白。(4)将纯化的SlMYB86蛋白免疫小白鼠获得抗体,利用该抗体进行Western blot分析发现,番茄中SlMYB86蛋白在缺氮胁迫后表达上调,表明番茄SlMYB86基因参与了缺氮胁迫的应答。  相似文献   

11.
Z Ge  D E Taylor 《Journal of bacteriology》1996,178(21):6151-6157
In this study, we isolated and sequenced a Helicobacter pylori gene, designated ftsH, coding for a 632-amino-acid protein which displayed striking similarity throughout its full length to FtsH proteins identified in Escherichia coli, Lactococcus lactis, and Bacillus subtilis. H. pylori FtsH also possessed approximately 200-amino-acid region containing a putative ATPase module which is conserved among members of the AAA protein family (AAA, ATPase associated with diverse cellular activities). The H. pylori ftsH product was overexpressed in E. coli and reacted immunologically with an anti-E. coli FtsH serum (T. Tomoyasu, K. Yamanaka, K. Murata, T. Suzaki, P. Bouloc, A. Kato, H. Niki, S. Hiraga, and T. Ogura, J. Bacteriol. 175:1352-1357, 1993). FtsH was also shown to be present in the membrane fraction of H. pylori, suggesting that it is membrane bound. Disruption of the ftsH gene led to the loss of viability of H. pylori, demonstrating that this gene is essential for cell growth. Overproduction of both H. pylori FtsH and E. coli FtsH together tremendously reduced the growth rate of the E. coli host cells, whereas the growth of the E. coli cells carrying the wild-type E. coli ftsH operon on the chromosome was not significantly affected by overproduction of H. pylori FtsH itself. This result suggests that the abnormal growth of cells results from interaction between H. pylori FtsH and E. coli FtsH.  相似文献   

12.
The Bradyrhizobium japonicum ftsH gene was cloned by using a set of widely applicable degenerated oligonucleotides. Western blot experiments indicated that the FtsH protein was produced under standard growth conditions and that it was not heat inducible. Attempts to delete the ftsH gene in B. japonicum failed, suggesting a pivotal cellular function of this gene. The expression of B. japonicum ftsH in an ftsH-negative Escherichia coli strain significantly enhanced the fitness of this mutant and reduced the steady-state level of sigma(32).  相似文献   

13.
The ftsH gene is essential for cell viability in Escherichia coli. We cloned and sequenced the wild-type ftsH gene and the temperature-sensitive ftsH1(Ts) gene. It was suggested that FtsH protein was an integral membrane protein of 70.7 kDa (644 amino acid residues) with a putative ATP-binding domain. The ftsH1(Ts) gene was found to have two base substitutions within the coding sequence corresponding to the amino acid substitutions Glu-463 by Lys and Pro-587 by Ala. Homology search revealed that an approximately 200-amino-acid domain, including the putative ATP-binding sequence, is highly homologous (35 to 48% identical) to the domain found in members of a novel, eukaryotic family of putative ATPases, e.g., Sec18p, Pas1p, CDC48p, and TBP-1, which function in protein transport pathways, peroxisome assembly, cell division cycle, and gene expression, respectively. Possible implications of these observations are discussed.  相似文献   

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The wine bacterium Oenococcus oeni has to cope with harsh environmental conditions, including an acidic pH, a high alcoholic content, nonoptimal growth temperatures, and growth-inhibitory compounds such as fatty acids, phenolic acids, and tannins. We describe the characterization and cloning of the O. oeni ftsH gene, encoding a protease belonging to the ATP binding cassette protein superfamily. The O. oeni FtsH protein is closest in sequence similarity to the FtsH homologue of Lactococcus lactis. The O. oeni ftsH gene proved to be stress-responsive, since its expression increased at high temperatures or under osmotic shock. O. oeni FtsH protein function was tested in an Escherichia coli ftsH mutant strain, and consistent with the O. oeni ftsH gene expression pattern, the O. oeni FtsH protein provided protection for the E. coli ftsH mutant against heat shock. O. oeni and Bradyrhizobium japonicum FtsH proteins also triggered E. coli resistance to wine toxicity. Genes homologous to O. oeni ftsH were detected in many other lactic acid bacteria found in wine, suggesting that this type of gene constitutes a well-conserved stress-protective molecular device.  相似文献   

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The spoVM gene encodes a 26-amino-acid polypeptide that is essential for spore formation in Bacillus subtilis. A transposon insertion within the spoVM open reading frame has been shown to encode a chimeric protein which is biologically inactive and produces a phenotype identical to that of a deletion and insertion mutation. A genetic approach was used to identify possible interacting proteins, and the membrane-bound FtsH protease was identified. Mutations in ftsH suppressed the sporulation defect of certain spoVM mutants but not others. However, production of the mother cell sigma factors, sigmaE and sigmaK, was abnormal in the suppressed strains, and mutations in either spoVM or ftsH alone impaired sigma factor production and sporulation gene expression. Using FtsH purified from Escherichia coli, we demonstrated that in vitro (i) SpoVM inhibits FtsH protease activity and (ii) SpoVM is a substrate for the FtsH protease. We propose that during sporulation, SpoVM serves as a competitive inhibitor of FtsH activity. This interaction appears to be important for completion of the prespore engulfment step of sporulation, based on the phenotype of certain spoVM ftsH double mutants.  相似文献   

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