MAPK基因家族成员在棉花抗黄萎病中的功能分析

作为植物中普遍存在的一类保守的丝氨酸/苏氨酸类蛋白激酶信号传导系统,促分裂原活化蛋白激酶(MAPK)级联信号传导途径,在调控植物生长发育,传导并响应各类生物和非生物逆境信号胁迫过程中都起着重要作用.鉴于对棉花(Gossypium hirsutum L.)MAPKs级联信号传导系统中最下游的激酶MAPK家族成员缺乏了解,更不明楚MAPK基因家族成员在棉花抗黄萎病中的功能和作用.本文利用已公布的雷蒙德氏棉(Gossypium raimondii)和亚洲棉(Gossypium arboreum)基因组数据,并从美国国家生物技术信息中心(NCBI)数据库中收集陆地棉的MAPK氨基酸序列,成功地对棉花MAPK基因家族成员进行了同源分析和聚类.结果显示,棉花MAPK基因家族各成员均具有特征性TEY磷酸化位点或TDY磷酸化位点结构特征,依据其蛋白序列可划分为A,B,C和D 4个族.进而从以上各族中分别选择两个MAPK基因成员,利用病毒介导的基因沉默(VIGS)技术,来研究MAPK基因家族成员在棉花抗黄萎病中的作用.结果发现,6个MAPK基因家族成员参与了棉花对黄萎病菌的抗性作用,这提示MAPKs级联信号传导途径是参与棉花抗黄萎病信号传导通路之一.     

三叶木通促分裂原活化蛋白激酶基因mapk3的克隆及表达分析

植物MAPK信号途径在植物生长发育以及多种逆境胁迫响应和激素调控过程中发挥着至关重要的作用。本文利用RACE-PCR技术克隆三叶木通促分裂原活化蛋白激酶基因mapk3的全长c DNA序列,并对其进行生物信息学分析和时空表达分析。克隆所得的Aktmapk3基因的ORF全长序列为1 164 bp,编码387个氨基酸,其编码的蛋白具有ATP结合位点,MAPK激酶保守结构和丝氨酸/苏氨酸蛋白激酶激活位点,推测其可能通过被磷酸化而激活以及通过磷酸化下游蛋白而执行生理功能。实时荧光定量PCR结果显示,Aktmapk3基因在三叶木通各组织器官均有表达,在芽成熟叶片以及花中表达量比较高,在茎、幼叶和果肉中的表达量最低,暗示该基因可能参与了三叶木通芽的形成和花的发育。     

植物MAPK信号转导组分的细胞定位与选择性剪接

促分裂原活化蛋白激酶(MAPK)级联途径在真核生物中是高度保守的,由MAPKs,MAPKKs,MAPKKKs组成,通过MAPKKK→MAPKK→MAPK逐级磷酸化传递细胞信号.已有大量研究表明,MAPK在植物响应生物与非生物胁迫,以及植物激素和细胞周期的信号转导中起重要作用.在植物响应各种逆境过程中激活的MAPK基因,细胞内的定位发生动态变化.选择性剪接是真核生物中调节基因表达的重要模式,能够影响蛋白的结合特性、胞内定位、酶的活性、蛋白的稳定性和翻译后的修饰.MAPK基因的选择性剪接能产生不同的转录异型并具有不同的亚细胞定位.本文综述这方面的研究进展.     

植物PP2C蛋白磷酸酶ABA信号转导及逆境胁迫调控机制研究进展

蛋白磷酸酶(protein phosphatase,PP)是蛋白质可逆磷酸化调节机制中的关键酶,而PP2C磷酸酶是一类丝氨酸/苏氨酸残基蛋白磷酸酶,是高等植物中最大的蛋白磷酸酶家族,包含76个家族成员,广泛存在于生物体中。迄今为止,在植物体内已经发现了4种PP2C蛋白磷酸酶。蛋白激酶和蛋白磷酸酶协同催化蛋白质可逆磷酸化,在植物体内信号转导和生理代谢中起着重要的调节作用,蛋白质的磷酸化几乎存在于所有的信号转导途径中。大量研究表明,PP2Cs参与多条信号转导途径,包括PP2C参与ABA调控,对干旱、低温、高盐等逆境胁迫的响应,参与植物创伤和种子休眠或萌发等信号途径,其调控机制不同,但酶催化活性都依赖于Mg2+或Mn2+的浓度。植物PP2C蛋白的C端催化结构域高度保守,而N端功能各异。文中还综述了高等植物PP2C的分类、结构、ABA受体与PP2Cs蛋白互作、PP2C基因参与ABA信号途径以及其他逆境信号转导途径的研究进展。     

谷子SiMAPK3基因的克隆和表达特性分析

丝裂原激活的蛋白激酶（MAPK）是一类丝氨酸/苏氨酸蛋白质激酶,在植物生长发育、响应逆境胁迫及激素信号转导等方面具有重要作用。以谷子品种豫谷1号为试材,克隆与拟南芥AtMAPK3同源性最高的谷子SiMAPK3基因,并系统利用生物信息学方法分析SiMAPK3蛋白理化性质、结构与功能;利用RT-qPCR技术检测SiMAPK3基因在谷子拔节前期不同组织和不同非生物逆境胁迫下的表达水平。结果表明,谷子SiMAPK3基因开放阅读框为1 128 bp,编码一个含有376个氨基酸的蛋白,预测蛋白分子量为43 427.85 Da,等电点为5.46。谷子SiMAPK3为不含信号肽的亲水性膜外蛋白,其二级结构包含44.27%的α螺旋、14.67%的β折叠、5.07%的延伸链及36.00%的无规则卷曲,其第44-328位氨基酸之间含有Pkinase保守结构域,属于MAPK蛋白激酶家族。谷子SiMAPK3三级结构与拟南芥MAPK具有很高的相似度,存在着11个丝氨酸、8个苏氨酸、4个酪氨酸及大量潜在磷酸化位点。RT-qPCR分析表明,SiMAPK3在拔节前期谷子根、茎和叶中均有表达,其在叶片中的表达量最高,约为...     

植物MAPK级联途径参与调控ABA信号转导

促分裂原活化蛋白激酶(MAPK)级联途径信号通路在真核生物细胞信号的转换和放大过程中起重要作用。MAPK级联途径由三个成员组成,分别是MAPK、MAPKK及MAPKKK,此三个信号组分按照MAPKKK-MAPKK-MAPK的方式依次磷酸化将外源信号级联放大向下传递。大量研究表明,植物MAPK级联途径参与调控脱落酸(ABA)信号转导。因此,该文就ABA和MAPK的生物学功能、ABA信号转导中的磷酸化与去磷酸化以及MAPK级联途径与ABA信号转导之间的关系等方面的研究进展进行综述,以便进一步认识MAPK和ABA信号转导的分子机制。     

植物蛋白激酶与作物非生物胁迫抗性的研究

干旱、盐碱、高温等非生物逆境胁迫严重影响作物生长发育、产量和品质。在遭受非生物逆境的威胁时,植物通过信号受体,可感知、转导胁迫信号,启动一系列抗逆相关基因的表达,最终缓解或抵御非生物逆境胁迫对植物造成的危害。其中,蛋白激酶和蛋白磷酸酯酶的磷酸化/去磷酸化作用在植物感受外界胁迫信号的分子传递过程中起到开关的作用。正常情况下,蛋白激酶磷酸化开启信号转导途径,启动相应的抗逆基因表达反应;当信号消失后,蛋白激酶去磷酸化将信号转导途径关闭,达到调控植物正常生长的目的。因此,蛋白激酶在调控感受胁迫信号、启动各种非生物逆境胁迫响应中起到了极其重要的作用。近年来,对植物蛋白激酶参与非生物胁迫响应的研究倍受关注。本文阐述了不同类型蛋白激酶在改良作物非生物胁迫抗性上的应用,为进一步研究提供资料。     

MAPK级联及其在植物抗病防卫反应中的研究进展

促分裂原活化蛋白激酶(MAPK)级联信号转导途径参与了生物体生长发育和抗逆胁迫生理。植物MAPK级联途径一般由三个丝氨酸/苏氨酸蛋白激酶组分构成:包括MAPKKK(MEKK、MAP3K)、MAPKK(MEK)和MAPK。植物在响应外界环境刺激时,MAPKKK首先被自磷酸化激活,依次通过磷酸化激活MAPKK和MAPK,进而将外界信号在细胞内传递从而调控目标基因的表达。MAPK级联途径参与植物激素、生物胁迫、非生物胁迫等过程的信号传递,本文就MAPK级联途径在植物抗病防卫反应中的研究进展进行综述。     

葡萄VvBAP1基因的克隆及表达特性分析

以抗性葡萄品种‘F-242’组培苗为材料, 利用同源克隆法克隆了葡萄VvBAP1基因。测序结果显示, VvBAP1扩增片段大小为531 bp, 可编码176个氨基酸序列。利用生物信息学分析VvBAP1基因编码的蛋白序列显示, 该蛋白分子量为19.43 kDa, 含有保守的钙离子依赖性的C2结构域; 等电点pI为9.42; 不稳定系数为37.09, 推测为稳定的亲水性蛋白; 含有多个丝氨酸/苏氨酸磷酸化位点。实时荧光定量PCR表明, 该基因在根茎叶中均有表达, 其中在叶片中表达量较高; 盐胁迫、低温等逆境因子及逆境相关的信号物质, 如水杨酸和一氧化氮均可诱导VvBAP1的表达, 其中低温对其表达量影响更为显著, 推测该基因参与了葡萄抵御逆境胁迫的过程, 尤其是与低温相关的过程。     

Rnc1:一种新的MAPK信号转导途径的负反馈调节蛋白

丝裂素活化蛋白激酶 (mitogenactivatedproteinkinase ,MAPK )磷酸酶可选择性地使MAPK分子中磷酸丝氨酸及苏氨酸残基脱磷酸化 ,导致MAPK失活 ,从而负反馈调节MAPK对细胞外信号的转导。SugluraR等在裂殖酵母中发现一种新的K homology (KH)RNA结合蛋白Rnc1,它可以结合并稳定Pmp1(一种负调控Pmk1 MAPK的磷酸酶 )的mRNA ,增加Pmp1的表达 ;而Pmk1可使Rnc1磷酸化 ,并加强和稳固其与Pmp1mRNA结合 ,因此Rnc1是MAPK信号转导途径负反馈调控环路的一个新的成员。此发现对MAPK信号转导途径调控机制的深入理解有重大意义Rnc1:一种新…     

Substrate discrimination among mitogen-activated protein kinases through distinct docking sequence motifs

Mitogen-activated protein kinases (MAPKs) mediate cellular responses to a wide variety of extracellular stimuli. MAPK signal transduction cascades are tightly regulated, and individual MAPKs display exquisite specificity in recognition of their target substrates. All MAPK family members share a common phosphorylation site motif, raising questions as to how substrate specificity is achieved. Here we describe a peptide library screen to identify sequence requirements of the DEF site (docking site for ERK FXF), a docking motif separate from the phosphorylation site. We show that MAPK isoforms recognize DEF sites with unique sequences and identify two key residues on the MAPK that largely dictate sequence specificity. Based on these observations and computational docking studies, we propose a revised model for MAPK interaction with substrates containing DEF sites. Variations in DEF site sequence requirements provide one possible mechanism for encoding complex target specificity among MAPK isoforms.     

CDNA cloning and characterization of the Ve homologue gene StVe from Solanum torvum Swartz.

Verticillium wilt is a disastrous disease causing significant yield losses of many crops. Isolation of verticillium wilt resistance gene is a fundamental work for controlling this disease through genetic engineering. In this report, we describe the cloning and characterization of a Ve like gene (StVe) from Solanum torvum Swartz. The nucleotide sequence of StVe is 3640 bp long with an open reading frame of 3414 bp encoding a protein precursor of 1138 aa. Sharing high homologies to tomato verticillium wilt disease resistance genes Ve1 and Ve2, the leucine rich (15.89%) protein StVe has a calculated molecular weight of 126.48kDa with an isoelectric point of 5.62. It possesses a hydrophobic N-terminal signal peptide of 20 aa and 38 predicted leucine-rich repeats containing 32 potential N-glycosylation sites (28 being significant). Fifty-seven predicted phosphorylation sites (36 for S, 8 for T and 13 for Y) distribute in StVe protein. A PEST-like sequence and a mammalian endocytosis signals YCVF are found within the C-terminal region. The C terminus of StVe concludes with the residues KKF similar to the KKX motif that confers endoplasmic reticulum localization in plants as well as mammals and yeast. The sequence analysis of the StVe gene implies that the StVe is a potential verticillium wilt disease resistance gene encoding a cell surface-like receptor protein.     

Isolation of the GSY1 gene encoding yeast glycogen synthase and evidence for the existence of a second gene

Glycogen synthase preparations from Saccharomyces cerevisiae contained two polypeptides of molecular weights 85,000 and 77,000. Oligonucleotides based on protein sequence were utilized to clone a S. cerevisiae glycogen synthase gene, GSY1. The gene would encode a protein of 707 residues, molecular mass 80,501 daltons, with 50% overall identity to mammalian muscle glycogen synthases. The amino-terminal sequence obtained from the 85,000-dalton species matched the NH2 terminus predicted by the GSY1 sequence. Disruption of the GSY1 gene resulted in a viable haploid with glycogen synthase activity, and purification of glycogen synthase from this mutant strain resulted in an enzyme that contained the 77,000-dalton polypeptide. Southern hybridization of genomic DNA using the GSY1 coding sequence as a probe revealed a second weakly hybridizing fragment, present also in the strain with the GSY1 gene disrupted. However, the sequences of several tryptic peptides derived from the 77,000-dalton polypeptide were identical or similar to the sequence predicted by the GSY1 gene. The data are explained if S. cerevisiae has two glycogen synthase genes encoding proteins with significant sequence similarity The protein sequence predicted by the GSY1 gene lacks the extreme NH2-terminal phosphorylation sites of the mammalian enzymes. The COOH-terminal phosphorylated region of the mammalian enzyme over-all displayed low identity to the yeast COOH terminus, but there was homology in the region of the mammalian phosphorylation sites 3 and 4. Three potential cyclic AMP-dependent protein kinase sites are located in this region of the yeast enzyme. The region of glycogen synthase likely to be involved in covalent regulation are thus more variable than the catalytic center of the molecule.     

Sequence Alignment Reveals Possible MAPK Docking Motifs on HIV Proteins

Over the course of HIV infection, virus replication is facilitated by the phosphorylation of HIV proteins by human ERK1 and ERK2 mitogen-activated protein kinases (MAPKs). MAPKs are known to phosphorylate their substrates by first binding with them at a docking site. Docking site interactions could be viable drug targets because the sequences guiding them are more specific than phosphorylation consensus sites. In this study we use multiple bioinformatics tools to discover candidate MAPK docking site motifs on HIV proteins known to be phosphorylated by MAPKs, and we discuss the possibility of targeting docking sites with drugs. Using sequence alignments of HIV proteins of different subtypes, we show that MAPK docking patterns previously described for human proteins appear on the HIV matrix, Tat, and Vif proteins in a strain dependent manner, but are absent from HIV Rev and appear on all HIV Nef strains. We revise the regular expressions of previously annotated MAPK docking patterns in order to provide a subtype independent motif that annotates all HIV proteins. One revision is based on a documented human variant of one of the substrate docking motifs, and the other reduces the number of required basic amino acids in the standard docking motifs from two to one. The proposed patterns are shown to be consistent with in silico docking between ERK1 and the HIV matrix protein. The motif usage on HIV proteins is sufficiently different from human proteins in amino acid sequence similarity to allow for HIV specific targeting using small-molecule drugs.     

Differential phosphorylation of c-Jun and JunD in response to the epidermal growth factor is determined by the structure of MAPK targeting sequences

MAPK phosphorylation of various substrates is mediated by the presence of docking sites, including the D domain and the DEF motif. Depending on the number and sequences of these domains, substrates are phosphorylated by specific subsets of MAPKs. For example, a D domain targets JNK to c-Jun, whereas a DEF motif is required for ERK phosphorylation of c-Fos. JunD, in contrast, contains both D and DEF domains. Here we show that these motifs mediate JunD phosphorylation in response to either ERK or JNK activation. An intact D domain is required for phosphorylation and activation of JunD by both subtypes of MAPK. The DEF motif acts together with the D domain to elicit efficient phosphorylation of JunD in response to the epidermal growth factor (EGF) but has no function on JunD phosphorylation and activation by JNK signaling. Furthermore, we show that conversion of a c-Jun sequence to a canonical DEF domain, as it is present in JunD, elicits c-Jun activation in response to EGF. Our results suggest that evolution of a particular modular system of MAPK targeting sequences has determined a differential response of JunD and c-Jun to ERK activation.