巴西橡胶树HbCMT的克隆与表达分析

通过PCR和RACE技术克隆了巴西橡胶树中一个染色质甲基化酶(Chromomethylase, CMT)基因（HbCMT）。HbCMT长2697 bp,含有2556 bp的阅读框,编码851个氨基酸。推测HbCMT分子量为95.67 KD,等电点为5.38,氨基酸序列与可可、葡萄、黄瓜、鹰嘴豆、番茄、拟南芥CMT家族成员的同源性分别为77％、74％、72%、67%、64%和52%。定量PCR分析表明HbCMT在巴西橡胶树的根、树皮、叶、胶乳中均有表达,其中在胶乳中表达量最低,此外HbCMT在橡胶树自根幼态无性系的胶乳中表达比老态无性系胶乳中的表达低。     

巴西橡胶树HbNAC1基因的克隆和表达分析

根据EST序列信息,利用RACE技术从巴西橡胶树(Hevea brasiliensis)中克隆了1个NAC类转录因子基因HbNAC1,其cDNA全长为1419 bp,含有完整的开放阅读框,编码309个氨基酸。推导的氨基酸序列含有1个NAM结构域,具有典型的NAC类蛋白的结构特征,与毛果杨(Populus trichocarpa)、蓖麻(Ricinus communis)、水稻(Oryza sativa)和拟南芥(Arabidopsisthaliana)中的相应氨基酸序列的同源性分别达79%、80%、57%和60%。聚类分析表明,HbNAC1属于NAC家族Ⅱ亚族。半定量RT-PCR分析表明,该基因在胶乳中的表达较丰富,经乙烯利刺激后其在胶乳中的表达量明显增加。     

巴西橡胶树HbCMT1的克隆与表达分析

通过PCR和RACE技术克隆获得了巴西橡胶树染色质甲基化酶(CMT,chromomethylase)基因(Hb CMT1)。Hb CMT1全长2697 bp,含有2556 bp的阅读框,编码851个氨基酸。推测Hb CMT1分子量为95.67 k D,等电点为5.38,氨基酸序列与可可、烟草、葡萄、黄瓜、鹰嘴豆和拟南芥等CMT家族成员的同源性分别为66%、51%、50%、56%、53%和50%。定量PCR分析表明Hb CMT1在巴西橡胶树的根、树皮、叶、胶乳中均有表达,其中在叶中表达量最高,在胶乳中表达量最低。此外Hb CMT1在橡胶树自根幼态无性系胶乳中的表达量比老态无性系胶乳中的低。     

巴西橡胶树 HbERFB4-1 基因的克隆和表达分析

根据EST序列信息,利用RACE技术分离了巴西橡胶树(Hevea brasiliensis)的1个AP2/ERF类基因,命名为HbERFB4-1。该基因cDNA全长732 bp,含有完整的开放阅读框架,编码147个氨基酸,不含内含子。推导的HbERFB4-1氨基酸序列与杨树(Populus trichocarpa)、蓖麻(Ricinus communis)、拟南芥(Arabidopsis thaliana)和大豆(Glycine max)中相应蛋白氨基酸序列的一致性分别为78%、71%、62%和55%,具有典型的AP2类蛋白结构特征。聚类分析表明,HbERFB4-1属于ERF家族B4亚族。半定量RT-PCR分析结果表明,该基因在胶乳中的表达量相对较低,但乙烯利刺激后其在胶乳中的表达量迅速增加,推测该基因可能参与了胶乳中乙烯信号的转导。     

巴西橡胶树4-羟基-3-甲基-2-(E)-丁烯基-4-磷酸还原酶基因(Hb-HDR)的克隆及表达分析

4-羟基-3-甲基-2-(E)-丁烯基-4-磷酸还原酶(4-hydroxy-3-methyl-2-(E)-butenyl-4-diphosphatereductase,HDR)是异戊烯基焦磷酸合成途径之一甲基赤藓糖磷酸(methylerythritol phosphate,MEP)途径中的最后一个酶,催化4-羟基-3-甲基-(2E)-丁烯基-4-磷酸生成异戊烯基焦磷酸.根据植物HDR的同源序列设计引物,通过RT-PCR结合RACE的方法在橡胶树中获得了与其相应的HDR基因,命名为HbHDR.序列分析表明HbHDR长1 627 bp,编码462个氨基酸,属于LYTB家族,该氨基酸序列与烟草、长春花、胡黄连、拟南芥、银杏和火炬松的HDR同源性为79.1%、78.4%、76.5%、75.3%、72.2%和70.9%.半定量RT-PCR结果显示,乙烯诱导胶乳HbHDR的表达.     

巴西橡胶树鲨烯合酶基因的克隆与序列分析

鲨烯合酶(SQS )是植物甾醇和三萜化合物生物合成途径中的关键酶。以巴西橡胶树为试验材料,提取胶乳总 RNA,利用 RT-PCR 以及 RACE 的方法克隆橡胶树鲨烯合酶 cDNA 编码区片段,并进行序列分析。结果表明：橡胶树鲨烯合酶 cDNA 编码区为1239 bp,编码413个氨基酸,命名为 HbSQS 。荧光定量分析表明鲨烯合酶基因在不同组织里表达水平存在明显差异,且受乙烯调控。     

巴西橡胶树一个编码含有C2结构域蛋白cDNA的克隆及表达分析

本研究根据从巴西橡树胶乳cDNA文库中获得的一个EST片段的序列信息设计引物,通过RACE的方法获得了橡胶树编码含有C2结构域蛋白的cDNA(命名为HbC2)。序列分析表明,HbC2长为1185bp,含有813bp的阅读框,140bp的5'-UTR和232bp的3'-UTR,编码270个氨基酸,分子量为30.9KD,等电点为6.29,含有保守的C2结构域。半定量RT-PCR分析表明HbC2在花、芽、叶、胶乳和树皮中都有表达,其中在胶乳中表达量最高。茉莉酸可抑制HbC2的表达,乙烯对HbC2的表达没有影响。此研究为进一步研究C2蛋白基因在橡胶树中的生物学功能奠定基础。     

巴西橡胶树液泡ATP酶F亚基基因克隆及表达

根据筛选文库时获得的EST序列信息,利用RACE技术分离了一个巴西橡胶树ATP酶 F亚基基因,命名为HbVHA-F.结果显示,该基因cDNA全长658 bp,含有完整的阅读框架,编码130个氨基酸.序列比对及结构预测分析表明,HbVHA-F编码的氨基酸序列与杨树、水稻、黄麻、小麦和香蕉中相应基因氨基酸序列的一致性分别达到88.46%、86.15%、84.62%、83.85%和74.62%,与杨树一致性最高,并与其他高等植物聚为一类.半定量RT-PCR分析显示,割胶(机械伤害)和乙烯利能够诱导HbVHA-F基因的表达.HbVHA-F基因可能通过转录表达调节参与了乙烯利刺激橡胶树增产的分子调控.     

巴西橡胶树HbHEV3基因的克隆和表达分析

根据EST序列信息,通过RT-PCR获得了一个编码橡胶素前体的基因,命名为HbHEV3。HbHEV3编码区cDNA长度为630bp,编码209个氨基酸。预测的HbHEV3蛋白包含1个信号肽,1个具有几丁质结合特性的Hevein结构域和1个Barwinn结构域,HbHEV3与橡胶树及其他植物中类似蛋白具有很高的同源性。分离获得了HbHEV3起始密码子上游1 050bp的启动子序列,该序列含有众多应答激素和胁迫信号元件。实时荧光定量PCR分析结果表明,HbHEV3在所检测的组织中均有表达,其中在胶乳中的表达量最高,乙烯诱导能显著上调胶乳中HbHEV3的表达。研究表明,HbHEV3可能参与了橡胶树乙烯介导的防御反应,并在橡胶凝集过程中具有重要功能。     

橡胶树DELLA蛋白编码基因HbGAI的克隆与表达分析

该研究采用RT-PCR与RACE技术,从橡胶树‘热研7-33-97’胶乳中克隆了1个DELLA蛋白编码基因HbGAI(GenBank登录号为KT696439)。HbGAI全长cDNA序列2 050bp,包含1个长1 842bp的完整开放阅读框。序列分析显示,HbGAI基因编码613个氨基酸,其推导的蛋白含有DELLA和GRAS结构域,分子量为66.476kD,理论等电点为5.19,无跨膜结构域,属于亲水性蛋白。进化树分析表明,HbGAI蛋白与其他植物中DELLA蛋白具有较高的相似性,与麻疯树JcGAI和蓖麻RcGAI亲缘关系较近。荧光定量PCR结果显示,割胶和茉莉酸甲酯处理下调胶乳中HbGAI基因的表达,乙烯利处理4h内显著上调胶乳中HbGAI基因的表达,表明HbGAI基因可能在橡胶树割胶、茉莉酸、乙烯响应中发挥作用。     

Two yeast genes encoding calmodulin-dependent protein kinases. Isolation, sequencing and bacterial expressions of CMK1 and CMK2

We have isolated two genes from Saccharomyces cerevisiae that both encode a calmodulin-dependent protein kinase (CaM kinase). The CMK1 gene has been cloned by hybridization using an oligonucleotide probe synthesized on the basis of the peptide sequence of purified yeast CaM kinase (Londesborough, J. (1989) J. Gen. Microbiol. 135, 3373-3383). The other gene, CMK2, which is homologous to CMK1, has been isolated by screening at low stringency with a CMK1 fragment as a probe. The CMK2 product expressed in bacteria shows Ca(2+)- and CaM-dependent protein kinase activity, indicating that CMK2 also encodes a CaM kinase. The CMK1 and CMK2 products expressed in bacteria were found to have different biochemical properties in terms of autoregulatory activity and preference for yeast CaM or bovine CaM for maximal activity. Antibody raised against a peptide fragment of the CMK1 protein cross-reacts with the CMK2 product. Immunoblotting with this antibody indicated that the CMK1 and CMK2 products have apparent molecular masses of 56 and 50 kDa, respectively, in yeast cells. The predicted amino acid sequences of the two CMK products exhibit highest similarity with mammalian calmodulin-dependent multifunctional protein kinase II (CaM kinase II): the similarity within the N-terminal catalytic domain is about 40%, whereas that within the rest of the sequence is 25%. These data indicate that yeast has two kinds of genes encoding CaM kinase isozymes whose structural and functional properties are closely related to those of mammalian CaM kinase II. Another gene may be substituted for function of the CMK1 and CMK2 kinase in vivo, since elimination of both kinase genes is not lethal.     

Multiple Ca2+/calmodulin-dependent protein kinase genes in a unicellular eukaryote.

We purified a Ca2+/calmodulin (CaM)-dependent protein kinase (CaM kinase) from the yeast Saccharomyces cerevisiae with properties similar to mammalian type II CaM kinases. Degenerate oligonucleotides designed on the basis of the amino acid sequence of tryptic peptides from the 55 kd subunit of the yeast CaM kinase were used to isolate its gene from a set of lambda gt11-yeast genomic DNA phage clones initially selected by the ability to bind 125I-labelled yeast CaM. The cloned gene (CMK1) encodes an open reading frame that is homologous to the sequences of vertebrate type II CaM kinases. Several criteria demonstrated that the CMK1 gene product is the 55 kd polypeptide. Neither over-production (11-fold) nor complete elimination of the CMK1 gene product had any detectably deleterious effect on yeast cell growth. Extracts from cmk1 delta cells, which lacked detectable p55 using an antiserum raised against a Staphylococcus aureus protein A-CMK1 fusion protein, possessed significant residual Ca2+/CAM-dependent protein kinase activity. Using the CMK1 gene as a probe at low stringency, a second gene (CMK2) encoding another CaM-dependent protein kinase with striking sequence similarity to CMK1 was cloned. Deletion of CMK2, or both CMK1 and CMK2, was not lethal, although loss of CMK2 caused a slow rate of spore germination.     

Developmental changes in phosphorylation of MAP-2 and synapsin I in cytosol and taxol polymerised microtubules from chicken brain

In cytosol, cyclic AMP stimulated phosphorylation of microtubule associated protein-2 (MAP-2) increased from 2 days to adult in proportion to the increase in the concentration of MAP-2. By contrast, the calmodulin stimulated phosphorylation of MAP-2 decreased in proportion to the decrease in the concentration of calmodulin stimulated protein kinase II (CMK II). Similarly, the cAMP stimulated phosphorylation of the site on synapsin I labeled by the cAMP stimulated protein kinase (PKA) changed little during development whereas the calcium/calmodulin stimulated phosphorylation of the CMK II site decreased dramatically in proportion to the decrease in the concentration of CMK II. The decrease in the concentration of CMK II which occurs in cytosol during synapse maturation was also observed in taxol polymerised microtubules and the effects of the change in the relative concentrations of CMK II and PKA on the phosphorylation of MAP-2 and synapsin I in this fraction were similar to that observed in the cytosol. These results are consistent with the hypothesis that the developmental changes in phosphorylation of endogenous substrates by PKA is controlled largely by changes in the concentration of those substrates, whereas the concentration of CMK II is limiting so that the developmental changes in the phosphorylation of endogenous substrates by CMK II are a function of the concentration of CMK II itself as well as the concentration of endogenous substrates. Some possible functional consequences of this during synapse maturation are discussed.Special issue dedicated to Dr. Lawrence Austin     

Mimicking direct protein–protein and solvent-mediated interactions in the CDP-methylerythritol kinase homodimer: a pharmacophore-directed virtual screening approach

The 2C-methylerythritol 4-phosphate (MEP) pathway for the biosynthesis of isopentenyl pyrophosphate and its isomer dimethylallyl pyrophosphate, which are the precursors of isoprenoids, is present in plants, in the malaria parasite Plasmodium falciparum and in most eubacteria, including pathogenic agents. However, the MEP pathway is absent from fungi and animals, which have exclusively the mevalonic acid pathway. Given the characteristics of the MEP pathway, its enzymes represent potential targets for the generation of selective antibacterial, antimalarial and herbicidal molecules. We have focussed on the enzyme 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol kinase (CMK), which catalyses the fourth reaction step of the MEP pathway. A molecular dynamics simulation was carried out on the CMK dimer complex, and protein–protein interactions analysed, considering also water-mediated interactions between monomers. In order to find small molecules that bind to CMK and disrupt dimer formation, interactions observed in the dynamics trajectory were used to model a pharmacophore used in database searches. Using an intensity-fading matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry approach, one compound was found to interact with CMK. The data presented here indicate that a virtual screening approach can be used to identify candidate molecules that disrupt the CMK–CMK complex. This strategy can contribute to speeding up the discovery of new antimalarial, antibacterial, and herbicidal compounds.     

球孢白僵菌FUS3/KSS1类MAPK同源基因(BbMPK1)的克隆及特征分析

根据几种丝状真菌FUS3/KSS1类MAPK的保守序列设计简并引物,从昆虫病原真菌球孢白僵菌中扩增出MAPK基因的部分片段,进而利用YADE法延伸该片段的上、下游邻接序列,获得MAPK基因的全长序列,命名为BbMPK1。用3′RACE扩增出BbMPK1的全长cDNA序列,该序列含有一个1071bp的ORF,编码356个氨基酸的多肽,推测分子量为41.2kDa,等电点为6.61。BbMPK1含有11个MAPK共有的蛋白激酶区域和1个MAPK激酶作用的磷酸化位点区域(TEY),其氨基酸序列与丝状真菌的TMKA、PMK1、CMK1、FMK1和BMP1等MAPK高度同源。系统聚类结果表明,BbMPK1属于酵母FUS3/KSS1类MAPK。Southern杂交表明,BbMPK1在球孢白僵菌基因组中以单拷贝形式存在。RT-PCR分析表明BbMPK1在分生孢子休眠阶段、萌发阶段和菌丝生长时期均表达。研究结果为阐明酵母FUS3/KSS1类MAPK同源基因在球孢白僵菌中的作用奠定了基础。     

The Colletotrichum lagenarium MAP kinase gene CMK1 regulates diverse aspects of fungal pathogenesis

The infection process of Colletotrichum lagenarium, the causal agent of cucumber anthracnose disease, involves several key steps: germination; formation of melanized appressoria; appressorial penetration; and subsequent invasive growth in host plants. Here we report that the C. lagenarium CMK1 gene encoding a mitogen-activated protein (MAP) kinase plays a central role in these infection steps. CMK1 can complement appressorium formation of the Pmk1 MAP kinase mutant of Magnaporthe grisea. Deletion of CMK1 causes reduction of conidiation and complete lack of pathogenicity to the host plant. Surprisingly, in contrast to M. grisea pmk1 mutants, conidia of cmk1 mutants fail to germinate on both host plant and glass surfaces, demonstrating that the CMK1 MAP kinase regulates conidial germination. However, addition of yeast extract rescues germination, indicating the presence of a CMK1-independent pathway for regulation of conidial germination. Germinating conidia of cmk1 mutants fail to form appressoria and the mutants are unable to grow invasively in the host plant. This strongly suggests that MAP kinase signaling pathways have general significance for infection structure formation and pathogenic growth in phytopathogenic fungi. Furthermore, three melanin genes show no or slight expression in the cmk1 mutant when conidia fail to germinate, suggesting that CMK1 plays a role in gene expression required for appressorial melanization.     

Effect of Zinc on Calmodulin-Stimulated Protein Kinase II and Protein Phosphorylation in Rat Cerebral Cortex

The effect of increasing concentrations of Zn2+ (1 microM-5 mM) on protein phosphorylation was investigated in cytosol (S3) and crude synaptic plasma membrane (P2-M) fractions from rat cerebral cortex and purified calmodulin-stimulated protein kinase II (CMK II). Zn2+ was found to be a potent inhibitor of both protein kinase and protein phosphatase activities, with highly specific effects on CMK II. Only one phosphoprotein band (40 kDa in P2-M phosphorylated under basal conditions) was unaffected by addition of Zn2+. The vast majority of phosphoprotein bands in both basal and calcium/calmodulin-stimulated conditions showed a dose-dependent inhibition of phosphorylation, which varied with individual phosphoproteins. Two basal phosphoprotein bands (58 and 66 kDa in S3) showed a significant stimulation of phosphorylation at 100 microM Zn2+ with decreased stimulation at higher concentrations, which was absent by 5 mM Zn2+. A few Ca2+/calmodulin-stimulated phosphoproteins in P2-M and S3 showed biphasic behavior; inhibition at less than 100 microM Zn2+ and stimulation by millimolar concentrations of Zn2+ in the presence or absence of added Ca2+/calmodulin. The two major phosphoproteins in this group were identified as the alpha and beta subunits of CMK II. Using purified enzyme, Zn2+ was shown to have two direct effects on CMK II: an inhibition of Ca2+/calmodulin-stimulated autophosphorylation and substrate phosphorylation activity at low concentrations and the creation of a new Zn(2+)-stimulated, Ca2+/calmodulin-independent activity at concentrations of greater than 100 microM that produces a redistribution of activity biased toward autophosphorylation and an alpha subunit with an altered mobility on sodium dodecyl sulfate-containing gels.     

The type II Ca/calmodulin-dependent protein kinases are involved in the regulation of cell wall integrity and oxidative stress response in Candida albicans

The type II Ca²⁺/calmodulin-dependent protein kinases (CaMKs) are thought to play a vital role in cellular regulation in mammalian cells. Two genes CMK1 and CMK2 in the Candida albicans genome encode homologues of mammalian CaMKs. In this work, we constructed the cmk1Δ/Δ, the cmk2Δ/Δ and the cmk1Δ/Δcmk2Δ/Δ mutants and found that CaMKs function in cell wall integrity (CWI) and cellular redox regulation. Loss of either CMK1 or CMK2, or both resulted in increased expression of CWI-related genes under Calcofluor white (CFW) treatment. Besides, CaMKs are essential for the maintenance of cellular redox balance. Disruption of either CMK1 or CMK2, or both not only led to a significant increase of intracellular ROS levels, but also led to a decrease of the mitochondrial membrane potential (MMP), suggesting the important roles that CaMKs play in the maintenance of the mitochondrial function.