AtPLC2, a gene encoding phosphoinositide-specific phospholipase C,is constitutively expressed in vegetative and floral tissues in Arabidopsis thaliana

A cDNA encoding a phosphoinositide-specific phospholipase C (PI-PLC) from the higher plant Arabidopsis thaliana was cloned and characterized.The gene corresponding to this cDNA is designated AtPLC2. The overall structure of the predicted AtPLC2 protein is similar to those of plant PI-PLCs and mammalian -type PI-PLCs. Northern blot analysis revealed that AtPLC2 is expressed constitutively whereas AtPLC1S, another gene for PI-PLC of Arabidopsis, is induced by environmental stresses such as dehydration and salinity, indicating that the function of AtPLC2 is distinct from that of AtPLC1S. The AtPLC2 mRNA was detected in vegetative and floral tissues. We determined the positions of these two PI-PLCs genes on Arabidopsis chromosomes by RFLP mapping using P1 genomic clones.     

Phosphoinositide-specific phospholipase C9 is involved in the thermotolerance of Arabidopsis

Intracellular calcium (Ca(2+)) increases rapidly after heat shock (HS) in the Ca(2+)/calmodulin (Ca(2+)/CaM) HS signal transduction pathway: a hypothesis proposed based on our previous findings. However, evidence for the increase in Ca(2+) after HS was obtained only through physiological and pharmacological experiments; thus, direct molecular genetic evidence is needed. The role of phosphoinositide-specific phospholipase C (PI-PLC) is poorly understood in the plant response to HS. In this work, atplc9 mutant plants displayed a serious thermosensitive phenotype compared with wild-type (WT) plants after HS. Complementation of atplc9 with AtPLC9 rescued both the basal and acquired thermotolerance phenotype of the WT plants. In addition, thermotolerance was even improved in overexpressed lines. The GUS staining of AtPLC9 promoter:GUS transgenic seedlings showed that AtPLC9 expression was ubiquitous. The fluorescence distribution of the fusion protein AtPLC9 promoter:AtPLC9:GFP revealed that the subcellular localization of AtPLC9 was restricted to the plasma membrane. The results of a PLC activity assay showed a reduction in the accumulation of inositol-1,4,5-trisphosphate (IP(3)) in atplc9 during HS and improved IP(3) generation in the overexpressed lines. Furthermore, the heat-induced increase in intracellular Ca(2+) was decreased in atplc9. Accumulation of the small HS proteins HSP18.2 and HSP25.3 was downregulated in atplc9 and upregulated in the overexpressed lines after HS. Together, these results provide molecular genetic evidence showing that AtPLC9 plays a role in thermotolerance in Arabidopsis.     

Characterization of a sulfurtransferase from Arabidopsis thaliana.

A database search for similarities between sequenced parts of the Arabidopsis thaliana genome with known sulfurtransferase sequences from Escherichia coli and mammals was undertaken to obtain information about plant sulfurtransferase-like proteins. One gene and several homologous EST clones were identified. One of the EST clones was used for screening an Arabidopsis cDNA library. The isolated full-length clone consists of 1134 bp and encodes a 42.6 kDa protein that includes a putative transit peptide sequence of about 7.1 kDa. Sequence comparisons with known sulfurtransferases from different organisms confirmed high homology between them and the existence of several highly conserved regions. Results of a Southern blot performed with genomic Arabidopsis DNA showed the occurrence of at least two sulfurtransferase-like isozymes in Arabidopsis. Recombinant proteins with and without the putative transit peptide were expressed in E. coli with an N-terminal His6-tag, purified by affinity chromatography and tested for enzyme activity using different sulfur donors and acceptors. Both recombinant proteins catalyzed the formation of SCN- from thiosulfate and cyanide as a rhodanese per definition; however, both recombinant proteins preferred 3-mercaptopyruvate to thiosulfate. A monospecific antibody produced by using the mature recombinant protein as an antigen recognized a single protein band in total extracts of Arabidopsis plants equating to the full-length protein size. A single band equating to the size of the mature protein was detected from purified Arabidopsis mitochondria, but there was no antigenic reaction with any protein from chloroplasts. The function of the protein is still speculative. Now tools are available to elucidate the roles and substrates of this sulfurtransferase in higher plants.     

Characterization of a plasma membrane-associated phosphoinositide-specific phospholipase C from soybean

Phosphoinositide-specific phospholipase C (PI-PLC) is a key signal transducing enzyme which generates the second messengers inositol trisphosphate and diacylglycerol in mammalian cells. A cDNA clone (PI-PLC1) encoding a phosphoinositide-specific phospholipase C was isolated from soybean by screening a cDNA expression library using an anti-(plasma membrane) serum. Genomic DNA gel blot analysis suggested that the corresponding gene is a member of a multigene family. The deduced amino acid sequence of the soybean PI-PLC1 isozyme contains the conserved X and Y regions, found in other PI-PLCs. It is closely related to mammalian δ-type PI-PLCs, Dictyostelium discoideum PI-PLC and yeast PI-PLC1 in terms of the arrangement of the conserved region. Unlike mammalian δ-type PI-PLCs and yeast PI-PLC1, the putative Ca²⁺-binding site of the soybean PI-PLC1 is located in the region spanning the X and Y domains, and the N-terminal region is truncated. FLAG epitope-tagged PI-PLC1 fusion protein purified from transgenic tobacco plants showed phosphoinositide-specific phospholipase C activity. Heterologous expression of the soybean PI-PLC1 cDNA in a yeast PI-PLC1 deletion mutant complemented the lethality phenotype of haploid PI-PLC1 disruptants. Immunoblot analysis of the cell fractions prepared from transgenic tobacco plants over-expressing the FLAG epitope-tagged PI-PLC1 fusion protein indicated that the protein encoded by the PI-PLC1 cDNA was localized in the cytosol and plasma membrane.     

An increase in phosphoinositide-specific phospholipase C activity precedes induction of C4 phosphoenolpyruvate carboxylase phosphorylation in illuminated and NH4Cl-treated protoplasts from Digitaria sanguinalis

A Ca2+-dependent phosphoinositide-specific phospholipase C (PI-PLC) activity has been characterized in the microsomal fraction of Digitaria sanguinalis mesophyll cell protoplasts. Microsomal PI-PLC was found to be inhibited in vitro by a mammalian anti-PLC-delta1 antibody and by the aminosteroide U-73122, an inhibitor of PI-PLC activity in animal cells. In Western blot experiments, the antibody recognized an 85 kDa protein in both microsomal protein extracts from mesophyll protoplasts and rat brain protein extracts containing the authentic enzyme. The involvement of the microsomal PI-PLC in the light-dependent transduction pathway leading to the phosphorylation of C4 phosphoenolpyruvate carboxylase (PEPC) was investigated in D. sanguinalis protoplasts. A transient increase in the PI-PLC reaction product inositol-1,4,5-trisphosphate (Ins(1,4, 5)P3) was observed in situ during early induction of the C4 PEPC phosphorylation cascade. U-73122, but not the inactive analogue U-73343, efficiently blocked the transient accumulation of Ins(1,4, 5)P3, and both the increase in C4 PEPC kinase activity and C4 PEPC phosphorylation in illuminated and weak base-treated protoplasts. Taken together, these data suggest that PI-PLC-based signalling is a committed step in the cascade controlling the regulation of C4 PEPC phosphorylation in C4 leaves.     

N-terminal EF-hand-like domain is required for phosphoinositide-specific phospholipase C activity in Arabidopsis thaliana

Phosphoinositide-specific phospholipase C's (PI-PLCs) are ubiquitous in eukaryotes, from plants to animals, and catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate into the two second messengers inositol 1,4,5-trisphosphate and diacylglycerol. In animals, four distinct subfamilies of PI-PLCs have been identified, and the three-dimensional structure of one rat isozyme, PLC-delta1, determined. Plants appear to contain only one gene family encoding PI-PLCs. The catalytic properties of plant PI-PLCs are very similar to those of animal enzymes. However, very little is known about the regulation of plant PI-PLCs. All plant PI-PLCs comprise three domains, X, Y and C2, which are also conserved in isoforms from animals and yeast. We here show that one PI-PLC isozyme from Arabidopsis thaliana, AtPLC2, is predominantly localized in the plasma membrane, and that the conserved N-terminal domain may represent an EF-hand domain that is required for catalytic activity but not for lipid binding.     

Isolation of a unique membrane protein from Naegleria fowleri

Naegleria fowleri, an amoeboflagellate, is the causative agent of Primary Amoebic Meningoencephalitis, a fulminating disease of the central nervous system. In order to elucidate the mechanisms of pathogenicity of this amoeba, a cDNA expression library was prepared from N. fowleri RNA. A specific protein was found to be expressed from a cDNA clone designated Mp2CL5. Northern blot analysis showed that the Mp2CL5 mRNA was expressed in pathogenic N. fowleri but was not expressed in non-pathogenic Naegleria species nor in Acanthamoeba. Western blot analysis using anti-N. fowleri antiserum demonstrated that IPTG-induced Escherichia coli Mp2CL5 expressed a 23-kDa recombinant protein. The Mp2CL5 recombinant protein was histidine-tagged and purified to homogeneity from E. coli. A polyclonal rabbit antiserum was prepared against the purified Mp2CL5 recombinant protein. This antibody was used to further characterize the Mp2CL5 native protein expressed by N. fowleri. Western blot analysis in conjunction with immunofluorescence microscopy demonstrated the presence of a native protein of 17 kDa on the plasma membrane of N. fowleri trophozoites. The native N. fowleri protein was expressed in the logarithmic phase of trophozoite growth and the production of this protein increased through the stationary phase of growth. Studies are in progress to examine further its role as a virulence factor.     

抗菌肽CM4与人可溶性B淋巴细胞因子hsBAFF在大肠杆菌中的融合表达

为进一步探讨抗菌肽CM4的原核表达及其生物学功能,本实验研究了抗菌肽CM4与人可溶性B淋巴细胞刺激因子hsBAFF的融合表达及抗菌肽CM4的生物学活性。运用PCR把B淋巴细胞因子hsBAFF和家蚕抗菌肽CM4进行基因融合,构建了融合表达载体pET28a (+)/CM4-hsBAFF,并在大肠杆菌中获得高可溶性表达的融合靶蛋白,且存在于超声破碎后的上清,经分子筛Sephadex G-75纯化后的重组融合蛋白用SDS-PAGE和Western blot分析鉴定.SDS-PAGE分析表明：可以通过分子筛一步纯化得到融合蛋白,该重组融合蛋白的分子量约22.0 KDa。Western blot结果显示该重组蛋白能与鼠抗人hsBAFF的抗体发生特异性反应.运用基因工程的方法获得CM4-hsBAFF重组融合蛋白,并具有很好的抑菌生物学活性。     

拟南芥中一种AtBAG4蛋白的抗体制备和特异性检测

以拟南芥AtBAG4的全长cDNA,构建pET-51780重组质粒,转化大肠杆菌BL21(DE3),IPTG可诱导融合蛋白高效表达,表达产物以可溶形式存在;经Ni-NTA层析柱分离纯化,可得到分子质量约为49kDa的PAGE纯蛋白。用纯化的融合蛋白pET-51780免疫家兔,可得到抗AtBAG4抗体。Western blot结果显示该抗体能特异识别原核系统内表达的抗原以及拟南芥自身的抗原。     

A proteomic analysis of arsenical drug resistance in Trypanosoma brucei

We have undertaken 2-DE and MS to identify proteins associated with arsenical drug resistance in Trypanosoma brucei. This parasite causes sleeping sickness in humans, and arsenical drug resistance is a significant potential problem. Comparative analysis of approximately 2000 spots resolved by 2-DE in the soluble proteomes of drug-sensitive and drug-resistant isogenic lines of T. brucei identified a protein spot whose absence associated with resistance to the arsenical drug, Cymelarsan. MS matched this protein to an identical pair of tandem genes Tb09.211.0120 and 0130 that encode a putative nascent polypeptide associated complex subunit. This protein also occurs as an isoform located in both resistant and sensitive lines at a similar molecular weight, but different pI. The difference between isogenic lines was confirmed by Western blot using an antibody against recombinant protein. Both genes were identical in sequence between drug-sensitive and drug-resistant lines and both were transcribed as determined by RT-PCR. We postulate that the missing protein isoform arose due to the lack of a PTM.     

草鱼Ⅲ型呼肠孤病毒NS66抗体制备及应用

[背景]草鱼Ⅲ型呼肠孤病毒(grass carp reovirus,GCRV genotypeⅢ) 104株可导致典型性草鱼出血病,对其编码片段的分析有望为临床免疫学检测提供依据。[目的]研究GCRV104株s6基因节段编码蛋白NS66的可能功能,制备良好的GCRV104株NS66蛋白多克隆抗体并分析其特异性。[方法]PCR方法扩增GCRV104株s6基因片段,并克隆至表达载体pGEX-4T-3,转化到大肠杆菌BL21后用IPTG诱导表达,其产物经SDS-PAGE鉴定分析后,通过纯化获得目的蛋白。然后用纯化的pGEX-4T-3-NS66重组蛋白免疫小鼠,获得Anti pGEX-4T-3-NS66多克隆抗体,Western blot测定抗体效价,Western blotting和间接免疫荧光试验(indirect immunofluorescence assay,IFA)鉴定抗体特异性。[结果]SDS-PAGE分析显示表达的重组蛋白约66 kD,大小与预期相符,主要存在于包涵体中;Western blotting测得制备的多克隆抗体效价大于1:50 000,Western blotting和IFA结果表明,制备的多克隆抗体能特异性识别GCRV104病毒。[结论]GCRV104病毒编码的非结构蛋白NS66可能参与了复制和组装过程,形成病毒包涵体,这为建立GCRV104免疫诊断方法及研究GCRV编码的NS66蛋白的功能奠定了前期基础。     

小鼠肝炎病毒N基因的原核表达和免疫活性初步分析

目的对小鼠肝炎病毒（mouse hapetitis virus）A59毒株核蛋白（N）基因进行了克隆、表达和重组蛋白的免疫活性分析。方法根据GenBank公布的MHV-A59 N基因序列（AY700211）,设计了一对特异性引物,通过RT-PCR扩增出N基因的主要抗原片段NS,将目的片段纯化后与pGEM-T-easy载体连接得到重组质粒pTN,双酶切回收目的基因片段克隆到原核表达载体pGEX-6p-1中,构建了重组质粒pGEX-NS,转化大肠杆菌BL21,用IPTG进行了诱导表达。对表达裂解物进行SDS-PAGE和Western-blotting验证。结果表达产物相对分子质量约56×103与预期相符;能与MHV阳性血清发生特异性反应,出现单一条带。结论原核表达的NP（S）蛋白有良好的抗原性,为检测小鼠肝炎病毒抗体的ELISA检测方法的研究奠定了基础。     

甲型H1N1流感病毒HA基因在昆虫细胞中的截短表达

目的:利用Bac-to-Bac Baculovirus Expression System表达重组HA蛋白,Western blot及IFA方法鉴定其表达。方法:采用PCR方法扩增A/California/04/2009(H1N1)HA基因,将其克隆到pFastBacHT A载体上,重组质粒pFastBacHT-HA经双酶切及测序鉴定正确后,转化阳性重组载体进入E.coli DH10Bac感受态细胞中,通过Bluo-gal蓝白斑筛选、PCR鉴定获得重组转座子rBacmid-HA。从重组转座子中提取rBacmid-HA质粒DNA转染sf 9昆虫细胞,制备重组杆状病毒。重组杆状病毒感染sf 9细胞表达重组蛋白,Western blot及IFA鉴定重组蛋白表达情况。结论:成功构建了甲型H1N1流感病毒HA基因的昆虫杆状病毒表达载体,该表达载体转染昆虫细胞后制备的重组杆状病毒病毒滴度较高,重组杆状病毒表达的重组蛋白经Western blot 及IFA 鉴定后具有良好的免疫反应原性。     

精子特异性蛋白Cabs1多克隆抗体的制备及多用途验证

制备抗小鼠Cabs1 (Calcium-binding and spermatid-specific protein 1)多克隆抗体,以便进一步利用小鼠模型解析Cabs1蛋白在精子变形与精子功能调控中的功能.以密码子的简并性和偏好性为原则,优化小鼠Cabs1基因编码序列,构建重组表达载体ET-30a(+)/Cabs1,...