Sequence analysis of keratin-like proteins and cloning of intermediate filament-like cDNA from higher plant cells

Two keratin-like proteins of 64 and 55 ku were purified from suspension cells of Daucus carota L.,and their partial amino acid sequences were determined.The homological analysis showed that the sequence from the 64 ku protein was highly homological to b -glucosidase,and that from the 55 ku protein had no significant homologue in GenBank.Using conservative sequence of animal IF proteins as primer,we cloned a cDNA fragment from Daucus carota L.Southern blot and Northern blot results indicated that this cDNA fragment was a single copy gene and expressed both in suspension cells and leaves.Homological analysis revealed that it had moderate homology to a variety of a -helical proteins.Our results might shed more light on molecular characterization of IF existence in higher plant.     

Isolation and molecular genetic characterization of the Bacillus subtilis gene (infB) encoding protein synthesis initiation factor 2.

Western blot (immunoblot) analysis of Bacillus subtilis cell extracts detected two proteins that cross-reacted with monospecific polyclonal antibody raised against Escherichia coli initiation factor 2 alpha (IF2 alpha). Subsequent Southern blot analysis of B. subtilis genomic DNA identified a 1.3-kilobase (kb) HindIII fragment which cross-hybridized with both E. coli and Bacillus stearothermophilus IF2 gene probes. This DNA was cloned from a size-selected B. subtilis plasmid library. The cloned HindIII fragment, which was shown by DNA sequence analysis to encode the N-terminal half of the B. subtilis IF2 protein and 0.2 kb of upstream flanking sequence, was utilized as a homologous probe to clone an overlapping 2.76-kb ClaI chromosomal fragment containing the entire IF2 structural gene. The HindIII fragment was also used as a probe to obtain overlapping clones from a lambda gt11 library which contained additional upstream and downstream flanking sequences. Sequence comparisons between the B. subtilis IF2 gene and the other bacterial homologs from E. coli, B. stearothermophilus, and Streptococcus faecium displayed extensive nucleic acid and protein sequence homologies. The B. subtilis infB gene encodes two proteins, IF2 alpha (78.6 kilodaltons) and IF2 beta (68.2 kilodaltons); both were expressed in B. subtilis and E. coli. These two proteins cross-reacted with antiserum to E. coli IF2 alpha and were able to complement in vivo an E. coli infB gene disruption. Four-factor recombination analysis positioned the infB gene at 145 degrees on the B. subtilis chromosome, between the polC and spcB loci. This location is distinct from those of the other major ribosomal protein and rRNA gene clusters of B. subtilis.     

植物细胞中NuMA类似蛋白的分布及其在细胞周期中的变化

免疫荧光染色结果说明植物细胞核内含有与抗动物ＮｕＭＡ多抗呈阳性交叉反应的多肽。选择性抽提并结合免疫荧光染色结果说明这种多肽位于核基质纤维蛋白网络上。免疫印迹反应显示胡萝卜（ＤａｕｃｕｓｃａｒｏｔａＬ．）悬浮培养细胞核基质蛋白与抗动物ＮｕＭＡ蛋白多抗的阳性反应条带为７４ｋＤ和７６ｋＤ。有丝分裂各期免疫荧光染色的结果表明植物细胞中的ＮｕＭＡ类似蛋白在有丝分裂过程中呈现有规律的变化。结合选择性抽提的有丝分裂各期的免疫荧光染色的结果表明核基质在此过程中也发生明显变化。应用选择性抽提并结合ＤＧＤ包埋去包埋电镜技术对植物细胞间期及有丝分裂期核基质的形态结构进行了观察。结果显示胡萝卜悬浮培养细胞间期核内存在一个非染色质性的纤维蛋白网络体系,而在正处于分裂的细胞中则未观察到。以上结果说明ＮｕＭＡ类似蛋白是核基质的组分之一并与有丝分裂密切相关。     

Isolation and Characterization of a cDNA Encoded an Embryogenic Cell Protein 63 Related to Embryogenesis from Carrot

cDNA library with about 6.0 x 108 plaques per μg phage from carrot ( Daucus carota L. cv. Hammaki) torpedo-shaped embryos was constructed by a eDNA cloning system (λgtl0). A fulllength cDNA for embryogenic cell protein 63 (ECP63), an embryogenic cell protein from carrot with a relative molecular weight of 63 000, was isolated from a eDNA library using PCR-amplified DNA as a probe. The nucleotide sequence of ECP63 cDNA was 1 989 bp in length. The cDNA encoded a polypeptide of 569 amino acids, and the calculated molecular weight of this pelypeptide was 62 000. The Northern blot analysis using labeled full-length ECP63 cDNA as a probe showed that the gene for ECP63 was expressed in embryogenic cells, globular, heart-, and torpedo-shaped embryos, but not in seedlings and non-embryogenic cells.     

番茄花粉特异表达的高赖氨酸蛋白基因(tsb)的克隆与特性分析

赖氨酸是人和单胃动物必需的八种氨基酸之一,食物蛋白中的必需氨基酸种类不全或数量不足,都会影响其它氨基酸的利用.玉米等主要禾谷类作物由于缺少赖氨酸,种子蛋白利用率还不到50%.在长期以玉米为口粮的地区,人们往往患有营养不足或糙皮病.     

Cytoplasmic intermediate filament proteins of invertebrates are closer to nuclear lamins than are vertebrate intermediate filament proteins; sequence characterization of two muscle proteins of a nematode.

The giant body muscle cells of the nematode Ascaris lumbricoides show a complex three dimensional array of intermediate filaments (IFs). They contain two proteins, A (71 kd) and B (63 kd), which we now show are able to form homopolymeric filaments in vitro. The complete amino acid sequence of B and 80% of A have been determined. A and B are two homologous proteins with a 55% sequence identity over the rod and tail domains. Sequence comparisons with the only other invertebrate IF protein currently known (Helix pomatia) and with vertebrate IF proteins show that along the coiled-coil rod domain, sequence principles rather than actual sequences are conserved in evolution. Noticeable exceptions are the consensus sequences at the ends of the rod, which probably play a direct role in IF assembly. Like the Helix IF protein the nematode proteins have six extra heptads in the coil 1b segment. These are characteristic of nuclear lamins from vertebrates and invertebrates and are not found in vertebrate IF proteins. Unexpectedly the enhanced homology between lamins and invertebrate IF proteins continues in the tail domains, which in vertebrate IF proteins totally diverge. The sequence alignment necessitates the introduction of a 15 residue deletion in the tail domain of all three invertebrate IF proteins. Its location coincides with the position of the karyophilic signal sequence, which dictates nuclear entry of the lamins. The results provide the first molecular support for the speculation that nuclear lamins and cytoplasmic IF proteins arose in eukaryotic evolution from a common lamin-like predecessor.     

The Existence of Intermediate Filament in Arabidopsis thaliana Cell

The network structure of cytoplasmic filaments of 10 nm in diameter was detected from callus cells of Arabidopsis thaliana (L.) Heynh by selective extraction combined with whole mount electron microscopy. Western blot analysis showed that the major filament components were 6 polypeptides, which reacted with keratin monoclonal antibody of AE1 or AE3 respectively. By indirect immunofluorescence technique, the AE1 and AE3-reactive antigens were localized throughout the cytoplasm in a diffused pattern. The 10 nm-plant filaments could be reassembled in vitro. These results demonstrated that keratin-like intermediate filaments exist in the cytoplasm of A. thaliana. Using conservative sequence of animal IF genes as primer, a cDNA fragment was further cloned from this model material by RT-PCR, which might shed more light on molecular characterization of IF existence in higher plant.     

The Lupinus albus class-III chitinase gene, IF3, is constitutively expressed in vegetative organs and developing seeds

 A cDNA fragment encoding a Lupinus albus. L. class-III chitinase, IF3, was isolated, using a cDNA probe from Cucumis sativus L., by in-situ plaque hybridization from a cDNA library constructed in the Uni-ZAP XR vector, with mRNAs isolated from mature lupin leaves. The cDNA had a coding sequence of 293 amino acids including a 27-residue N-terminal signal peptide. A class-III chitinase gene was detected by Southern analysis in the L. albus genome. Western blotting experiments showed that the IF3 protein was constitutively present during seed development and in all the studied vegetative lupin organs (i.e., roots, hypocotyls and leaves) at two growth stages (7- and 20-d-old plants). Accumulation of both the IF3 mRNA and IF3 protein was triggered by salicylic acid treatment as well as by abiotic (UV-C light and wounding) and biotic stress conditions (Colletotrichum gloeosporioides infection). In necrotic leaves, IF3 chitinase mRNA was present at a higher level than that of another mRNA encoding a pathogenesis-related (PR) protein from L. albus (a PR-10) and that of the rRNAs. We suggest that one role of the IF3 chitinase could be in the defense of the plant against fungal infection, though our results do not exclude other functions for this protein. Received: 15 March 1999 / Accepted: 12 July 1999     

拟南芥细胞中存在中间纤维的研究

利用整装电镜制样与选择性抽提技术,在拟南芥（Ａｒａｂｉｄｏｐｓｉｓｔｈａｌｉａｎａ （Ｌ．） Ｈｅｙｎｈ） 愈伤组织细胞质中观察到直径１０ ｎｍ 左右的纤维网络结构。免疫印迹分析表明纤维的主要成分是６ 种多肽,它们分别与动物角蛋白单克隆抗体ＡＥ１ 、ＡＥ３ 有免疫交叉反应。利用间接免疫荧光技术,与ＡＥ１ 和ＡＥ３ 反应的抗原呈弥散状定位于整个细胞质中,而且１０ ｎｍ 纤维可以在体外重新组装。以上结果表明,在拟南芥细胞质中存在类角蛋白的中间纤维。以动物中间纤维基因的保守序列为引物,采用ＲＴ＿ＰＣＲ技术,进一步从这一模式植物中克隆到一个ｃＤＮＡ片段,这可能为从分子水平上证明植物中间纤维的存在提供了一个线索     

菜豆病程相关蛋白基因在重金属胁迫下的表达分析

为探讨植物抗重金属的分子机理 ,差别筛选了 Hg Cl2 胁迫的菜豆 ( Phaseolus vulgaris L.)叶片 c DNA库 ,分离出一个重金属胁迫响应基因 Pv SR4克隆 .c DNA和氨基酸序列分析表明 Pv SR4编码一种细胞内病程相关蛋白 ,该蛋白具有 RNase活性 .Northern blot分析表明 Pv SR4基因在正常生长条件下的叶片中不表达 ,重金属 ( Hg、Cd、As、Zn和 Cu等 )和水杨酸能强烈地诱导其基因的表达 ,受伤也能促进该基因的转录 ,而热胁迫几乎没有调节作用 .推测 Pv SR4蛋白在诱导植物的抗逆性和抵抗重金属胁迫方面有重要作用     

The Expression of Mycobacterium tuberculosis MPT64 Protein in Transgenic Carrots

Oral vaccines produced by transgenic plants would change the traditional means of production and inoculation of vaccines and the cost of vaccine production would be reduced greatly. In the experiments, hypocotyls and cotyledons of carrot (Daucus carota L.var.sativa)were infected with Agrobacterium tumefaciens (Smith et Townsend) Conn LBA4404 containing Mycobacterium tuberculosis (Zopf) Lehmann et Neumann MPT64 gene under the control of the 35S promoter of cauliflower mosaic virus. After two days coculture, the explants were transferred to MS selection media which contained different concentrations of kanamycin and carbenicillin. The regenerated plants with kanamycin resistance were obtained through somatic embryogenesis from the embryogenic calli formed on the selection media. Some of the plants have been transplanted and grew well in phytotron. PCR and Southern blot analyses of carrot DNA confirmed that the MPT64 gene has been introduced into the plant genome. The results of Western blot showed that the MPT64 protein have been expressed in some transgenic plants. Therefore, the transgenic plants should provide a valuable tool for the development of edible oral vaccines.     

二化螟钙黏蛋白CAD1的原核表达及与Cry1Ac、Cry2Aa蛋白的结合

【目的】二化螟是水稻的重要害虫之一,钙黏蛋白(cadherin,CAD)是一类重要的Bt杀虫蛋白受体,在获得二化螟钙黏蛋白基因(Cs CAD1)的基础上,明确Cs CAD1蛋白与Cry1Ac和Cry2Aa蛋白的结合能力。【方法】利用PCR技术克隆Cs CAD1基因片段,将构建的p ET-28a-(+)-Cs CAD1重组质粒转入原核表达菌株BL21(DE3)中,IPTG诱导表达。目的蛋白经Ni柱亲和纯化后SDS-PAGE电泳检测,利用western blot和ligand blot技术分析其与Cry1Ac和Cry2Aa蛋白的结合能力。【结果】重组载体可在表达菌株BL21中表达一个约44 ku的蛋白,原核表达载体构建成功。SDS-PAGE显示该蛋白条带单一,且纯度较好。Ni柱亲和层析纯化该目的蛋白后进行Ligand blot分析,结果显示Cs CAD1重组蛋白可以与Cry1Ac和Cry2Aa蛋白结合。【结论】Cs CAD1蛋白可以与Cry1Ac和Cry2Aa蛋白结合,是潜在的Cry蛋白受体,所得结果有助于阐明Cry1Ac和Cry2Aa蛋白对二化螟的作用机制。     

Expression of <Emphasis Type="Italic">Lupinus albus</Emphasis> PR-10 proteins during root and leaf development

Based on the NH₂-terminal sequence of three PR-10 isoforms previously identified in Lupinus albus leaves and a conserved amino-acid region in the PR-10 proteins from leguminosae, a pair of oligonucleotides was designed and used to amplify the corresponding cDNA fragment from a L. albus leaves cDNA library. A fragment of DNA of 200 bp was isolated from the polymerase chain reaction (PCR) mixture and subsequently used to screen the cDNA library. A cDNA coding for a PR-10 protein of 158 amino acid residues was cloned and sequenced. Subsequent studies involving Northern and Western blot analysis have shown that the PR-10 protein isoforms are differentially expressed during the development of the healthy lupin plant. High mRNA and protein contents were detected in roots and hypocotyls of both 7- and 20-d-old plants. In young leaves, the mRNA and protein contents were low and increasead in mature leaves. Tissue printing experiments with root sections suggest that the proteins are extracellular and are mainly associated with the vascular tissues in mature roots.     

Evidence for phosphorylation of tubulin in carrot suspension cells

Two-dimensional gels of phosphoproteins from carrot ( Daucus carota L. var. Juwarot) suspension cells labeled in vivo or in vitro revealed phosphoproteins that comigrate with carrot tubulin. A polyclonal antiserum to hibiscus tubulin immunoprecipitated an in vivo labeled phosphoprotein of 50 kDa. Cell-free extracts of carrot suspension cells phosphorylated both purified carrot and bovine brain tubulins in the presence of gamma-labeled adenosine triphosphate. This tubulin phosphorylating activity was reduced 2-fold in extracts from globular stage embryos and approximately 10-fold in extracts from heart/torpedo stage embryos. These data suggest that carrot cells phosphorylate tubulin, and that tubulin phosphorylating activity may be developmentally regulated