不结球白菜BcLRK01基因对芜菁花叶病毒及胁迫诱导的响应

通过cDNA-AFLP技术,从芜菁花叶病毒(TuMV)侵染的不结球白菜幼叶中分离到一条差异表达的基因片段,克隆获得其cDNA全长为2 124bp,编码707个氨基酸的富亮氨酸重复类受体激酶,命名为BcLRK01。利用实时定量PCR研究了该基因在TuMV侵染及高盐、冷胁迫、水杨酸(SA)、茉莉酸(JA)、乙烯(ET)等处理下的表达情况,结果显示,TuMV侵染、高盐、冷胁迫、SA、JA和ET等均能诱导BcLRK01不同程度的表达,说明该基因可能是不结球白菜病毒病的病程相关基因,同时也参与高盐和冷胁迫以及SA、JA、ET等的信号途径。     

Characterization of Geranium (Pelargonium graveolens) Chloroplast EF-Tu cDNA

A geranium (Pelargonium graveolens) chloroplast translational elongation factor EF-Tu (tufA) cDNA was isolated. The geranium tufA cDNA is 1,584 bp long with 20 bp of 5 untranslated region (UTR) and 139 bp of 3 UTR. It encodes 474 amino acids including a putative chloroplast transit peptide of 65 amino acids. The deduced polypeptides of the geranium tufA cDNA contains four GTP binding sequences in its N-terminal region and two chloroplast EF-Tu signature regions in the C-terminal region. The predicted molecular weight of the mature geranium chloroplast EF-Tu protein was about 45,000 and its amino acid sequence identity with the chloroplast EF-Tu proteins of tobacco, pea, Arabidopsis, rice, and soybean ranges from 85% to 91%. The geranium tufA appears to exist as a single copy gene like Arabidopsis and rice, whereas other known dicot plants have more than one copy in their nuclear genomes.     

Purification of chloroplast elongation factor Tu and cDNA analysis in tobacco: the existence of two chloroplast elongation factor Tu species

We have purified a chloroplast elongation factor Tu (EF-Tu) from tobacco (Nicotiana tabacum) and determined its N-terminal amino acid sequence. Two distinct cDNAs encoding EF-Tu were isolated from a leaf cDNA library of N. sylvestris (the female progenitor of N. tabacum) using an oligonucleotide probe based on the EF-Tu protein sequence. The cDNA sequence and genomic Southern analyses revealed that tobacco chloroplast EF-Tu is encoded by two distinct genes in the nuclear genome of N. sylvestris. We designated the corresponding gene products EF-Tu A and B. The mature polypeptides of EF-Tu A and B are 408 amino acids long and share 95.3% amino acid identity. They show 75–78% amino acid identity with cyanobacterial and chloroplast-encoded EF-Tu species.     

Complete nucleotide sequences of seven eubacterial genes coding for the elongation factor Tu: functional,structural and phylogenetic evaluations

The nucleotide sequences of cloned genes coding for the elongation factor Tu of seven eubacteria have been determined. These genes were fiom Anacystis nidulans, Bacillus subtilis, Bacteroides fragilis, Deinonema spec., Pseudomonas cepacia, Shewanella putrefaciens and Streptococcus oralis. The primary structures of the genes were compared to the available sequences of prokaryotic elongation factors Tu and eukaryotic elongation factors 1 alpha. A conservation profile was determined for homologous amino acid residues. Sites of known or putative functions are usually located at highly conserved positions or within highly conserved sequence stretches. The aligned 24 amino acid sequences were used as basis for a phylogenetic analysis. The phylogenetic tree corroborates the kingdom as well as phylum concept deduced from 16S rRNA data.Abbreviations EF-Tu elongation factor Tu - GDP guanosine 5-diphosphate - GTP guanosine 5-triphosphate; tuf gene, gene coding for elongation factor Tu     

Unusually strong immunological cross-reaction between elongation factor Tu of Escherichia coli and Bacillus subtilis

Polyclonal antibodies were prepared against the purified elongation factor Tu (EF-Tu) of Escherichia coli and Bacillus subtilis. Using the methods of Western blotting and microcomplement fixation the cross-reactivities of EF-Tu of 19 different prokaryotes were determined. The immunological distance were compared with the results of 16S rRNA oligonucleotide analysis. An unexpectedly high cross-reactivity was revealed between the EF-Tu of B. subtilis and the antiserum against the EF-Tu of E. coli. A comparison of the predicted amino acid sequences from the tuf-genes of E. coli and B. subtilis yielded two identical peptide fragments that are likely candidates for antibody binding sites.Abbreviations EF-Tu elongation factor Tu - GDP guanosine 5-diphosphate - GTP guanosine 5-triphosphate - MCF microcomplement fixation - T type strain     

Molecular cloning and characterization of a PR-5 like protein gene from <Emphasis Type="Italic">Brassica campestris</Emphasis> ssp. <Emphasis Type="Italic">chinensis</Emphasis>

Downy mildew caused by Hyaloperonospora parasitica is a serious fungal disease in non-heading Chinese cabbage (Brassica campestris L. ssp. chinensis Makino). Pathogenesis-related 5 (PR-5) genes play an important role in plant resistance to disease invasion. In this study, a gene encoding pathogenesis-related 5-like (PR-5L) protein, named BcPR-5L, was successfully cloned from non-heading Chinese cabbage. The cDNA sequence of BcPR-5L was 747 bp in length. It encoded a protein of molecular mass of 25.78 kDa, an isoelectric point of 4.42, and containing 248 amino acids. Multiple sequence alignment indicated that BcPR-5L protein was highly homologous to other PR-5L proteins identified in 13 different species, with the highest homology to Brassica rapa. We analyzed the subcellular localization of BcPR-5L protein by using onion epidermal cells and found that it was localized in the membrane. Real time quantitative PCR analyses revealed that the expression of BcPR-5L gene was significantly upregulated after H. parasitica infection, and the expression in the resistant cultivar was higher than that in the susceptible cultivar. In summary, our data suggest that BcPR-5L gene may play an important role in the resistance of non-heading Chinese cabbage to H. parasitica infection.     

Kirromycin-resistant elongation factor Tu from wild-type of Lactobacillus brevis

Properties of the elongation factor Tu from Lactobacillus brevis which is naturally insensitive to kirromycin are described. The protein is characterized by an unusual nucleotide-binding site with increased affinity for GTP and extreme heat stability. EF-Tu is sensitive to pulvomycin in the assay of polyphenylalanine synthesis. However, the failure of the protein to display pulvomycin-dependent GDP-binding and GTPase activity indicates that pulvomycin action in L. brevis differs from that in E. coli.     

Elongation factor Tu of the extreme thermophilic hydrogen oxidizing bacterium Calderobacterium hydrogenophilum

Protein synthesis elongation factor Tu has been purified from an extreme thermophilic hydrogen oxidizing bacterium Calderobacterium hydrogenophilum. The molecular mass of EF-Tu. GDP is 51,000. The factor is heat stable and loses only 50% of its activity after heating for 5 min at 80 degrees C. Under mild conditions trypsin cleaved EF-Tu. GDP to four main fragments. Only one fragment of Mr = 20,000 had a mobility similar to the trypsin fragment "B" of Escherichia coli EF-Tu. Other peptide fragments of E. coli and C. hydrogenophilum EF-Tu differed in size, but native preparations of both factors are immunologically similar.     

Structure-based sequence alignment of elongation factors Tu and G with related GTPases involved in translation

The G domain and domain II in the crystal structure of Thermus thermophilus elongation factor G (EF-G) were compared with the homologous domains in Thermus aquaticus elongation factor Tu (EF-Tu). Sequence alignment derived from the structural superposition was used to define conserved sequence elements in domain II. These elements and previously known conserved sequence elements in the G domain were used to guide the alignment of the sequences of Sulfolobus acidocaldarius elongation factor 2, human elongation factor 2, and Escherichia coli initiation factor 2 and release factor 3 to the aligned sequences of EF-G and EF-Tu. This alignment, which deviates from previously published alignments, has evolutionary implications and leads to alternative interpretations of biochemical data concerning the interaction of elongation factors with the -sarcin/ricin region of the ribosome. A single conserved sequence motif in domain II was identified and used to further characterize the GTPase subfamily of translation factors and related proteins. It was shown that the motif is found in most if not all the members of the family. Apparently, the common characteristic of these GTPases is an extensive consensus structural unit that possibly accounts for a similar interaction with the ribosome and is composed of two domains homologous to the G domain and domain II in EF-Tu and EF-G.     

Isolation,expression, and evolution of the gene encoding mitochondrial elongation factor Tu in Arabidopsis thaliana

We have characterized a second nuclear gene (tufM) in Arabidopsis thaliana that encodes a eubacterial-like protein synthesis elongation factor Tu (EF-Tu). This gene does not closely resemble the previously described Arabidopsis nuclear tufA gene, which encodes the plastid EF-Tu, and does not contain sequence elements found in all cyanobacterial and plastid tufA genes. However, the predicted amino acid sequence includes an N-terminal extension which resembles an organellar targeting sequence and shares three unique sequence elements with mitochondrial EF-Tu's, from Saccharomyces cerevisiae and Homo sapiens, suggesting that this gene encodes the Arabidopsis mitochondrial EF-Tu. Consistent with this interpretation, the gene is expressed at a higher level in flowers than in leaves. Phylogenetic analysis confirms the mitochondrial character of the sequence and indicates that the human, yeast, and Arabidopsis tufM genes have undergone considerably more sequence divergence than their cytoplasmic counterparts, perhaps reflecting a cross-compartmental acceleration of gene evolution for components of the mitochondrial translation apparatus. As previously observed for tufA, the tufM gene is present in one copy in Arabidopsis but in several copies in other species of crucifers.     

Cross-reactivity studies on the interaction between elongation factors Tu and Ts from Streptomyces aureofaciens and Escherichia coli in the GDP exchange reaction

The method of purification of elongation factor Ts from Streptomyces aureofaciens is described. Purified elongation factors Ts from S. aureofaciens and Escherichia coli were tested in cross-reactivity studies with elongation factors Tu from both species in a GDP exchange reaction under equilibrium and non-equilibrium conditions. Experiments have revealed that slower spontaneous release of GDP from S. aureofaciens EF-Tu is compensated for by higher affinity of homologous EF-Ts towards EF-Tu and thus the initial rates of EF-Ts catalysed GDP exchange can be kept the same in both E. coli and S. aurefaciens in vitro systems.     

Molecular cloning and characterization of ascorbate oxidase gene in non-heading Chinese cabbage

The cDNA clone of ascorbate oxidase gene was isolated from non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino, cv. Suzhouqing) and characterized. Sequence analysis showed that there was a high similarity between this sequence (named BcAO) and its homologues in other plant species. Southern blotting indicated that more than one nuclear gene encoded this enzyme in non-heading Chinese cabbage. The mRNA level of the BcAO gene in leaves was monitored by real-time PCR at different developmental stages and under different stress conditions. Results showed that the expression of BcAO was upregulated by light, and the BcAO gene responded to copper stress as well. After inoculation with Alternaria brassicae, the expression of BcAO in the leaves was increased in general and peaked at 12 and 72 h post inoculation, with much higher expression at the later date. Cloning the BcAO gene will enable us to further understand its function and would provide useful information for resistance breeding program for non-heading Chinese cabbage.     

The inhibition of eucaryotic protein synthesis by procaryotic elongation factor Tu

The activity of elongation factor Tu (EF-Tu) from $\text{Escherichia}\text{coli}$ in eucaryotic protein synthesis systems was investigated. EF-Tu was found to inhibit polyphenylalanine synthesis when incubated with $\text{Artemia}$ 80S ribosomes, purified rabbit reticulocyte elongation factor Tu (eEF-Tu) and partially purified reticulocyte translocase enzyme, eEF-G. The inhibition could be overcome by supplying the system with additional eEF-Tu. EF-Tu also inhibited protein synthesis in rabbit reticulocyte lysates. Data presented in this report indicate that inhibition by EF-Tu results from the accumulation of ternary complexes of the protein factor, GTP and aminoacyl-tRNA which do not interact with the ribosomal A-site of 80S ribosomes under physiological conditions.     

Expression analysis and functional characterization of a cold-responsive gene COR15A from Arabidopsis thaliana

The cold-responsive (COR) genes play an important role in cold acclimation of higher plants. Here, a tight correlation between chloroplast functionality and COR15A expression, and the functional characterization of Arabidopsis COR15A involved in salt/osmotic stress, were revealed. COR15A gene is light inducible and expressed in light-grown seedlings. The expression level of COR15A was reduced when chloroplasts were damaged by norflurazon treatment. By using several albino mutants, seca1, secy1, and tic20, all of which exhibited severe defects in both structure and function of chloroplast, it was shown that the accumulation of COR15A mRNA depends on chloroplast functionality. Real-time RT-PCR and GUS-staining assays demonstrated that COR15A was induced by salt/osmotic stress partially via ABA. Overexpression of COR15A in Arabidopsis resulted in the seedlings displaying hypersensitivity to salt/osmotic stress. All these results suggest that plant acquire the ability to fully express COR15A only after the development of functional chloroplasts, COR15A may be involved in response to salt/osmotic stress during early stages of plant development.     

Analysis of transgenic wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) harboring a maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) gene for plastid EF-Tu: segregation pattern,expression and effects of the transgene

We previously reported that transgenic wheat (Triticum aestivum L.) carrying a maize (Zea mays L.) gene (Zmeftu1) for chloroplast protein synthesis elongation factor, EF-Tu, displays reduced thermal aggregation of leaf proteins, reduced injury to photosynthetic membranes (thylakoids), and enhanced rate of CO₂ fixation following exposure to heat stress (18 h at 45°C) [Fu et al. in Plant Mol Biol 68:277–288, 2008]. In the current study, we investigated the segregation pattern and expression of the transgene Zmeftu1 and determined the grain yield of transgenic plants after exposure to a brief heat stress (18 h at 45°C). We also assessed thermal aggregation of soluble leaf proteins in transgenic plants, testing the hypothesis that increased levels of EF-Tu will lead to a non-specific protection of leaf proteins against thermal aggregation. The transgenic wheat displayed a single-gene pattern of segregation of Zmeftu1. Zmeftu1 was expressed, and the transgenic plants synthesized and accumulated three anti-EF-Tu cross-reacting polypeptides of similar molecular mass but different pI, suggesting the possibility of posttranslational modification of this protein. The transgenic plants also showed better grain yield after exposure to heat stress compared with their non-transgenic counterparts. Soluble leaf proteins of various molecular masses displayed lower thermal aggregation in transgenic than in non-transgenic wheat. The results suggest that overexpression of chloroplast EF-Tu can be beneficial to wheat tolerance to heat stress. Moreover, the results also support the hypothesis that EF-Tu contributes to heat tolerance by acting as a molecular chaperone and protecting heat-labile proteins from thermal aggregation in a non-specific manner.