木薯14-3-3蛋白家族成员MeGRF3基因的原核表达及多克隆抗体制备

14-3-3蛋白是植物体内重要的信号转导调节分子,在碳代谢、逆境胁迫响应、生长发育等过程中发挥重要调控作用。为了深入解析14-3-3蛋白家族在木薯中的生物学功能,该研究采用酶切连接方法构建木薯14-3-3蛋白家族成员MeGRF3的原核表达载体,用热激法转化大肠杆菌Rosetta(DE3)株系,诱导表达带有MeGRF3的融合蛋白;使用纯化后的MeGRF3融合蛋白免疫新西兰公兔制备多克隆抗体,并检测抗体的效价和特异性。结果显示:(1)成功构建了木薯MeGRF3的原核表达载体pET-30a-MeGRF3,并诱导表达得到带有MeGRF3和6个His标签的融合蛋白。(2)MeGRF3融合蛋白主要以包涵体的形式存在,相对分子质量约为33 kD。(3)间接酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)测定的抗体效价为1024000。(4)Western blot分析显示,木薯叶片、茎尖、茎皮和块根中均检测到与MeGRF3蛋白大小一致的条带,说明MeGRF3在这4个组织器官中均表达,但主要是在茎尖和块根中大量积累,表明该研究成功制备的MeGRF3多克隆抗体的特异性较好。     

Evolution and isoform specificity of plant 14-3-3 proteins

The 14-3-3 proteins, once thought of as obscure mammalian brain proteins, are fast becoming recognized as major regulators of plant primary metabolism and of other cellular processes. Their presence as large gene families in plants underscores their essential role in plant physiology. We have examined the Arabidopsis thaliana 14-3-3 gene family, which currently is the largest and most complete 14-3-3 family with at least 12 expressed members and 15 genes from the now completed Arabidopsis thaliana genome project. The phylogenetic branching of this family serves as the prototypical model for comparison with other large plant 14-3-3 families and as such may serve to rationalize clustering in a biological context. Equally important for ascribing common functions for the various 14-3-3 isoforms is determining an isoform-specific correlation with localization and target partnering. A summary of localization information available in the literature is presented. In an effort to identify specific 14-3-3 isoform location and participation in cellular processes, we have produced a panel of isoform-specific antibodies to Arabidopsis thaliana 14-3-3s and present initial immunolocalization studies that suggest biologically relevant, discriminative partnering of 14-3-3 isoforms.     

Induced mutations in cassava using somatic embryos and the identification of mutant plants with altered starch yield and composition

A cyclic somatic embryogenic system was used to induce mutations in cassava variety PRC 60a in vitro. Globular-stage somatic embryos were selected as suitable experimental materials, and 50 Gy of -rays was determined to be the optimal dose for inducing mutations. During subsequent field trials, more than 50% of the regenerated mutant lines varied morphologically from wild-type plants. Consequently, we used this approach to induce genetic variability for obtaining novel cassava cultivars. Among the different mutant lines obtained, lines S14 and S15 showed large morphological variations. In 10-month-old S14 and S15 mutant lines, storage root yield was reduced 17-fold and 60-fold, respectively, compared to wild-type plants, while the storage roots of S15 mutant plants also exhibited an almost 50% decrease in starch content and a significant reduction (30%) in amylose content. These two features were observed throughout the different developmental stages of the storage roots in S15 plants.Abbreviations BA 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - EMS Ethylmethanesulfonate - IBA Indole-3-butyric acid     

A unigene catalogue of 5700 expressed genes in cassava

Two economically important characters, starch content and cassava bacterial blight resistance, were targeted to generate a large collection of cassava ESTs. Two libraries were constructed from cassava root tissues of varieties with high and low starch contents. Other libraries were constructed from plant tissues challenged by the pathogen Xanthomonas axonopodis pv.manihotis. We report here the single pass sequencing of 11 954 cDNA clones from the 5’ ends, including 111 from the 3’ ends. Cluster analysis permitted the identification of a unigene set of 5700 sequences. Sequence analyses permitted the assignment of a putative functional category for 37% of sequences whereas ~ 16% sequences did not show any significant similarity with other proteins present in the database and therefore can be considered as cassava specific genes. A group of genes belonging to a large multigene family was identified. We characterize a set of genes detected only in infected libraries putatively involved in the defense response to pathogen infection. By comparing two libraries obtained from cultivars contrasting in their starch content a group of genes associated to starch biosynthesis and differentially expressed was identified. This is the first large cassava EST resource developed today and publicly available thus making a significant contribution to genomic knowledge of cassava.     

TFT6 and TFT7, two different members of tomato 14-3-3 gene family, play distinct roles in plant adaption to low phosphorus stress

14‐3‐3 proteins are a large family of proteins but exact roles of their members in plant response to abiotic stresses are not clear, especially under nutrient deficiency. We investigated the expressions of all the tomato 14‐3‐3 gene family members (TFT1–TFT12) under low phosphorus stress (LP) and found that TFT6 belongs to the later responsive gene while TFT7 belongs to the early responsive gene. When the two genes were separately introduced into Arabidopsis and overexpressed, their plant growth under LP was much enhanced compared with wild‐type plant. TFT6 overexpressing plants showed reduced starch synthase activity, reduced starch content but enhanced sucrose loading into phloem in the shoot under LP. TFT7 overexpressing plants had much enhanced H⁺ flux along their root tip and activity of plasma membrane H⁺‐ATPase in the roots under LP. Our results suggest that TFT6 and TFT7 play different roles in plant adaption to LP. TFT6 acts mainly in leaves and is involved in the systemic response to LP by regulating leaf carbon allocation and increasing phloem sucrose transport to promote root growth, while TFT7 directly functions in root by activating root plasma membrane H⁺‐ATPase to release more protons under LP.     

Identification and expression analysis of four 14-3-3 genes during fruit ripening in banana (<Emphasis Type="Italic">Musa acuminata</Emphasis> L. AAA group,cv. Brazilian)

To investigate the regulation of 14-3-3 proteins in banana (Musa acuminata L. AAA group, cv. Brazilian) fruit postharvest ripening, four cDNAs encoding 14-3-3 proteins were isolated from banana and designated as Ma-14-3-3a, Ma-14-3-3c, Ma-14-3-3e, and Ma-14-3-3i, respectively. Amino acid sequence alignment showed that the four 14-3-3 proteins shared a highly conserved core structure and variable C-terminal as well as N-terminal regions with 14-3-3 proteins from other plant species. Phylogenetic analysis revealed that the four 14-3-3 genes belong to the non-ε groups. They were differentially and specifically expressed in various tissues. Real-time RT-PCR analysis indicated that these four genes function differentially during banana fruit postharvest ripening. Three genes, Ma-14-3-3a, Ma-14-3-3c, and Ma-14-3-3e, were significantly induced by exogenous ethylene treatment. However, gene function differed in naturally ripened fruits. Ethylene could induce Ma-14-3-3c expression during postharvest ripening, but expression patterns of Ma-14-3-3a and Ma-14-3-3e suggest that these two genes appear to be involved in regulating ethylene biosynthesis during fruit ripening. No obvious relationship emerged between Ma-14-3-3i expression in naturally ripened and 1-MCP (1-methylcyclopropene)-treated fruit groups during fruit ripening. These results indicate that the 14-3-3 proteins might be involved in various regulatory processes of banana fruit ripening. Further studies will mainly focus on revealing the detailed biological mechanisms of these four 14-3-3 genes in regulating banana fruit postharvest ripening.     

Members of two gene families encoding ubiquitin-conjugating enzymes, AtUBC1-3 and AtUBC4-6, fromArabidopsis thaliana are differentially expressed

Covalent attachment of ubiquitin to other intracellular proteins is essential for many physiological processes in eukaryotes, including selective protein degradation. Selection of proteins for ubiquitin conjugation is accomplished, in part, by a group of enzymes designated E2s or ubiquitin-conjugating enzymes (UBCs). At least six types of E2s have been identified in the plantArabidopsis thaliana; each type is encoded by a small gene family. Previously, we described the isolation and characterization of two three-member gene families, designatedAtUBC1-3 andAtUBC4-6, encoding two of these E2 types. Here, we investigated the expression patterns, of theAtUBC1-3 andAtUBC4-6 genes by the histochemical analysis of transgenicArabidopsis containing the corresponding promoters fused to the -glucuronidase-coding region. Staining patterns showed that these genes are active in many stages of development and some aspects of cell death, but are not induced by heat stress. Within the two gene families, individual members exhibited both overlapping and complementary expression patterns, indicating that at least one member of each gene family is expressed in most cell types and at most developmental stages. Different composite patterns of expression were observed between theAtUBC1-3 andAtUBC4-6 families, suggesting distinct biochemical and/or physiological functions for the encoded E2s inArabidopsis.     

Sequence analysis and expression profiling of 14-3-3 genes from the extremophile <Emphasis Type="Italic">Thelungiella salsuginea</Emphasis>, ecotype Yakutsk

Members of the 14-3-3 protein family are known to be important regulators of plant primary metabolism, hormonal signal transduction, and ion homeostasis. We identified nine isoforms of 14-3-3 genes of Thellungiella salsuginea, an extremophile relative of Arabidopsis thaliana. All the identified isoforms were designated according to their Arabidopsis orthologs: Chi, Omega, Psi, Phi, Upsilon, Lambda, Mu, Epsilon, and Omicron. Comparison of the deduced amino acid sequences reveals high degree of identity between the members of this protein family. Isoforms, designated as Ts14-3-3 Chi, Omicron, and Mu, display noticeable differences in their C-terminal domain as compared to their Arabidopsis homologs. Phylogenetic analysis demonstrated that the identified isoforms split into two groups, epsilon and non-epsilon, according to the common classification of the 14-3-3 family genes. The Thellungiella 14-3-3 isoforms are differentially expressed in various plant tissues, and real-time RT-PCR revealed that most of the isoforms are highly expressed even under normal growth conditions. In response to abiotic stress, low temperatures and high concentrations of salts, 14-3-3 genes exhibited different expression patterns. Our data suggest that, due to the high expression levels of the 14-3-3 genes, Thellungiella plants are likely pre-adapted to the stress conditions. Differences between the C-terminal domains of some Thellungiella 14-3-3 proteins and their Arabidopsis homologs may result in differences in target protein specificity.     

An ordered EST catalogue and gene expression profiles of cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis>) at key growth stages

A cDNA library was constructed from the root tissues of cassava variety Huanan 124 at the root bulking stage. A total of 9,600 cDNA clones from the library were sequenced with single-pass from the 5′-terminus to establish a catalogue of expressed sequence tags (ESTs). Assembly of the resulting EST sequences resulted in 2,878 putative unigenes. Blastn analysis showed that 62.6% of the unigenes matched with known cassava ESTs and the rest had no ‘hits’ against the cassava database in the integrative PlantGDB database. Blastx analysis showed that 1,715 (59.59%) of the unigenes matched with one or more GenBank protein entries and 1,163 (40.41%) had no ‘hits’. A cDNA microarray with 2,878 unigenes was developed and used to analyze gene expression profiling of Huanan 124 at key growth stages including seedling, formation of root system, root bulking, and starch maturity. Array data analysis revealed that (1) the higher ratio of up-regulated ribosome-related genes was accompanied by a high ratio of up-regulated ubiquitin, proteasome-related and protease genes in cassava roots; (2) starch formation and degradation simultaneously occur at the early stages of root development but starch degradation is declined partially due to decrease in UDP-glucose dehydrogenase activity with root maturity; (3) starch may also be synthesized in situ in roots; (4) starch synthesis, translocation, and accumulation are also associated probably with signaling pathways that parallel Wnt, LAM, TCS and ErbB signaling pathways in animals; (5) constitutive expression of stress-responsive genes may be due to the adaptation of cassava to harsh environments during long-term evolution.     

Soybean 14-3-3 gene family: identification and molecular characterization

The 14-3-3s are a group of proteins that are ubiquitously found in eukaryotes. Plant 14-3-3 proteins are encoded by a large multigene family and are involved in signaling pathways to regulate plant development and protection from stress. Recent studies in Arabidopsis and rice have demonstrated the isoform specificity in 14-3-3s and their client protein interactions. However, detailed characterization of 14-3-3 gene family in legumes has not been reported. In this study, soybean 14-3-3 proteins were identified and their molecular characterization performed. Data mining of soybean genome and expressed sequence tag databases identified 18 14-3-3 genes, of them 16 are transcribed. All 16 SGF14s have higher expression in embryo tissues suggesting their potential role in seed development. Subcellular localization of all transcribed SGF14s demonstrated that 14-3-3 proteins in soybean have isoform specificity, however, some overlaps were also observed between closely related isoforms. A comparative analysis of SGF14s with Arabidopsis and rice 14-3-3s indicated that SGF14s also group into epsilon and non-epsilon classes. However, unlike Arabidopsis and rice 14-3-3s, SGF14s contained only one kind of gene structure belonging to each class. Overall, soybean consists of the largest family of 14-3-3 proteins characterized to date. Our results provide a solid framework for further investigations into the role of SGF14s and their involvement in legume-specific functions.     

14-3-3 proteins activate a plant calcium-dependent protein kinase (CDPK)

Plants and protozoa contain a unique family of calcium-dependent protein kinases (CDPKs) which are defined by the presence of a carboxyl-terminal calmodulin-like regulatory domain. We present biochemical evidence indicating that at least one member of this kinase family can be stimulated by 14-3-3 proteins. Isoform CPK-1 from the model plant Arabidopsis thaliana was expressed as a fusion protein in E. coli and purified. The calcium-dependent activity of this recombinant CPK-1 was shown to be stimulated almost twofold by three different 14-3-3 isoforms with 50% activation around 200 nM. 14-3-3 proteins bound to the purified CPK-1, as shown by binding assays in which either the 14-3-3 or CPK-1 were immobilized on a matrix. Both the 14-3-3 binding and activation of CPK-1 were specifically disrupted by a known 14-3-3 binding peptide LSQRQRSTpSTPNVHMV (IC₅₀=30 μM). These results raise the question of whether 14-3-3 can modulate the activity of CDPK signal transduction pathways in plants.