Small GTP-binding Protein Genes Research Articles

Analysis of the salt-stress responsive element of the promoter of peanut small GTP binding protein gene AhRabG3f

To study the molecular mechanism of salt stress response of peanut small GTP binding protein gene AhRabG3f, a 1 914 bp promoter fragment upstream of the start codon of AhRabG3f gene (3f-P) from peanut was cloned. Subsequently, five truncated fragments (3f-P1-3f-P5) with lengths of 1 729, 1 379, 666, 510 and 179 bp were obtained through deletion at the 5' end, respectively. Plant expression vectors where these six promoter fragments were fused with the gus gene were constructed and transformed into tobacco by Agrobacterium-mediated method, respectively. GUS expression in transgenic tobacco and activity analysis were conducted. The gus gene expression can be detected in the transgenic tobacco harboring each promoter segment, among which the driving activity of the full-length promoter 3f-P was the weakest, while the driving activity of the promoter segment 3f-P3 was the strongest. Upon exposure of the transgenic tobacco to salt stress, the GUS activity driven by 3f-P, 3f-P1, 3f-P2 and 3f-P3 was 3.3, 1.2, 1.9 and 1.2 times compared to that of the transgenic plants without salt treatment. This suggests that the AhRabG3f promoter was salt-inducible and there might be positive regulatory elements between 3f-P and 3f-P3 in response to salt stress. The results of GUS activity driven by promoter fragments after salt treatment showed that elements included MYB and GT1 between 1 930 bp and 1 745 bp. Moreover, a TC-rich repeat between 682 bp and 526 bp might be positive cis-elements responsible for salt stress, and an MYC element between 1 395 bp and 682 bp might be a negative cis-element responsible for salt stress. This study may facilitate using the induced promoter to regulate the salt resistance of peanut.

Characterization of a small GTP-binding protein gene TaRab18 from wheat involved in the stripe rust resistance

The stripe rust resistance gene, Yr26, is commonly used in wheat production. Identification of Yr26 resistance related genes is important for better understanding of the resistance mechanism. TaRab18, a putative small GTP-binding protein, was screened as a resistance regulated gene as it showed differential expression between the Yr26-containing resistant wheat and the susceptible wheat at different time points after Pst inoculation. TaRab18 contains four typical domains (GI to GIV) of the small GTP-binding proteins superfamily and five domains (RabF1 to RabF5) specific to the Rab subfamily. From the phylogenetic tree that TaRab18 was identified as belonging to the RABC1 subfamily. Chromosome location analysis indicated that TaRab18 and its homeoalles were on the homeologous group 7 chromosomes, and the Pst induced TaRab18 was on the 7 B chromosome. Functional analysis by virus induced gene silencing (VIGS) indicated that TaRab18 was positively involved in the stripe rust resistance through regulating the hypersensitive response, and Pst can develop on the leaves of TaRab18 silenced 92R137. However, over-expression of TaRab18 in susceptible Yangmai158 did not enhance its resistance dramatically, only from 9 grade in Yangmai158 to 8 grade in the transgenic plant. However, histological observation indicated that the transgenic plants with over-expressed TaRab18 showed a strong hypersensitive response at the early infection stage. The research herein, will improve our understanding of the roles of Rab in wheat resistance.

Wheat TaRab7 GTPase Is Part of the Signaling Pathway in Responses to Stripe Rust and Abiotic Stimuli

Small GTP-binding proteins function as regulators of specific intercellular fundamental biological processes. In this study, a small GTP-binding protein Rab7 gene, designated as TaRab7, was identified and characterized from a cDNA library of wheat leaves infected with Puccinia striiformis f. sp. tritici (Pst) the wheat stripe rust pathogen. The gene was predicted to encode a protein of 206 amino acids, with a molecular mass of 23.13 KDa and an isoeletric point (pI) of 5.13. Further analysis revealed the presence of a conserved signature that is characteristic of Rab7, and phylogenetic analysis demonstrated that TaRab7 has the highest similarity to a small GTP binding protein gene (BdRab7-like) from Brachypodium distachyon. Quantitative real-time PCR assays revealed that the expression of TaRab7 was higher in the early stage of the incompatible interactions between wheat and Pst than in the compatible interaction, and the transcription level of TaRab7 was also highly induced by environmental stress stimuli. Furthermore, knocking down TaRab7 expression by virus induced gene silencing enhanced the susceptibility of wheat cv. Suwon 11 to an avirulent race CYR23. These results imply that TaRab7 plays an important role in the early stage of wheat-stripe rust fungus interaction and in stress tolerance.

Small GTP-Binding Protein Gene Is Associated with QTL for Submergence Tolerance in Rice

Small GTP-binding proteins play critical roles in signal transduction in mammalian and plant systems. In this study, sequence variation of a small GTP-binding protein identified in the subgenomic region was analyzed. The major quantitative trait locus (QTL) controlling submergence tolerance on the 6.5-cM region of chromosome 9 was previously mapped, sequenced, and annotated. One of the most interesting candidate genes located in this QTL was a 5.2-kb sequence, which included a coding sequence consisting of two exons and a promoter. The deduced amino acid sequence corresponded to a 24.8 kD protein consisting of 226 amino acids, with 98% identity to RGP1, a small GTP-binding protein involved in a signal pathway responding to hormones, such as cytokinin and ethylene. According to the amino acid sequence, a putative small G-protein was classified as a small Ras-related GTP-binding protein. DNA gel blot analysis showed that the putative gene encoding the Ras-related GTP-binding protein was present as a single copy in the rice genome. Comparison of genomic sequences from several rice cultivars tolerant to submergence identified single nucleotide polymorphisms located in the TATA box of the Ras promoter region. Linkage analysis showed that the putative gene for GTP-binding protein was tightly linked to the peak of the QTL previously mapped on the long arm of chromosome 9. The single strand conformation polymorphism of the putative GTP-binding protein gene can be used for allele discrimination and marker assisted selection for tolerance to flash flooding.

Identification of a new small GTP-binding protein gene OsRab5a, genomic organization, and expression pattern analysis during nitrate supply and early nutrient starvation in rice ( Oryza sativa L .) root

A new nitrate-induced gene OsRab5a in rice was isolated using suppression subtractive hybridization (SSH) method. OsRab5a has 1125 nucleotides with an open reading frame of 612 nucleotides encoding a small GTP-binding protein of Rab family. OsRab5a had a higher expression in root, weak expression in shoot, flower and grain, and visually not in stem and leaf. Southern and mapping analysis indicated that OsRab5a would be a single copy gene at locus of 108.8 cM of chromosome 12. Comparison of genomic organization and tissue distribution showed well-conserved genomic organizations and similar expression patterns between OsRab5a gene in rice and Rha1 gene in Arabidopsis during evolution. OsRab5a was quickly induced by nitrate in a split-root treatment, also up-regulated at early nitrogen and phosphorus starvation in root. Acceleration of root growth was observed under nitrate supply, early nitrogen and phosphorous starvation, which was consistent with the expression pattern of OsRab5a. These results suggest that OsRab5a may function in nutrient-regulated root growth.

Expression of Rab small GTPases in epithelial Caco-2 cells: Rab21 is an apically located GTP-binding protein in polarised intestinal epithelial cells

Rab proteins belong to a subfamily of small GTP-binding protein genes of the Ras superfamily and play an important role in intracellular vesicular targeting. The presence of members of this protein family was examined in Caco-2 cells by a PCR-based strategy. Twenty-five different partial cDNA sequences were isolated, including 18 Rab protein family members. Seven novel human sequences, representing Rab2B, Rab6A', Rab6B, Rab10, Rab19B, Rab21 and Rab22A, were identified. For one clone, encoding Rab21, full-length cDNA was isolated from a Caco-2 cDNA library. Northern blot analysis showed a ubiquitous expression pattern of Rab21. To study Rab21 protein expression in Caco-2 cells, polyclonal antibodies were raised against GST-Rab21 fusion protein and characterised. The antibodies recognised Rab21 as a protein of approximately 25 kDa. Interestingly, the protein shows a general ER-like staining in nonpolarised Caco-2 cells in contrast to an apically located vesicle-like staining in polarised Caco-2 cells. Furthermore, immunohistochemical staining on human jejunal tissue showed a predominant expression of Rab21 in the epithelial cell layer with high expression levels in the apical region, whereas stem cells in the crypts were negative. We therefore suggest an alternative role for Rab21 in the regulation of vesicular transport in polarised intestinal epithelial cells.

CDNA cloning and molecular analysis of papaya small GTP-binding protein, pgp1.

In the course of papaya EST collection, one clone (pRA4-3) encoding partial sequence of papaya small GTP-binding protein gene, pgp1, was obtained. Based on the sequence information of pRA4-3, the entire coding region of pgp1 was cloned using the 3'RACE PCR technique. ORF of pgp1 is 636bp long and deduced molecular weight of the protein is 23,311. Phylogenetic analysis showed that PGP1 belongs to YPT/RAB group of the small GTP-binding protein and is a homologue of RAB2. Southern analysis showed that there are several pgp1-related genes in papaya genome. Northern analysis showed that pgp1 was expressed equally in stems of seedlings that were grown under light and dark conditions. This result shows that PGP1 is not involved in the phytochrome-mediated signal transduction as an auxin signal transducer in stems of papaya seedlings.

Differential Display Analysis of the Early Compatible Interaction Between Soybean and the Soybean Cyst Nematode

The marked cellular changes during feeding site formation of the soybean cyst nematode (Heterodera glycines) indicate major changes in soybean gene expression. We used differential display of mRNA to detect host gene expression changes during the early compatible interaction between soybean and H. glycines. Fifteen cDNA clones corresponding to mRNAs with different abundances in H. glycines-infected versus uninfected roots were identified. Differential display results indicated that abundances of five mRNAs increased in infected roots, whereas abundances of 10 mRNAs decreased. Transcripts for nine of these 15 cDNAs could be detected on RNA blots, and their hybridization signals confirmed the differential display results for eight of these nine cDNAs. Sequence analyses identified five cDNAs with decreased mRNA levels in infected roots as corresponding to two putative aldolase genes, a transcription-factor TFIIA homologue, the soybean small GTP-binding protein gene sra1, and the soybean auxin down-regulated gene ADR12. RNA blot analyses of other auxin down-regulated genes revealed a decrease in their mRNA abundances in H. glycines-infected roots as well.

RAB GTPases expressed in human melanoma cells

The expression of small GTP-binding protein genes of the ras superfamily was examined in pigmented human melanoma cells by a PCR-based strategy. Twenty six different partial cDNA sequences were isolated, including 17 members of the rab subfamily, of which 9 represented novel genes. Some rabs expressed in melanoma cells overlapped with those of platelets; this should prove relevant to the investigation of murine and human disorders characterized by the combination of pigment dilution and a platelet storage pool defect.

Phenotypic instability of transgenic tobacco plants and their progenies expressing Arabidopsis thaliana small GTP-binding protein genes.

Chimeric genes consisting of the cauliflower mosaic virus 35S promoter, a cDNA encoding a small GTP-binding protein from Arabidopsis thaliana (ara-2 or ara-4) and the terminator of the nopaline synthase gene were cloned into a binary vector. Tobacco leaf tissues were transformed with this plasmid via Agrobacterium-mediated transformation. Transgenic plants possessing either ara-2 or ara-4 occasionally showed morphological abnormalities in leaves and other organs. However, such alterations were not always associated with co-transferred characters, such as kanamycin tolerance, and they arose in no more than 10% of the transgenic plants. Such phenomena were also observed in the progenies of the primary transgenic plants. Despite such unusual inheritance of the phenotypic abnormalities, GTP-binding activity of the inserted ara gene products was detected in all plants tested.

Characterization of cDNAs encoding small GTP-binding proteins from maize.

To date, more than 100 genes encoding small GTP-binding proteins (21-25 kD) have been identified from various eukaryotic organisms, including mammals, insects, yeasts, and higher plants (Hall, 1990; Bourne et al., 1991; Terryn et al., 1993). These proteins, which contain highly homologous regions that have been conserved during evolution, are conveniently classified into three major groups; ras, rho, and rab/ypt families (Kahn et al., 1992). Although the physiological functions of these proteins are not fully understood, increasing evidence suggests that whereas the ras-related proteins are involved in the signal transduction pathways, the rab/ypt-related proteins are associated with intracellular transport and cell growth (Bednarek and Raikhel, 1992). From the plant kingdom, about 30 small GTP-binding protein genes have been identified in Arabidopsis thaliana, rice, tobacco, maize, pea, and others (Terryn et al., 1993). Here, we report the isolation and characterization of two new cDNAs, mgpl and mgp2, that encode small GTPbinding proteins in maize (Table I). The mgpl sequence of 941 bp contains a 633-bp open reading frame that encodes a protein of 211 amino acids with a calculated M, of 22,400. The mgp2 sequence consists of 769 bp with a 648-bp open reading frame encoding a protein of 216 amino acids and a calculated M, of 23,800. Both proteins contain four conserved regions necessary for GTP binding and an effector loop that interacts with specific effector proteins such as GAP. The mgpl and mgp2 proteins show the highest sequence similarities with rabll and the marine ray ora3 proteins followed by various rab/ypt family proteins. This indicates that the mgpl and mgp2 proteins belong to the rab/ypt subfamily, although they are rather distantly related to the only other maize small GTP-binding proteins, YPTml and YPTm2, reported to date (Palme et al., 1992). Northern hybridization analyses revealed that, although the mgpl and mgp2 mRNAs were detected in a11 tissues examined, the highest transcript levels of mgpl and mgp2 were in the tassel and ear tissues, respectively. The mRNA levels of mgpl and mgp2 were also examined during the various developmental stages of the plant at 14, 24,50, 70, and 90 d after emergence. Both mgpl and mgp2 mRNAs

Expressed sequence tags from cultured cells of rice (Oryza sativa L.) under stressed conditions: analysis of transcripts of genes engaged in ATP-generating pathways

Large-scale sequencing of randomly selected cDNA clones was used to isolate numerous genes in rice (Oryza sativa L.). Total RNA used for cDNA synthesis was prepared from suspension-cultured cells of rice grown under stressed conditions, such as in saline or nitrogen-starvation conditions. A total of 780 cDNA clones were partially sequenced and about 15% could be identified as putative genes. In the library constructed under saline conditions, we identified several genes associated with signal transduction, such as protein kinase and small GTP-binding protein genes. Many stress-related genes were isolated from both the saline and nitrogen-starvation libraries. These results indicate that stress treatment of suspension-cultured cells makes it possible to efficiently isolate various types of plant genes. To examine the usefulness of such tagged cDNAs for the study of gene expression in a specific metabolic pathway, we analyzed mRNA levels of genes engaged in the ATP-generating pathways in cultured cells of rice under different stresses, such as 20% sucrose, salt stress, cold stress and nitrogen-starvation stress. The results suggest that the coordinated induction of several genes in key steps under stressed conditions may be essential for activation of the entire energy-producing pathway to maintain homeostasis in rice cells. Expressed sequence tags identified by random cDNA sequencing provide the opportunity to generate a transcript map of rice genes.

Molecular structure of ras-related small GTP-binding protein genes of rice plants and GTPase activities of gene products in Escherichia coli

We isolated two rice cDNA clones (rid and ric2) encoding proteins homologous to the ras-related small GTP-binding protein. The amino acid sequences of rid and ric2 are conserved in four regions involved in GTP binding and hydrolysis which are characteristic in the ras and ras-related small GTP-binding protein genes. In addition, two consecutive cysteine residues near the carboxyl-terminal end required for membrane anchoring are also present in rid and ric2. The rid and ric2 proteins synthesized in Escherichia coli possessed GTPase activity (i.e. hydrolysis of GTP to GDP).