Cloning of TPS gene from eelgrass species Zostera marina and its functional identification by genetic transformation in rice.

The full-length cDNA sequence (2613 bp) of the trehalose-6-phosphate synthase (TPS) gene of eelgrass Zostera marina (ZmTPS) was identified and cloned. Z. marina is a kind of seed-plant growing in sea water during its whole life history. The open reading frame (ORF) region of ZmTPS gene encodes a protein of 870 amino acid residues and a stop codon. The corresponding genomic DNA sequence is 3770 bp in length, which contains 3 exons and 2 introns. The ZmTPS gene was transformed into rice variety ZH11 via Agrobacterium-mediated transformation method. After antibiotic screening, molecular characterization, salt-tolerance and trehalose content determinations, two transgenic lines resistant to 150 mM NaCL solutions were screened. Our study results indicated that the ZmTPS gene was integrated into the genomic DNA of the two transgenic rice lines and could be expressed well. Moreover, the detection of the transformed ZmTPS gene in the progenies of the two transgenic lines was performed from T1 to T4 generations; and results suggested that the transformed ZmTPS gene can be transmitted from parent to the progeny in transgenic rice.

Cloning of galactinol synthase gene from Ammopiptanthus mongolicus and its expression in transgenic Photinia serrulata plants.

A cold induced galactinol synthase gene (AmGS) and its promoter sequence were identified and cloned from the cold-tolerant tree Ammopiptanthus mongolicus by using cDNA-AFLP, RACE-PCR and TAIL-PCR strategies combined with its expression pattern analysis after cold inducing treatment. Accession number of the AmGS gene in GenBank is DQ519361. The open reading frame (ORF) region of the AmGS gene is 987 nucleotides encoding for 328 amino acid residues and a stop codon. The genomic DNA sequence of AmGS gene contains 3 exons and 2 introns. Moreover, a variety of temporal gene expression patterns of AmGS was detected, which revealed the up-regulation of AmGS gene in stresses of cold, ABA and others. Then the AmGS gene was transformed into Photinia serrulata tree by Agrobacterium-mediated transformation, and the transgenic plants exhibited higher cold-tolerance comparing with non-transformed plants.

Cloning and comparative studies of seaweed trehalose-6-phosphate synthase genes.

The full-length cDNA sequence (3219 base pairs) of the trehalose-6-phosphate synthase gene of Porphyra yezoensis (PyTPS) was isolated by RACE-PCR and deposited in GenBank (NCBI) with the accession number AY729671. PyTPS encodes a protein of 908 amino acids before a stop codon, and has a calculated molecular mass of 101,591 Daltons. The PyTPS protein consists of a TPS domain in the N-terminus and a putative TPP domain at the C-terminus. Homology alignment for PyTPS and the TPS proteins from bacteria, yeast and higher plants indicated that the most closely related sequences to PyTPS were those from higher plants (OsTPS and AtTPS5), whereas the most distant sequence to PyTPS was from bacteria (EcOtsAB). Based on the identified sequence of the PyTPS gene, PCR primers were designed and used to amplify the TPS genes from nine other seaweed species. Sequences of the nine obtained TPS genes were deposited in GenBank (NCBI). All 10 TPS genes encoded peptides of 908 amino acids and the sequences were highly conserved both in nucleotide composition (>94%) and in amino acid composition (>96%). Unlike the TPS genes from some other plants, there was no intron in any of the 10 isolated seaweed TPS genes.

Characterization of multiple cold induced genes from Ammopiptanthus mongolicus and functional analyses of gene AmEBP1.

In comparison to herbaceous plants, studies of cold responsive genes and cold signaling in woody perennials are limited. Since most woody plants have evolved freezing tolerance (FT) in winter, together with similar lignified structures and winter adaptive mechanisms, it is more likely to find cold resistant genes in woody plants growing in temperate and arctic regions. In this study, Ammopiptanthus mongolicus, an evergreen, broadleaf and leguminous shrub was selected as a model to study cold tolerance in woody plants. Thirteen cold up-regulated cDNAs (including eight full-length cDNAs and five partial cDNAs) were cloned from A. mongolicus. One of these genes, AmEBP1, confers enhanced cold tolerance to E. coli and obvious increased freezing survival to Arabidopsis. In transgenic Arabidopsis expressing AmEBP1, transcript levels of some downstream genes in cold signaling exhibit increased accumulation upon cold treatment. Together with structural information, sub-cellular location, and promoter analysis data, it is suggested that AmEBP1 enhances plants cold tolerance by accelerating ribosome biogenesis and the concomitant translation of cold induced transcription factors and downstream protective proteins under cold stress.

Cloning and characterization of a second form of the rice adenine phosphoribosyl transferase gene (OsAPT2) and its association with TGMS.

A rice gene, OsAPT2, which encodes a putative adenine phosphoribosyl transferase (APRT), was cloned and characterized. Analysis of the cDNA and genomic sequences revealed seven exons and six introns in the OsAPT2. The deduced amino acid sequence of OsAPT2 is highly homologous to those of previously isolated APRTs. RT-PCR analysis indicated that the OsAPT2 transcript in the young panicles of 'Annong S-1' is down-regulated at 29 degrees C, the critical temperature for induction of 'Annong S-1' fertility conversion. Since the panicle is likely the thermo-sensitive organ at the early stages of pollen fertility alternation, the observed heat-induced change in the OsAPT2 expression pattern in young panicles may mediate, at least in part, thermo-sensitive genic male sterility (TGMS) in 'Annong S-1'. An antisense strategy was used to suppress the expression of the OsAPT2 homolog in Arabidopsis, and the obtained homozygous transgenic plants contained lower AMP content, displayed lower pollen germination rates and exhibited some abnormalities in leaf phenotypes and flowering timing. These data suggest that OsAPT2 is likely to be involved in TGMS in the rice line 'Annong S-1'.

Mutations in PurBox1 of the Bacillus subtilis pur operon control site affect adenine-regulated expression in vivo.

Transcription of the Bacillus subtilis pur operon is regulated by a purine repressor (PurR)-DNA control site interaction. The pur operon control site has two PurBoxes that are required for high-affinity PurR binding. An upstream, strong-binding PurBox1 is at position -81 to -68 relative to the transcription start site and a downstream weak-binding PurBox2 is at position -49 to -36. We constructed three PurBox1 mutations and the effects on binding of PurR to the control region in vitro and on regulation of pur operon expression in vivo were investigated. The mutations significantly reduced the binding of PurR to control region DNA. In strains with G-75A, G-75T and a five bp deletion (delta5) pur operon repression was defective in vivo. In addition in vivo PurR titration was used to confirm that sequences flanking PurBox1 and PurBox2 are required for PurR binding to the puroperon control site.

lambdaN gene expression regulated by translation termination in ribosome L24 mutant.

Besides transcription regulation, gene expression is also regulated at translation level. Although translation regulation is mainly mediated by translation initiation, an abundance of evidence shows that the termination phase of translation is also important for gene expression. The expression of lambdaN gene is down regulated at translation level in L24 mutant, however the precise mechanism still remains unknown. We report here that in an L24 mutant strain, the expression of lac-lambdaN and GST-lambdaN is decreased to 25% and 50% of that in wild type T83 strain respectively. Strikingly, the yield of GST-lambdaN fusion protein in L24 mutant can be restored to the level as in T83 wild type strain by changing the two codons upstream lambdaN stop codon. These findings imply that the stop codon and its context are involved in the translation regulation. The possible reason is that the translation termination complex containing L24 mutant ribosome may not dissociate properly in stop code region. This failure of disengagement from mRNA will slow down the process of following ribosomes, and consequently decrease the efficiency of lambdaN gene expression.