Molecular cloning of <Emphasis Type="Italic">Ve</Emphasis> promoters from <Emphasis Type="Italic">Gossypium barbadense</Emphasis> and <Emphasis Type="Italic">G. hirsutum</Emphasis> and functional analysis in Verticillium wilt resistance

Global warming is affecting Mediterranean ecosystems, in which Arbutus unedo represents an important species. Mesophyll protoplasts are convenient material to study plant DNA integrity, nevertheless their release from mature plants along the year has not been reported in sclerophyll species. In this respect, in the present study the chance to isolate protoplasts from leaf tissues of A. unedo in different seasons was investigated. The digestion was obtained using Macerozyme R10 (1%) and Cellulase Onozuka RS (2%), with 0.6 M of mannitol, incubated for 4 h at 25 °C in the dark. In spring, soft leaves of different ages were studied to identify the most suitable material and protoplast yields were significantly influenced, with the still expanding 4th leaf characterized by the highest amount. Protoplast release decreased during the growing season when leaves become partially hardened, while their obtainment was quite impossible in summer and winter, due to leaf hardening. In November, an increase of incubation time (16 h) and of Cellulase RS (4%) or mannitol concentration (0.8 M) was needed with chilled leaves from the field; conversely, a good source for protoplasts were the green soft leaves coming from detached and pruned branches placed in controlled environment at spring temperatures. The employment of polyvinylpyrrolidone-40 during isolation procedure is discussed. The isolated protoplasts were employed to assess the DNA integrity, by using single cell gel electrophoresis (SCGE). The X-irradiated nucleoids were significantly damaged confirming that the present approach could be used in the selection of elite material of A. unedo devoted to fruit plantation.     

Molecular cloning and expression analysis of a novel <Emphasis Type="Italic">CONSTANS</Emphasis>-like gene from potato

A full-length cDNA of a StCONSTANS-like (StCOL) gene was cloned from potato (Solanum tuberosum L.) by RTPCR and RACE. The predicted amino acid sequence of this cDNA has a high degree of identity with other homologous members of the CO or COL family. Analysis of mRNA levels for StCOL shows that it is highly expressed in leaves and becomes weaker during tuberization; moreover, is independent of gibberellin A₃ and sucrose. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 11, pp. 1525–1531.     

Molecular cloning and expression analysis of <Emphasis Type="Italic">Bmrbp1</Emphasis>, the <Emphasis Type="Italic">Bombyx mori</Emphasis> homologue of the <Emphasis Type="Italic">Drosophila</Emphasis> gene <Emphasis Type="Italic">rbp1</Emphasis>

RBP1 is an important splicing factor involved in alternative splicing of the pre-mRNA of Drosophila sex-determining gene dsx. In this work, the Bombyx mori homologue of the rbp1 gene, Bmrbp1, was cloned. The pre-mRNA of Bmrbp1 gene is alternatively spliced to produce four mature mRNAs, named Bmrbp1-PA, Bmrbp1-PB, Bmrbp1-PC and Bmrbp1-PD, with nucleotide lengths of 799 nt, 1,316 nt, 894 nt and 724 nt, coding for 142 aa, 159 aa, 91 aa and 117 aa, respectively. BmRBP1-PA and BmRBP1-PD contain a N terminal RNA recognization motif (RRM) and a C terminal arginine/serine-rich domain, while BmRBP1-PB and BmRBP1-PC only share a RRM. Amino acid sequence alignments showed that BmRBP1 is conserved with its homologues in other insects and with other SR family proteins. The RT-PCR showed that Bmrbp1-PA was strongly expressed in all examined tissues and development stages, but Bmrbp1-PB was weakly expressed in these tissues and stages. The expression of both Bmrbp1-PA and Bmrbp1-PB showed no obvious sex difference. While the Bmrbp1-PC and Bmrbp1-PD were beyond detection by RT-PCR very likely due to their tissue/stage specificity. These results suggested that Bmrbp1 should be a member of SR family splicing factors, whether it is involved in the sex-specific splicing of Bmdsx pre-mRNA needs further research.     

Molecular cloning and characterization of cold-responsive gene <Emphasis Type="Italic">Cbrci35</Emphasis> from <Emphasis Type="Italic">Capsella bursa-pastoris</Emphasis>

A new rare cold-inducible (RCI) gene designated Cbrci35 was cloned from Capsella bursa-pastoris, an edible wild herb, using the rapid amplification of cDNA ends (RACE) method. The full-length cDNA of Cbrci35 (Database Accession No.: AY566573) was 1300 bp and contained a 978 bp ORF encoding a precursor of 326 amino acid residues with a 23 amino acids signal peptide. The predicted Cbrci35 protein contained a peroxidase active site and proximal heme-ligand signatures, an RGD cell attachment sequence motif and two leucine zipper pattern motifs. Bioinformatics analysis revealed that Cbrci35 has a high level of similarity with RCI genes from Arabidopsis thaliana and peroxidases genes from other plants. RT-PCR analysis revealed that Cbrci35 expressed only in root. A cold acclimation assay showed that Cbrci35 was expressed immediately after cold triggering, but this expression was transient, suggesting that it concerns cold acclimation. But expression was not induced exposed to dehydration, salt stress or abscisic acid, indicating that it might be subjected specifically to cold regulation. These results indicate that Cbrci35 is an analogue of RCI genes and may participate in cold-response or increasing the freezing tolerance of plants.     

Isolation and characterization of a <Emphasis Type="Italic">GAI/RGA</Emphasis>-like gene from <Emphasis Type="Italic">Gossypium hirsutum</Emphasis>

The aim of the investigation reported here was to assess the role of gibberellin in cotton fiber development. The results of experiments in which the gibberellin (GA) biosynthesis inhibitor paclobutrazol (PAC) was tested on in vitro cultured cotton ovules revealed that GA is critical in promoting cotton fiber development. Plant responses to GA are mediated by DELLA proteins. A cotton nucleotide with high sequence homology to Arabidopsis thaliana GAI (AtGAI) was identified from the GenBank database and analyzed with the BLAST program. The full-length cDNA was cloned from upland cotton (Gossypium hirsutum, Gh) and sequenced. A comparison of the putative protein sequence of this cDNA with all Arabidopsis DELLA proteins indicated that GhRGL is a putative ortholog of AtRGL. Over-expression of this cDNA in Arabidopsis plants resulted in the dwarfed phenotype, and the degrees of dwarfism were related to the expression levels of GhRGL. The deletion of 17 amino acids, including the DELLA domain, resulted in the dominant dwarf phenotype, demonstrating that GhRGL is a functional protein that affects plant growth. Real-time quantitative PCR results showed that GhRGL mRNA is highly expressed in the cotton ovule at the elongation stage, suggesting that GhRGL may play a regulatory role in cotton fiber elongation.     

Molecular cloning and characterization of the <Emphasis Type="Italic">Dicer-like</Emphasis> 2 gene from <Emphasis Type="Italic">Brassica rapa</Emphasis>

Dicer-like proteins (DCLs) are involved in small RNA-mediated development and viral defense in plants. In model plants, at least four DCLs have been found and a number of studies have helped to understand their function. However, the function of the Dicer or DCLs in other plants is still unclear. Here, we report the full-length cDNA sequence of Brassica rapa ssp. chinensis DCL2 (BrDCL2) gene, which contains a 4,179 bp open reading frame (ORF) encoding a protein of 1,392 amino acids. At the 3′ end of BrDCL2, clones with three different lengths of 3′ untranslated region were found. An alternative splice variant of BrDCL2, BrDCL2sv, in which one intron was retained between exon9 and exon10, was also cloned. Because of a change in the coding sequence resulting in a premature terminal codon, BrDCL2sv was expected to translate a short peptide containing the whole DEXHc domain.     

Molecular cloning and expression analysis of two <Emphasis Type="Italic">FAD2</Emphasis> genes from chia (<Emphasis Type="Italic">Salvia hispanica</Emphasis>)

FATTY ACID DESATURASE 2 (FAD2, EC 1.3.1.35), also known as delta-12 oleate desaturase, is a key enzyme for linoleic acid and α-linolenic acid biosynthesis. Chia (Salvia hispanica) seeds contain the highest known proportion of α-linolenic acid in any plant sources. In this study, two full-length FAD2 genes, named as ShFAD2-1 and ShFAD2-2, were isolated from S. hispanica based on RACE method. Both ShFAD2-1 and ShFAD2-2 proteins possess strong transmembrane helices, three histidine motifs and a C-terminal ER-located signal (YNNKL). Phylogenetic analysis showed that both ShFAD2-1 and ShFAD2-2 are grouped with constitutive plant FAD2s. Heterologous expression in Saccharomyces cerevisiae indicated that ShFAD2-1 and ShFAD2-2 genes both encode a bio-functional delta-12 oleate desaturase. ShFAD2-2 was mainly expressed in flowers and early-stage seeds while ShFAD2-1 expression was almost constitutive in different organs. qRT-PCR results demonstrated that ShFAD2-1 and ShFAD2-2 show a cold-induced and heat-repressed expression pattern, whereas they also were differentially up-regulated or repressed by other abiotic stresses. This is the first cloning and function characterization of FAD2 from S. hispanica, which can provide insights into molecular mechanism of high ALA traits of S. hispanica and enrich our understanding of the roles of FAD2 genes in various abiotic stresses.     

Molecular cloning and characterization of a profilin gene <Emphasis Type="Italic">BnPFN</Emphasis> from <Emphasis Type="Italic">Brassica</Emphasis><Emphasis Type="Italic">nigra</Emphasis> that expressing in a pollen-specific manner

Brassica nigra is a newly found invasive species in Zhejiang Province, China. It distributes alongside the roads, in vegetable fields and on riversides. When it blooms, some natives there will suffer from allergic rhinitis. We designed gene-specific primer pairs according to reported profilin genes and successfully isolated their homolog from flower bud cDNA of B. nigra. The gene, designated BnPFN, was submitted to GenBank under accession number EU004073. BnPFN was 405 bp in length encoding 134 amino acids. Expression analysis of BnPFN gene was carried out by means of RT-PCR. The results showed that BnPFN express only in anthers and pollens, and there was no detection in roots, leaves, stems, sepals, petals and pistils. We suggest that BnPFN is a pollen-specific gene and may be responsible for pollen anaphylactic reactions in those invading areas when B. nigra blooms.