Some properties of a microsomal oleate desaturase from leaves.

1. When [1-14C]oleoyl-CoA was incubated with a pea-leaf homogenate oleate was both incorporated into microsomal 3-sn-phosphatidylcholine and released as the unesterified fatty acid. The proportion of oleate incorporated into this phospholipid was dependent on the relative amounts of thiol ester and microsomal preparation present in reactions. 2. At the concentrations of microsomal preparation and [14C]oleoyl-CoA used to study oleate desaturation the metabolism of the thiol ester was essentially complete after 5 min incubation, but the loss of label from 3-sn-phosphatidylcholine oleate and the concomitant increase in radioactivity in the linoleate of this phospholipid proceeded at approximately linear rates over a 60 min period. The kinetics of labelling of unesterified linoleate was consistent with the view that this labelled fatty acid was derived from 3-sn-phosphatidylcholine. 3. Oleate desaturation required oxygen and with unwashed microsomal fractions was stimulated either by NADPH or by the 105 000g supernatant. Washed microsomal preparations did not catalyse desaturation, but actively was restored by the addition of NADPH, 105 000G supernatant or Sephadex-treated supernatant. NADPH could be replaced by NADH or NADP+, but not by NAD+. 4. Microsomal fractions from mature and immature maize lamina and expanding spinach leaves also rapidly incorporated oleate from ([14C]oleoyl-CoA into 3-sn-phosphatidylcholine, but desaturation of 3-sn-phosphatidylcholine oleate was detected only with microsomal preparations from immature maize lamina. 5. It is proposed that leaf microsomal preparations posses an oleate desaturase for which 3-sn-phosphatidylcholine oleate is either the substrate or an immediate precursor of the substrate.     

Molecular cloning and characterization of soybean peroxidase gene families

Plant peroxidases play major roles in many physiological processes. A soybean seedbud (21 days after flowering) Uni-ZAP XR cDNA library was screened with a peroxidase-specific probe. The probe was generated by 3′ rapid amplification of cDNA ends with soybean seedbud total RNA and a degenerate primer derived from a plant peroxidase conserved amino acid region (distal heme ligand). Positive clones were recovered by PCR using the degenerate peroxidase-specific primer and the vector primer T₇ flanking the cloning site. Four cDNAs, designated GmEpa1, GmEpa2, GmEpb1, and GmEpb2, contained 1298, 1326, 1171, and 1145 nucleotides, excluding poly(A) tail, and encoded mature proteins of 303, 303, 292, and 292 amino acids, respectively. The four predicted amino acid sequences showed homology to other peroxidases. GmEpa1 and GmEpa2 exhibited 97% amino acid identity, GmEpb1 and GmEpb2 exhibited 93% amino acid identity, and GmEpa1 and GmEpb1 exhibited 47% amino acid identity. GmEPa1 and GmEPb1 were expressed as fusion proteins in Escherichia coli. The recombinant fusion proteins were sequestered in inclusion bodies and active forms of the two denatured proteins were recovered after in vitro folding in a medium containing hemin, urea and Ca²⁺. GmEpa1 and GmEpa2 messages were detected in developing seed and root, while GmEpb1 and GmEpb2 messages were present in root, leaf, stem and seed pod. These cDNAs and cDNA-specific primers will allow investigations into peroxidase’s role in development, stress response and in other physiological processes.     

Molecular cloning and characterization of a novel stem-specific gene from Camptotheca acuminata

In higher plants, P450s participate in the biosynthesis of many important secondary metabolites. Here we reported for the first time the isolation of a new cytochrome P450 cDNA that expressed in a stem-specific manner from Camptotheca acuminata (designated as CaSS), a native medicinal plant species in China, using RACE-PCR. The full-length cDNA of CaSS was 1735 bp long containing a 1530 bp open reading frame (ORF) encoding a polypeptide of 509 amino acids. Bioinformatic analysis revealed that CASS contained a heme-binding domain PFGXGRRXCX and showed homology to other plant cytochrome P450 monooxygenases and hydroxylases. Southern blotting analysis revealed that there was only one copy of the CaSS present in the genome of Camptotheca acuminata. Northern blotting analysis revealed that CaSS expressed, in a tissuespecific manner, highly in stem and lowly in root, leaf and flower. Our study suggests that CaSS is likely to be involved in the phenylpropanoid pathway.     

Molecular cloning and characterization of a novel WRKY gene from Brassica chinensis

A new WRKY gene was cloned from Brassica chinensis by rapid amplification of cDNA ends (RACE). The full-length cDNA of BcWRKY was 1175 bp long and contained a 924 bp open reading frame (ORF) encoding a putative W-box-binding protein of 308 amino acids. The predicted BcWRKY protein was found to have a potential bipartite nuclear localization sequence (NLS-BP) in its N-terminal region followed by a WRKY DNA-binding domain. Bioinformatic analysis revealed that BcWRKY resembled other WRKY domain-containing proteins from Arabidopsis (AtWRKY18), tobacco (WIZZ), parsley (PcWRKY4) and wild oat (ABF2). Expression of the BcWRKY gene could be induced by salicylic acid (SA), and influenced by Pseudomonas syringae pv. tomato strain DC3000 infection and wounding treatment. Our study implies that BcWRKY might have similar functions possessed by other WRKY genes such as inducing the expression of some defense-related genes and increasing plant's disease resistance ability.     

Molecular cloning and characterization of a novel WRKY gene from Brassica chinensis

A new WRKY gene was cloned from Brassica chinensis by rapid amplification of cDNA ends (RACE). The full-length cDNA of BcWRKY was 1175 bp long and contained a 924-bp open reading frame (ORF) encoding a putative W-box-binding protein of 308 amino acids. The predicted BcWRKY protein was found to have a potential bipartite nuclear localization sequence (NLS-PB) in its N-terminal region followed by a WRKY DNA-binding domain. Bioinformatic analysis revealed that BcWRKY resembled other WRKY domain-containing proteins from Arabidopsis thaliana (AtWRKY18), tobacco (WIZZ), parsley (PcWRKY4), and wild oat (ABF2). Expression of the BcWRKY gene could be induced by salicylic acid (SA) and influenced by Pseudomonas syringae pv. tomato strain DC3000 infection and wounding treatment. Our study implies that BcWRKY might have similar functions possessed by other WRKY genes, such as inducing the expression of some defense-related genes and increasing plants’ disease resistance ability. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 5, pp. 816–824. The text was submitted by the authors in English.     

Molecular cloning and characterization of a novel cold-regulated gene from Capsella bursa-pastoris.

A novel cor gene was cloned from Capsella bursa-pastoris (designated Cbcor15b) by RACE-PCR. The full-length cDNA of Cbcor15b was 652bp and contained a 417bp open reading frame (ORF) encoding a 139-amino acid hydrophilic protein. Multiple alignments showed that Cbcor15b had high similarity with other cold-regulated genes from Arabidopsis thaliana (cor15b, cor15a), Brassica napus (bn115, bn19 and bn26) and genes encoding late embryogenesis abundant (LEA) proteins. The predicted CbCOR15B protein was found to have a potential chloroplast signal sequence cleavage site, two cAMP- and cGMP-dependent protein kinase (PKA and PKG) phosphorylation sites. Cold acclimation assay showed that Cbcor15b was relevant to cold acclimation. Our study implies that Cbcor15b might have similar functions possessed by other cor genes in increasing plants' freezing tolerance.     

Molecular cloning and characterization of a novel mannose-binding lectin gene from Amorphophallus konjac

A new lectin gene was cloned from Amorphophallus konjac. The full-length cDNA of Amorphophallus konjac agglutinin (aka) was 736 bp and contained a 474 bp open reading frame encoding a 158 amino acid protein. Homology analysis revealed that the lectin from this Araceae species belonged to the superfamily of monocot mannose-binding proteins. Molecular modeling of AKA indicated that the three-dimensional structure of AKA strongly resembles that of the snowdrop lectin. Southern blot analysis of the genomic DNA revealed that aka belonged to a low-copy gene family. Northern blot analysis demonstrated that aka expression was tissue-specific with the strongest expression being found in root.     

Molecular cloning and characterization of a novel cbl-family gene, cbl-c

We have cloned a novel gene, cbl-c, of mammalian cbl-family. The cbl-c gene is predicted to encode a protein of 52 kDa that has a phosphotyrosine-binding domain, a RING finger and a proline-rich region. Cbl-c shows 50% homology to the amino-terminal sequences of Cbl and Cbl-b, but a sequence corresponding to the carboxy-terminal half of Cbl and Cbl-b is largely missing in Cbl-c. The expression of cbl-c mRNA is distinct from that of cbl and cbl-b mRNAs, being high in the colon and small intestine, but undetectable in brain and lymphoid tissues. The cbl-c gene is mapped in 19q13.2-13.3. Finally, the 52 kDa Cbl-c protein binds to the EGF receptor and Fyn tyrosine kinase. We conclude that Cbl-c is a novel Cbl-family adaptor protein that would regulate intracellular signaling mediated by various tyrosine kinases.     

Molecular cloning of two novel stearoyl-acyl desaturase genes from winterness wheat

Using RT-PCR and rapid amplification of cDNA ends, two new full-length cDNAs of SAD (TaSAD1 and TaSAD2) were obtained from a hardiest winter wheat cultivar (Mironovskaya808). Sequence comparison analysis showed that the deduced amino acid sequences of TaSAD1 and TaSAD2 had high similarity to those of other reported SAD proteins. They were also different each other by some substitutions, insertions and/or deletions involving single amino acid residues or motifs. Based on evolution analysis, it was clear that all SAD genes from Poaceae were closer than those from other genus such as Arabidopsis, Glycine, Triadica, Brassica, Sesamum and Bassia. All SAD genes clustered into two major groups in Poaceae. Meanwhile, TaSAD1 and TaSAD2 were clustered into different groups. The tertiary structure prediction indicated that both TaSAD1 and TaSAD2 proteins were a compact globular protein and their model structures almost were the same.     

Molecular cloning and characterization of a novel CD1 gene from the pig.

Much effort is underway to define the immunological functions of the CD1 multigene family, which encodes a separate lineage of Ag presentation molecules capable of presenting lipid and glycolipid Ags. To identify porcine CD1 homologues, a cosmid library was constructed and screened with a degenerate CD1 alpha3 domain probe. One porcine CD1 gene (pCD1.1) was isolated and fully characterized. The pCD1.1 gene is organized similarly to MHC class I and other CD1 genes and contains an open reading frame of 1020 bp encoding 339 amino acids. Expression of pCD1.1 mRNA was observed in CD3- thymocytes, B lymphocytes, and tissue macrophages and dendritic cells. The pCD1.1 cDNA was transfected into Chinese hamster ovary cells, and subsequent FACS analysis demonstrated that mAb 76-7-4, previously suggested to be a pig CD1 mAb, recognizes cell surface pCD1.1. Structurally, the pCD1.1 alpha1 and alpha2 domains are relatively dissimilar to those of other CD1 molecules, whereas the alpha3 domain is conserved. Overall, pCD1.1 bears the highest similarity with human CD1a, and the ectodomain sequences characteristically encode a hydrophobic Ag-binding pocket. Distinct from other CD1 molecules, pCD1.1 contains a putative serine phosphorylation motif similar to that found in human, pig, and mouse MHC class Ia molecules and to that found in rodent, but not human, MHC class-I related (MR1) cytoplasmic tail sequences. Thus, pCD1.1 encodes a molecule with a conventional CD1 ectodomain and an MHC class I-like cytoplasmic tail. The unique features of pCD1.1 provoke intriguing questions about the immunologic functions of CD1 and the evolution of Ag presentation gene families.     

Molecular cloning and characterization of a group II chaperonin delta-subunit from soybean

Molecular characterization of plant group II chaperonin (CCT, c-cpn, or TriC) still remains elusive. By PCR-based cloning techniques using soybeans, we have made a successful attempt to clone a delta-subunit homologue of CCT (CCTdelta). This subunit is responsible for the binding of an in vivo substrate, alpha-actin, by assisting the correct folding of the cytoskeletal protein in mouse, and the occurrence of the subunit homologue in plant CCT was unclear. As the cloning strategy, a putative amino acid segment, NH(2)-Gly-Gly-Gly-Ala-Pro-Glu-COOH, which is tightly conserved in all known animal and yeast CCTdelta subunits, was chosen for designing a degenerate primer of the PCR-cloning. The resultant 1881-bp cDNA was found to have an open-reading frame of 533 amino acids with a calculated molecular mass of 57,677 Da and to share about 58-65% identity overall at the amino acid level with the corresponding subunits known to date. Using antibodies raised against Escherichia coli-produced soybean insoluble CCTdelta as a monitoring tool, we purified soybean CCT from the extract of its immature seeds. STEM images demonstrated that the molecular shape of soybean CCT is a double eight-membered ring, which resembles the known group II chaperonins. The CCT also reactivated a denatured firefly luciferase with a significant, but limited level of the native enzymic activity in an in vitro system. Northern blot analysis showed that soybean CCTdelta gene, which is intronless and composed of a small family, was only expressed at a very early stage of seed development of soybean.     

Molecular cloning, characterization and expression of a novel jasmonate-dependent defensin gene from Ginkgo biloba

A novel defensin gene was isolated from Ginkgo biloba. The full-length cDNA of G. biloba defensin (designated as Gbd) was 534bp. The cDNA contained a 240-bp open reading frame encoding an 80-amino acid protein of 5.68 kDa with a potential 30 aa signal peptide. The putative GbD mature protein showed striking similarity to other plant defensins, representing low molecular size antimicrobial polypeptides. Eight cysteine sites conserved in plant defensins were also found in GbD at similar positions. Three-dimensional structure modeling showed that GbD strongly resembled defensin from tobacco (NaD1) and consisted of an alpha-helix and a triple-strand antiparallel beta-sheet that were stabilized by four intramolecular disulfide bonds, implying GbD may have functions similar to NaD1. The genomic DNA gel blot indicated that Gbd belonged to a multigene family. Expression analysis revealed that Gbd was up-regulated by wounding and methyl jasmonate treatments, suggesting that Gbd is potentially involved in plant resistance or tolerance to pathogens during wounding.