Examination of camptothecin and 10-hydroxycamptothecin in Camptotheca acuminata plant and cell culture, and the affected yields under several cell culture treatments

Camptothecin and its derivatives are monoterpenoid indole alkaloids exhibiting significant anti-tumor actions. With the aim of improving the production of these pharmaceuticals, the contents of camptothecin and 10-hydroxycamptothecin in different tissues including roots, stems, leaves, young flower buds, opening flowers, fading flowers and seeds from Camptotheca acuminata, were investigated. The young flower buds had the highest alkaloid concentrations (camptothecin, 2.46 mg/g of dry weight; 10-hydroxycamptothecin, 1.41 mg/g of dry weight). Callus showed lower concentrations but it should also be considered as a potential source of these pharmaceuticals. In the present study, the growth rate of Camptotheca acuminata cells in culture did not correlate with contents of camptothecin and 10-hydroxycamptothecin. Alkalo id accumulation by cells under various treatments (heavy metal ions, UV-B), methyl-jasmonate, abscisic acid, salicylic acid and hydrogen peroxide was examined, and the most notable effects appeared in the cells induced by UV-B light (which showed an 11-fold increase in camptothecin concentration) and by salicylic acid (which showed a 25-fold increase in 10-hydroxycamptothecin concentration). These results are significant in the context of the production of both pharmaceuticals.

Identifi cation and expression profi le of GbAGL2, a C-class gene from Gossypium barbadense.

An AGAMOUS (AG)-like gene, GbAGL2, was isolated from Gossypium barbadense and characterized. Alignment and phylogenetic analysis indicated that GbAGL2 shared high homology with AG-subfamily genes and belonged to a C-class gene family. DNA gel blot analysis showed that GbAGL2 belonged to a low-copy gene family. Reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qPCR) revealed that GbAGL2 was highly expressed in reproductive tissues including ovules and carpels, but barely expressed in vegetative tissues. In addition, GbAGL2 expression in a cotton cultivar XuZhou142 (wt) (XZ142, G. hirsutum L.) and its fi breless mutant XZ142 (fl ) was examined. RNA in situ hybridization analysis indicated that GbAGL2 transcripts were preferentially restricted to outer ovule integuments, carpels and fi bres. These expression patterns implied that GbAGL2 might participate in the development of the carpel and ovule. Furthermore, Arabidopsis transformation was performed and modifi cations occurred in fl owers, and the silique length of transgenic plants also increased slightly, suggesting that the GbAGL2 gene may have a positive effect on the development of the ovary or ovule. Our fi ndings suggest that GbAGL2 might not only specify the identity of fl oral organs but also play a potential key role in ovary or fi bre development in cotton.

Generation of tobacco lines with widely different reduction in nicotine levels via RNA silencing approaches.

Issues related to the nicotine content of tobacco have been public concerns.Several reports have described decreasing nicotine levels by silencing the putrescine N-methyltransferase (PMT) genes, but the reported variations of nicotine levels among transgenic lines are relatively low in general. Here we describe the generation in tobacco (Nicotiana tabacum) lines with widely different, reduced nicotine levels using three kinds of RNA-silencing approaches.The relative efficacies of suppression were compared among the three approaches regarding the aspect of nicotine level in tobacco leaves.By suppressing expression of the PMT genes, over 200 transgenic lines were obtained with nicotine levels reduced by 9.1-96.7%. RNA interference (RNAi) was the most efficient method of reducing the levels of nicotine,whereas cosuppression and antisense methods were less effective. This report gives clues to the efficient generation of plants with a variety of metabolite levels, and the results demonstrate the relative efficiencies of various RNA-silencing methods.

cDNA cloning and expression analysis of a mannose-binding lectin from Pinellia pedatisecta.

Pinellia pedatisecta agglutinin (PPA)is a very basic protein that accumulates in the tuber of P.pedatisecta .PPA is a hetero-tetramer protein of 40 kDa,composed of two polypeptide chains A (about 12 kDa)and two polypeptides chains B (about 12 kDa).The full-length cDNA of PPA was cloned from P.pedatisecta using SMART RACE-PCR technology; it was 1146 bp and contained a 771 bp open reading frame (ORF)encoding a lectin precursor of 256 amino acid residues with a 24 amino acid signal peptide.The PPA precursor contained 3 mannose-binding sites (QXDXNXVXY) and two conserved domains of 43% identity,PPA-DOM 1 (polypeptides A)and PPA-DOM 2 (polypeptides B).PPA shared varying identities,ranging from 40% to 85%,with mannose-binding lectins from other species of plant families such as Araceae, Alliaceae, Iridaceae, Liliaceae, Amaryllidaceae and Bromeliaceae. Southern blot analysis indicated that ppa belonged to a multi-copy gene family. Expression pattern analysis revealed that ppa expressed in most tested tissues, with high expression being found in spadix,spathe and tuber.Cloning of the ppa gene not only provides a basis for further investigation of its structure,expression and regulatory mechanism,but also enables us to test its potential role in controlling pests and fungal diseases by transferring the gene into plants in the future.

Overexpression of GbERF confers alteration of ethylene-responsive gene expression and enhanced resistance to Pseudomonas syringae in transgenic tobacco.

GbERF belongs to the ERF (ethylene responsive factor) family of transcription factors and regulates the GCC-box containing pathogen-related (PR) genes in the ethylene signal transduction pathway. To study the function of GbERF in the process of biotic stress, transgenic tobacco plants expressing GbERF were generated. Overexpression of GbERF did not change transgenic plant's phenotype and endogenous ethylene level. However, the expression profile of some ethylene-inducible GCC-box and non-GCC-box containing genes was altered, such as PR1b, PR2, PR3, PR4, Osmotin, CHN50, ACC oxidase and ACC synthase genes. These data indicate that the cotton GbERF could act as a transcriptional activator or repressor to regulate the differential expression of ethylene-inducible genes via GCC and non-GCC cis-elements. Moreover, the constitutive expression of GbERF in transgenic tobacco enhanced the plant's resistance to Pseudomonas syringae pv tabaci infection. In conclusion, GbERF mediates the expression of a wide array of PR and ethylene-responsive genes and plays an important role in the plant's response to biotic stress.

Genomic cloning and characterization of a PPA gene encoding a mannose-binding lectin from Pinellia pedatisecta

A gene encoding a mannose-binding lectin, Pinellia pedatisecta agglutinin (PPA), was isolated from leaves of Pinellia pedatisecta using genomic walker technology. The ppa contained an 1140-bp 5'-upstream region, a 771-bp open reading frame (ORF) and an 829-bp 3'-downstream region. The ORF encoded a precursor polypeptide of 256 amino acid residues with a 24-amino acid signal peptide. There were one putative TATA box and six possible CAAT boxes lying in the 5'-upstream region of ppa. The ppa showed significant similarity at the nucleic acid level with genes encoding mannose-binding lectins from other Araceae species such as Pinellia ternata, Arisaema heterophyllum, Colocasia esculenta and Arum maculatum. At the amino acid level, PPA also shared varying homology (ranging from 40% to 85%) with mannose-binding lectins from other plant species, such as those from Araceae, Alliaceae, Iridaceae, Lillaceae, Amaryllidaceae and Bromeliaceae. The cloning of the ppa gene not only provides a basis for further investigation of PPA's structure, expression and regulation mechanism, but also enables us to test its potential role in controlling pests and fungal diseases by transferring the gene into tobacco and rice in the future

Enhancement of resistance to aphids by introducing the snowdrop lectin gene gna into maize plants.

In order to enhance the resistance to pests, transgenic maize (Zea mays L.) plants from elite inbred lines containing the gene encoding snowdrop lectin (Galanthus nivalis L. agglutinin; GNA) under control of a phloem-specific promoter were generated through the Agrobacterium tumefaciens-mediated method. The toxicity of GNA-expressing plants to aphids has also been studied. The independently derived plants were subjected to molecular analyses. Polymerase chain reaction (PCR) and Southern blot analyses confirmed that the gna gene was integrated into maize genome and inherited to the following generations. The typical Mendelian patterns of inheritance occurred in most cases. The level of GNA expression at 0.13%-0.28% of total soluble protein was observed in different transgenic plants. The progeny of nine GNA-expressing independent transformants that were derived separately from the elite inbred lines DH4866, DH9942, and 8902, were selected for examination of resistance to aphids. These plants synthesized GNA at levels above 0.22% total soluble protein, and enhanced resistance to aphids was demonstrated by exposing the plants to corn leaf aphid (Rhopalosiphum maidis Fitch) under greenhouse conditions. The nymph production was significantly reduced by 46.9% on GNA-expressing plants. Field evaluation of the transgenic plants supported the results from the inoculation trial. After a series of artificial self-crosses, some homozygous transgenic maize lines expressing GNA were obtained. In the present study, we have obtained new insect-resistant maize material for further breeding work.

Molecular cloning and characterization of a novel mannose-binding lectin cDNA from Zantedeschia aethiopica

Using RNA extracted from Zantedeschia aethiopica young leaves and primers designed according to the conservative regions of Araceae lectins, the full-length cDNA of Z. aethiopica agglutinin (ZAA) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of zaa was 871 bp and contained a 417 bp open reading frame (ORF) encoding a lectin precursor of 138 amino acids. Through comparative analysis of zaa gene and its deduced amino acid sequence with those of other Araceae species, it was found that zaa encoded a precursor lectin with signal peptide. Secondary and three-dimensional structure analyses showed that ZAA had many common characters of mannose-binding lectin superfamily and ZAA was a mannose-binding lectin with three mannose-binding sites. Southern blot analysis of the genomic DNA revealed that zaa belonged to a multi-copy gene family

cDNA cloning and characterization of a mannose-binding lectin from Zingiber officinale Roscoe (ginger) rhizomes.

Using RNA extracted from Zingiber officinale rhizomes and primers designed according to the conservative regions of monocot mannose-binding lectins, the full-length cDNA of Z. officinale agglutinin (ZOA) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of zoa was 746 bp and contained a 510 bp open reading frame (ORF) encoding a lectin precursor of 169 amino acids with a signal peptide. ZOA was a mannose-binding lectin with three typical mannose-binding sites (QDNY). Semi-quantitative RT-PCR analysis revealed that zoa expressed in all the tested tissues of Z. officinale including leaf, root and rhizome, suggesting it to be a constitutively expressing form. ZOA protein was successfully expressed in Escherichia coli with the molecular weight expected. To our knowledge, this is the first mannose-binding lectin cDNA cloned from the family Zingiberaceae. Our results demonstrate that monocot mannose-binding lectins also occur within the family Zingiberaceae.

Isolation and characterization of a new mannose-binding lectin gene from Taxus media.

In this paper, we report the cloning and characterization of the first mannose-binding lectin gene from a gymnosperm plant species, Taxus media. The full-length cDNA of T. media agglutinin (TMA) consisted of 676 bp and contained a 432 bp open reading frame (ORF) encoding a 144 amino acid protein. Comparative analysis showed that TMA had high homology with many previously reported plant mannose-binding lectins and that tma encoded a precursor lectin with a 26-aa signal peptide. Molecular modelling revealed that TMA was a new mannosebinding lectin with three typical mannose-binding boxes like lectins from species of angiosperms. Tissue expression pattern analyses revealed that tma is expressed in a tissue-specific manner in leaves and stems, but not in fruits and roots. Phylogenetic tree analyses showed that TMA belonged to the structurally and evolutionarily closely related monocot mannose-binding lectin superfamily. This study provides useful information to understand the molecular evolution of plant lectins.