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 ofT. 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 thattma 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 thattma 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.

Using RNA extracted fromZingiber officinale rhizomes and primers designed according to the conservative regions of monocot mannose-binding lectins, the full-length cDNA ofZ. officinale agglutinin (ZOA) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA ofzoa 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 thatzoa expressed in all the tested tissues ofZ. officinale including leaf, root and rhizome, suggesting it to be a constitutively expressing form. ZOA protein was successfully expressed inEscherichia 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

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 theAgrobacterium 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 thegna 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.

Water stress is by far the leading environmental stress limiting crop yields worldwide. Genetic engineering techniques hold great promise for developing crop cultivars with high tolerance to water stress. In this study, theBrassica oleracea var.acephala BoRS1 gene was transferred into tobacco throughAgrobacterium- mediated leaf disc transformation. The transgenic status and transgene expression of the transgenic plants was confirmed by polymerase chain reaction (PCR) analysis, Southern hybridization and semi-quantitative one step RT-PCR analysis respectively. Subsequently, the growth status under water stress, and physiological responses to water stress of transgenic tobacco were studied. The results showed that the transgenic plants exhibited better growth status under water stress condition compared to the untransformed control plants. In physiological assessment of water tolerance, transgenic plants showed more dry matter accumulation and maintained significantly higher levels of leaf chlorophyll content along with increasing levels of water stress than the untransformed control plants. This study shows thatBoRS1 is a candidate gene in the engineering of crops for enhanced water stress tolerance.

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 expressingGbERF were generated. Overexpression ofGbERF 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 asPR1b, 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-GCCcis-elements. Moreover, the constitutive expression ofGbERF in transgenic tobacco enhanced the plant’s resistance toPseudomonas syringae pvtabaci infection. In conclusion,GbERF mediates the expression of a wide array ofPR and ethylene-responsive genes and plays an important role in the plant’s response to biotic stress.

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-DOM1 (polypeptides A) and PPA-DOM₂ (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.

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.

The E8 gene is related to ethylene biosynthesis in plants. To explore the effect of the expression pattern of the E8 gene on different E8 promoters, the molecular evolution of E8 promoters was investigated. A total of 16 E8 promoters were cloned from 16 accessions of seven tomato species, and were further analysed. The results from 19 E8 promoters including three previously cloned E8 promoters (X13437, DQ317599 and AF515784) showed that the size of the E8 promoters varied from 2101 bp (LA2150) to 2256 bp (LA2192); their sequences shared 69.9% homology and the average A/T content was 74.9%. Slide-window analysis divided E8 promoters into three regions – A, B and C – and the sequence identity in these regions was 72.5%, 41.2% and 70.8%, respectively. By searching the cis-elements of E8 promoters in the PLACE database, mutant nucleotides were found in some functional elements, and deletions or insertions were also found in regions responsible for ethylene biosysnthesis (–1702 to –1274) and the negative effect region (–1253 to –936). Our results indicate that the size of the functional region for ethylene biosynthesis in the E8 promoter could be shortened from 429 bp to 113 bp (–1612 to –1500). The results of molecular evolution analysis showed that the 19 E8 promoters could be classified into four clade groups, which is basically consistent with evolution of the tomato genome. Southern blot analysis results showed that the copy number of E8 promoters in tomato and some other wild species changed from 1 to 4. Taken together, our study provides important information for further elucidating the E8 gene expression pattern in tomato, analysing functional elements in the E8 promoter and reconstructing the potent E8 promoter.

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 fibreless mutant XZ142 (fl) was examined. RNA in situ hybridization analysis indicated that GbAGL2 transcripts were preferentially restricted to outer ovule integuments, carpels and fibres. These expression patterns implied that GbAGL2 might participate in the development of the carpel and ovule. Furthermore, Arabidopsis transformation was performed and modifications occurred in flowers, 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 findings suggest that GbAGL2 might not only specify the identity of floral organs but also play a potential key role in ovary or fibre development in cotton.