Yuanlei Hu

Inducible excision of selectable marker gene from transgenic plants by the Cre/lox site-specific recombination system

In a plant transformation process, it is necessary to use marker genes that allow the selection of regenerated transgenic plants. However, selectable marker genes are generally superfluous once an intact transgenic plant has been established. Furthermore, they may cause regulatory difficulties for approving transgenic crop release and commercialization. We constructed a binary expression vector with the Cre/lox system with a view to eliminating a marker gene from transgenic plants conveniently. In the vector, recombinase gene cre under the control of heat shock promoter and selectable marker gene nptII under the control of CaMV35S promoter were placed between two lox P sites in direct orientation, while the gene of interest was inserted outside of the lox P sites. By using this vector, both cre and nptII genes were eliminated from most of the regenerated plants of primary transformed tobacco through heat shock treatment, while the gene of interest was retained and stably inherited. This autoexcision strategy, mediated by the Cre/lox system and subjected to heat shock treatment to eliminate a selectable marker gene, is easy to adopt and provides a promising approach to generate marker-free transgenic plants.

Molecular cloning and characterization of a gene encoding RING zinc finger ankyrin protein from drought-tolerant Artemisia desertorum

A RING zinc finger ankyrin protein gene, designated AdZFP1, was isolated from drought-tolerant Artemisia desertorum Spreng by mRNA differential display and RACE. Its cDNA was 1723 bp and encoded a putative protein of 445 amino acids with a predicted molecular mass of 47.9 kDa and an isoelectric point (pI) of 7.49. A typical C3HC4-type RING finger domain was found at the C-terminal region of the AdZFP1 protein, and several groups of ankyrin repeats were found at the N-terminal region. Alignments of amino acid sequence showed that AdZFP1 was 66% identical to the Arabidopsis thaliana putative RING zinc finger ankyrin protein AAN31869. Transcriptional analysis showed that AdZFP1 was inducible under drought stress in root, stem and leaf of the plant. Semi-quantitative reverse-transcriptase-polymerase chain reaction (RT-PCR) analysis showed that the transcript of AdZFP1 was strongly induced by exogenous abscisic acid (ABA) and also by salinity, cold and heat to some extent. Overexpression of the AdZFP1 gene in transgenic tobacco enhanced their tolerance to drought stress.

Efficient Transformation of the Medicinal Mushroom Ganoderma lucidum

Ganoderma lucidum is a well-known and important medicinal mushroom, but its genetic modification has not been reported. We developed an efficient procedure for isolation and regeneration of protoplasts fromG. lucidum. To construct a vector for high-level expression of heterologous genes inG. lucidum, the 1.4-kb regulatory region of the glyceraldehyde-3-phosphate dehydrogenase gene (GPD) was isolated from the genomic DNA ofLentinus edodes, and theGPD promoter was fused to the β-glucuronidase (GUS) and bialaphos resistance (bar) genes. Using the resulting construct, p301-bG1, an efficient transformation system based on electroporation was established forG. lucidum. GUS expression was observed among transformants conferring bialaphos resistance, indicating that theL. edodes GPD promoter can be used for expression of exogenous genes inG. lucidum. We also studied green fluorescent protein (GFP) as another reporter for transformation ofG. lucidum. TheL. edodes GPD promoter was fused respectively to theGFP andbar genes, and the resulting construct, p301-bg, was introduced intoG. lucidum. StableGFP expression in transformants was detectable by fluorescence microscopy, suggesting the suitability ofGFP as a reporter system in transformation of this mushroom. This is the first report of an efficient transformation system forG. lucidum using different reporters, paving the way for genetic modification of this famous medicinal mushroom.

CaMV 35S promoter directs β-glucuronidase expression in Ganoderma lucidum and Pleurotus citrinopileatus

The cauliflower mosaic virus (CaMV) 35S promoter has been most commonly used in plant transformation studies, but its activity in mushrooms has not been reported. p301-b is a binary vector containing a bialaphos resistance gene driven by the promoter of Lentinus edodes glyceraldehyde-3-phosphate dehydrogenase (GPD) gene. CaMV 35S-GUS was inserted into p301-b, and the resulting construct p301-bG was transformed to protoplasts of Ganoderma lucidum and basidiospores of Pleurotus citrinopileatus. GUS activity was observed in the transformants, indicating that CaMV 35S promoter can direct expression of exogenous gene in the mushrooms. This is the first report on the application of CaMV 35S promoter in genetic modification of mushrooms.

Increase of sulphur-containing amino acids in transgenic potato with 10 ku zein gene from maize

The 10 ku zein gene of maize was under control of patatin class I promoter of potato and transferred into potato genome by the leaf-disc method. The expression of 10 ku zein was determined in the tuber of transgenic plants by RT-PCR. Furthermore, the sulphur-containing amino acids in transgenic tuber increase remarkably.

Ectopic expression of a c1-I allele from maize inhibits pigment formation in the flower of transgenic tobacco

MYB-related proteins play a key role in regulating the biosynthesis of anthocyanins in plants at the transcriptional level. An MYB gene designated cl-I-2K1 (GenBank accession no. AY237128) was cloned from maize line “2K1 purple,” and except for a deletion of nine nucleotides encoding three amino acids right at the carboxyl terminal end of the encoded protein, it was identical to the previously characterized c1-I gene. Flowers of transgenic tobacco overexpressing this c1-I allele showed severe reduction in pigmentation, whereas the pigmentation patterns of flowers of tobacco transformed with both c1-I-2K1 and R homologue gene r-2K1 showed no obvious change. Thus, this c1-I allele appears to act as a repressor in pigment formation and function through titration of a bHLH factor.

Expression of NtPT5 Is Correlated with the Degree of Colonization in Tobacco Roots Inoculated with Glomus etunicatum

An important characteristic of arbuscular mycorrhizal (AM) symbiosis is the transfer of phosphate (Pi) from AM fungi to plants, and this transfer is facilitated by plasma membrane-spanning phosphate transporter (PT) proteins. Five Nicotiana tabacum PT genes (NtPT), members of Pht1 family, express in the root. However, the ways these genes contribute to the development of AM is unclear. In this study, transcription analysis was performed to study the expression levels of NtPT1–5 genes in tobacco roots. To investigate the expression pattern of these five genes during the development of AM from no inoculation to high colonization, we screened for differentiating transcriptional responses of non-mycorrhizal roots and AM roots by real-time PCR. Our results indicate that only NtPT5 was mycorrhiza-specific phosphate transporter during arbuscular mycorrhizal symbiosis in tobacco roots, and the induction was tightly correlated with the degree of root colonization by Glomus etunicatum. In addition, based on an alignment and analysis of the evolution of Pht1 family members in six solanaceous plants, we developed the evolutionary pattern of the Pht1 gene family members in six solanaceous plants.

Enhanced late blight resistance of transgenic potato expressing glucose oxidase under the control of pathogen-inducible promoter

To engineer crop disease resistance by utilizing natural defense mechanism that was expressed in the incompatible host-pathogen interactions is expected to result in a durable and broad-spectrum resistance. In order to prove this viewpoint, we amplified the coding region of the glucose oxidase (GO) gene fromAspergillus niger via PCR and fused it to the pathogen-inducible promoter, Prp1-1. The chimeric gene was cloned into a plant expression vector and conjugated intoAgrobacterium. Twenty-three transgenic potato plants were obtained byAgrobacterium-mediated transformation. The integration ofGO gene was confirmed by Southern hybridization and theGO gene expression was identified with Kl-starch color reaction.Phytophthora infestans inoculation revealed that the expression of the chimeric transgene was induced by pathogen infection. Most of the transgenic plants exhibited various degrees of enhanced disease resistance. Four of them had lesion sizes reduced to less than half of the non-transgenic controls. One plant showed disease resistance of the hypersensitive response. These results testified the feasibility of our strategy of expressingGO transgene under the control of the disease-inducible promoter in engineering crop disease resistance.

Computational analysis of the relationship between allergenicity and digestibility of allergenic proteins in simulated gastric fluid.

BackgroundSafety assessment of genetically modified (GM) food, with regard to allergenic potential of transgene-encoded xenoproteins, typically involves several different methods, evaluation by digestibility being one thereof. However, there are still debates about whether the allergenicity of food allergens is related to their resistance to digestion by the gastric fluid. The disagreements may in part stem from classification of allergens only by their sources, which we believe is inadequate, and the difficulties in achieving identical experimental conditions for studying digestion by simulated gastric fluid (SGF) so that results can be compared. Here, we reclassify allergenic food allergens into alimentary canal-sensitized (ACS) and non-alimentary canal-sensitized (NACS) allergens and use a computational model that simulates gastric fluid digestion to analyze the digestibilities of these two types.ResultsThe model presented in this paper is as effective as SGF digestion experiments, but more stable and reproducible. On the basis of this model, food allergens are satisfactorily classified as ACS and NACS types by their pathways for sensitization; the former are relatively resistant to gastric fluid digestion while the later are relatively labile.ConclusionThe results suggest that it is better to classify allergens into ACS and NACS types when understanding the relationship between their digestibility and allergenicity and the digestibility of a target foreign protein is a parameter for evaluating its allergenicity during safety assessments of GM food.

Promoter of the rolC gene of Agrobacterium rhizogenes can be strongly regulated in glandular cell of transgenic tobacco

A 577-bp promoter segment of Agrobacterium rhizogenes rolC, previously known as the phloem-specific gene expression promoter, was fused to the 5′ end of a reporter gene, β-glucuronidase (GUS), uidA. This rolC-promoter-driven expression of the GUS gene was found to be significantly strong in glandular cells in transgenic tobacco plants. Analysis of this segment of the promoter sequence revealed a myb response element.