We investigated the microRNA172 (miR172)-mediated regulatory network for the perception of changes in external and endogenous signals to identify a universally applicable floral regulation system in ornamental plants, manipulation of which could be economically beneficial. Transgenic gloxinia plants, in which miR172 was either overexpressed or suppressed, were generated using Agrobacterium-mediated transformation. They were used to study the effect of altering the expression of this miRNA on time of flowering and to identify its mRNA target. Early or late flowering was observed in transgenic plants in which miR172 was overexpressed or suppressed, respectively. A full-length complementary DNA (cDNA) of gloxinia (Sinningia speciosa) APETALA2-like (SsAP2-like) was identified as a target of miR172. The altered expression levels of miR172 caused up- or down-regulation of SsAP2-like during flower development, which affected the time of flowering. Quantitative real-time reverse transcription PCR analysis of different gloxinia tissues revealed that the accumulation of SsAP2-like was negatively correlated with the expression of miR172a, whereas the expression pattern of miR172a was negatively correlated with that of miR156a. Our results suggest that transgenic manipulation of miR172 could be used as a universal strategy for regulating time of flowering in ornamental plants.
Arabidopsis/genetics* ; Arabidopsis Proteins/metabolism* ; Cloning, Molecular ; DNA, Complementary/metabolism* ; Flowers/physiology* ; Gene Expression Profiling ; Gene Expression Regulation, Plant ; Homeodomain Proteins/metabolism* ; Lamiales/physiology* ; MicroRNAs/metabolism* ; Nuclear Proteins/metabolism* ; Plants, Genetically Modified/physiology* ; Plasmids/metabolism* ; Polymerase Chain Reaction ; Transgenes

OBJECTIVETo evaluate the health effects of parental dietary exposure to GM rice TT51 on the male reproductive system of rat off spring.

METHODSRice-based diets, containing 60% ordinary grocery rice, MingHui63, or TT51 by weight, were given to parental rats (15 males/30 females each group) for 70 days prior mating and throughout pregnancy and lactation. After weaning, eight male offspring rats were randomly selected at each group and fed with diets correspondent to their parents' for 70 days. The effects of exposure to TT51 on male reproductive system of offspring rats were assessed through sperm parameters, testicular function enzyme activities, serum hormones (FSH, LH, and testosterone levels), testis histopathological examination, and the relative expression levels of selected genes along the hypothalamic-pituitary- testicular (HPT) axis.

RESULTSNo significant differences were observed in body weight, food intake, organ/body weights, serum hormone, sperm parameters, testis function enzyme ACP, LDH, and SDH activities, testis histopathological changes, and relative mRNA expression levels of GnRH-R, FSH-R, LH-R, and AR along the HPT axis.

CONCLUSIONThe results of this study demonstrate that parental dietary exposure to TT51 reveals no significant differences on the reproductive system of male offspring rats compared with MingHui63 and control.

Animals ; Diet ; adverse effects ; Female ; Genitalia, Male ; physiology ; Male ; Oryza ; chemistry ; Plants, Genetically Modified ; adverse effects ; chemistry ; Random Allocation ; Rats ; Rats, Wistar

We describe a genetic transformation method of secondary somatic embryogenesis in alfalfa through cotyledon-stage somatic embryos of alfalfa infected by Agrobacterium strain GV3101. The Agrobacterium strain GV3101 contained binary vector pCAMBIA2301 that had gus gene as reporter and npt II gene as selectable marker. The infected primary embryos were induced through series of medium under 75 mg/L kanamycin selection. We obtained the transgenic alfalfa plants. Then, GUS expression in different tissue of transgenic alfalfa was tested by GUS histochemical analysis. Further, the stable integration and transformation efficiency were tested by polymerase chain reaction and Southern blotting hybridization. The result showed that GUS expression was different in different organs of transgenic alfalfa; the copy number of integrated npt II gene was from 1 to 4; the transformation efficiency via primary somatic embryogenesis was 65.82%.
Agrobacterium ; genetics ; Medicago sativa ; embryology ; genetics ; physiology ; Plant Somatic Embryogenesis Techniques ; Plants, Genetically Modified ; embryology ; genetics ; Tissue Culture Techniques ; Transformation, Genetic

Polyamine is an important physiological regulation substance functioning in a wide variety of biological processes, such as plant growth, development, senescence and adversity stress tolerance, which widely exist in all living organisms. Their biosynthetic pathways have already been revealed, and their physiological roles are being elucidated gradually. Previous work on polyamines biosynthetic deficiency mutants and various transgenic plants facilitates improved understanding of the important roles of polyamines and biosynthetic enzymes in plant growth and development. This paper summarizes researches in the biosynthetic pathways of polyamines in plants, focusing on research advances on functions of genes involved in polyamine metabolism. In addition, the potential research directions, especially the application of the genes in the genetic engineering of plant stress tolerance were also discussed.
Biosynthetic Pathways ; physiology ; Gene Expression Regulation, Plant ; Genes, Plant ; genetics ; Plant Physiological Phenomena ; Plants ; metabolism ; Plants, Genetically Modified ; Polyamines ; metabolism ; Stress, Physiological

To study the possibilities for improvement of the ornamental character and production of secondary metabolites by using Wedelia trilobata hairy roots, we investigated the induction of W. trilobata L. hairy roots and its consumption changes of carbon resource, nitrogen resource, phosphate and calcium in the medium during liquid culture. The results showed that hairy roots could be incited from the cut edges of leaf explants 7 days after inoculation with Agrobacterium rhizogenes ATCC15834 and could have an autonomous growth on the medium without phytohormones. The PCR amplification showed that rol genes of Ri plasmid of A. rhizogenes was integrated and expressed into the genome of transformed hairy roots. The hairy root line grew very slowly in 0-7 days, very fast from 7 to 21 days. During the liquid culture of hairy roots, sucrose, NO3(-)-N, PO4(3-) and Ca2+ in the medium could be gradually absorbed and utilized with time. The content of NO3(-)-N in the medium was 5.8% of the initial amount at day 7, while sucrose content was about 50% of the initial amount. At day 35, the NO3(-)-N and sucrose content in the medium was 1.82% and 3.39% of the initial amount, respectively. In combination with Ca2+ consumption, PO4(3-) of the medium was rapidly absorbed and utilized. At day 7, the content of PO4(3-) in the spent medium was only 1.76% of the initial amount; but even at day 35, the content of Ca2+ in the spent medium was still 61.3% of the initial amount. The results presented here had provided the possibilities on improvement the ornamental character and how to prepare optimum medium for large scale cultivation and production of secondary metabolites from W. trilobata L. hairy roots.
Culture Techniques ; methods ; Plant Roots ; cytology ; growth & development ; Plants, Genetically Modified ; genetics ; growth & development ; metabolism ; Rhizobium ; genetics ; physiology ; Transformation, Genetic ; Wedelia ; genetics ; growth & development ; microbiology

OBJECTIVETo transform the antimicrobial peptide fusion gene of cecropin B and rabbit NP-1(CN) into Houttuynia cordata to improve its antimicrobic capability.

METHODThe fusion gene of CN designed and synthesized artificially was recombined with expression vector pBI121. The recombined vector was transformed to Agrobacterium tumefaciens LBA4404, by which CN gene was transformed to the explants of H. cordata. The transgenic regeneration plantlets were selected by kanamycin and rapid screening PCR. The transgenic plants were identified by PCR-Southern of genomic DNA and RT-PCR. The disease resistances were detected by antibacterial zone trail of leaf extracts to E. coli K12 and infection by Rhizoctonia solani.

RESULTGene of interesting CN was inserted into genomic DNA and expressed in transformed H, cordata, whose resistance to E. coli K12 and Rh. solani was stronger than that of the non-transformed control.

CONCLUSIONThe fusion gene CN can improve antimicrobic capability of transformed H. cordata.

Animals ; Anti-Bacterial Agents ; immunology ; pharmacology ; C-Reactive Protein ; genetics ; metabolism ; pharmacology ; Houttuynia ; genetics ; immunology ; microbiology ; Immunity, Innate ; Insect Proteins ; genetics ; immunology ; pharmacology ; Nerve Tissue Proteins ; genetics ; metabolism ; pharmacology ; Plant Diseases ; immunology ; microbiology ; Plants, Genetically Modified ; genetics ; immunology ; microbiology ; Rabbits ; Recombinant Fusion Proteins ; genetics ; immunology ; pharmacology ; Rhizoctonia ; physiology ; Transformation, Genetic

We constructed an expression cassette of the organophosphorus pesticide degrading (opd) gene under the control of the E8 promoter. Then opd was transformed into tomato fruit using an agroinfiltration transient expression system. beta-Glucuronidase (GUS) staining, reverse transcription-polymerase chain reaction (RT-PCR), wavelength scanning, and fluorescent reaction were performed to examine the expression of the opd gene and the hydrolysis activity on coumaphos of organophosphorus hydrolase (OPH) in tomato fruit. The results show that the agroinfiltrated tomato fruit-expressed OPH had the maximum hydrolysis activity of about 11.59 U/mg total soluble protein. These results will allow us to focus on breeding transgenic plants that could not only enhance the degrading capability of fruit and but also hold no negative effects on pest control when spraying organophosphorus pesticides onto the seedlings in fields.
Aryldialkylphosphatase ; genetics ; physiology ; Coumaphos ; pharmacology ; Insecticides ; pharmacology ; Lycopersicon esculentum ; genetics ; metabolism ; Plants, Genetically Modified ; Promoter Regions, Genetic

A novel vacuolar Na+/H+ exchanger, CgNHX1, was cloned from a halophytic species Chenopodium glaucum by using reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) technique. Sequence alignment and phylogenetic analysis of 22 NHX genes from GenBank as well as the new CgNHX1 gene indicate that NHX genes shared a great degree of similarity, regardless of their glycophytic or halophytic origin. Expression of the CgNHX1 gene was induced by NaCl and peaked at 400 mmol/L NaCl. Overexpression of NHX1 genes in rice enhanced their tolerance to salt stress. However, there is no significant difference in salt tolerance among the transgenic rice plants overexpressing the NHX1 genes from either glycophytic or halophytic species. The Na+ content of both the wild type (WT) and transgenic plants increased when exposed to 50 and 100 mmol/L NaCl, and the Na+ concentration in transgenic plants was marginally higher than that of WT. Our data demonstrate that the overexpression of the NHX1 gene from either glycophytic or halophytic species resulted in the enhanced tolerance to salt stress at a similar level, suggesting that NHX gene per se might not be the reason accounting for the difference in salt tolerance between glycophytes and halophytes.
Amino Acid Sequence ; Cloning, Molecular ; DNA, Complementary ; metabolism ; Gene Expression Regulation, Plant ; Molecular Sequence Data ; Oryza ; genetics ; Phylogeny ; Plant Proteins ; genetics ; Plants ; genetics ; metabolism ; Plants, Genetically Modified ; Protein Structure, Tertiary ; Reverse Transcriptase Polymerase Chain Reaction ; Salts ; pharmacology ; Sequence Homology, Amino Acid ; Sodium Chloride ; pharmacology ; Vacuolar Proton-Translocating ATPases ; chemistry ; physiology

Obtaining marker-free plants with high efficiency will benefit the environmental release of transgenic crops. To achieve this point, a binary vector pNB35SVIP1 with three T-DNAs was constructed by using several mediate plasmids, in which one copy of bar gene expression cassette and two copies of VIP1 gene expression cassette were included. EHA101 Agrobacterium strain harboring the final construct was applied to transform soybean (Glycine max) cotyledon nodes. Through 2 - 3 months regeneration and selection on 3 - 5mg/L glufosinate containing medium, transgenic soybean plants were confirmed to be obtained at 0.83% - 3.16%, and co-transformation efficiency of both gene in the same individual reached up to 86.4%, based on southern blot test. By the analysis of PCR, southern blot and northern blot combining with leaf painting of herbicide in T1 progenies, 41 plants were confirmed to be eliminated of bar gene with the frequency of 7.6% . Among the T1 populations tested, the loss of the alien genes happened in 22.7% lines, the silence of bar gene took place in 27.3% lines, and VIP1 gene silence existed in 37.1% marker-free plants. The result also suggested that the plasmid with three T-DNAs might be an ideal vector to generate maker-free genetic modified organism.
Aminobutyrates ; pharmacology ; Blotting, Northern ; Blotting, Southern ; Cotyledon ; drug effects ; genetics ; physiology ; DNA, Bacterial ; genetics ; Gene Expression Regulation, Plant ; Genes, Plant ; genetics ; Genetic Vectors ; genetics ; Herbicide Resistance ; genetics ; Herbicides ; pharmacology ; Plant Leaves ; drug effects ; genetics ; physiology ; Plants, Genetically Modified ; drug effects ; genetics ; physiology ; Regeneration ; drug effects ; genetics ; physiology ; Rhizobium ; genetics ; Soybeans ; drug effects ; genetics ; physiology ; Transformation, Genetic

The Arabidopsis thaliana tonoplast Na+ /H+ antiporter gene, AtNHX1, was transferred into buckwheat by Agrobacterium-mediated method. Transgenic buckwheat plants were regenerated and selected on MS basal medium supplemented with 2.0mg/L 6-BA, 1.0mg/L KT, 0.lmg/L IAA, 50mg/L kanamycin and 500mg/L carbenicillin. 426 seedlings from 36 resistant calli originated from 864 explants (transformed about at 4.17 percentage) exhibited resistance to kanamycin. The transformants were confirmed by PCR, Southern blotting, RT-PCR and Northern blotting analysis. After stress treatment for 6 weeks with 200mmol/L NaCl, transgenic plants survived, while wild-type plants did not. After 3 days of stress treatment through different concentrations of NaCl, transgenic plants accumulated higher concentration of Na+ and proline than the control plants. However, the K+ concentration of transgenic plants declined in comparison with the control plants. Moreover, the rutin content of the roots, stems and leaves of transgenic buckwheat increased than those of the control plants. These results showed that it could be possible to improve the salt-tolerance of crops with genetic technology.
Adaptation, Physiological ; drug effects ; genetics ; physiology ; Arabidopsis Proteins ; genetics ; physiology ; Blotting, Northern ; Blotting, Southern ; Cation Transport Proteins ; genetics ; physiology ; Fagopyrum ; genetics ; metabolism ; physiology ; Plant Roots ; genetics ; metabolism ; physiology ; Plant Stems ; genetics ; metabolism ; physiology ; Plants, Genetically Modified ; genetics ; metabolism ; physiology ; Potassium ; metabolism ; Proline ; metabolism ; Regeneration ; Reverse Transcriptase Polymerase Chain Reaction ; Rutin ; metabolism ; Sodium ; metabolism ; Sodium Chloride ; pharmacology ; Sodium-Hydrogen Exchangers ; genetics ; physiology ; Transformation, Genetic