Mosaic expression pattern of the nptII gene in transgenic tobacco plants Nu 21

Mosaic expression pattern of the nptII gene in transgenic tobacco Nu 21 leaf somatic cells was demonstrated. Inheritance of this phenotype (in T1-T4 and F1 backcrosses) was revealed. Three plant groups were distinguished, with low frequency of variegation manifestation (0-21.8%), with the high frequency of mosaic progeny (63.1 to 100%), and the intermediate type, where the frequency of the appearance of mosaic plants varied in a wide range, from 0 to 100%. The data obtained suggested the existence of two metastable states of a transgene in the leaf disk somatic cells (active and silenced), which could be associated with DNA modification, i.e., methylation of cytosine within the nptII gene sequence.     

Analysis of mosaic expression of the nptII gene in transgenic tobacco plant lines contrasting in mosaicism

Differences in the mosaic plants occurence frequency between the Nu5 and Nu6 lines of transgenic tobacco (Nicotiana tabacum) plants remained irrespective of the nptII marker gene allelic state. nptII gene transition from the hemizygous state (T₃) to the homozygous one (T₄) was accompanied by an increase in the frequency of mosaics in both lines. Transition from the homozygous state (T₄) into the hemizygous one (F₁) resulted in a further increase in the frequency of mosaic plants in the Nu5 line, whereas this parameter remained at a high level in the Nu6 line. pMAS promoter hypermethylation in plants of both lines, as well as differences in the 5??-part truncated nptII gene copy cytosin methylation level between the Nu5 and Nu6 lines, pointed to nptII gene mosaic expression epigenetic regulation.     

Variability of organ-specific gene expression in transgenic tobacco plants

Variability of expression of introduced marker genes was analysed in a large number of tobacco regenerants from anAgrobacterium-mediated transformation. In spite of standardization of sampling, considerable variation of GUS and NPTII expression was observed between individual transformants at different times of analysis and in different parts of the same plant. Organ-specificity of root versus leaf expression conferred by the par promoter from the haemoglobin gene ofParasponia andersonii in front of thegus gene showed a continuous spectrum. GUS expression in roots was found in 128 out of 140 plants; expression in leaves was found in 46 plants, and was always lower than in the corresponding roots. NPTII expression regulated by the nos promoter also showed a continuous spectrum. Expression levels were generally higher in roots than in leaves. Plants with high GUS expression in leaves showed high NPTII activity as well. A positive correlation between the level of NPTII expression and the numbers of integrated gene copies was noted. Chromosomal position effects and physiological determination are suggested as triggers for the variations. The transformed regenerated tobacco plants were largely comparable to clonal variants.     

Stability of expressing the nptII gene in transgenic tobacco plants (nicotiana tabacum L.) with multiple T-DNA insertions

The stability of nptII gene expression was assessed in transgenic tobacco plants with multiple T-DNA insertions. The plants were obtained by self-pollination in the first (T1) and second (T2) generations and also in F1 from crossing T1 plants. The multiple copies showed stable Mendelian inheritance.     

The expression of a heat-inducible chimeric gene in transgenic tobacco plants

Summary A chimeric gene under the control of the hsp70 promoter of Drosophila is heat regulated in roots, stems and leaves, but not in pollen of transgenic tobacco plants. For these and other parameters, it behaves similarly to plant heat-shock genes.     

Consistent and stable expression of the nptII, uidA and bar genes in transgenic Pinus radiata after Agrobacterium tumefaciens-mediated transformation using nurse cultures

An Agrobacterium tumefaciens-mediated transformation protocol has been developed for embryogenic cell cultures of Pinus radiata. Transgenic lines were only produced when embryogenic tissue was placed on nurse tissue during the Agrobacterium co-cultivation and recovery stages of the procedure. Plantlets were regenerated via somatic embryogenesis from ten of the 11 transgenic lines tested and at least 20 of each line were planted in a GMO glasshouse. Expression of the nptII, uidA and bar genes in up to ten plants of each individual transgenic line was evaluated by molecular, biochemical and functional analysis. As expected, expression of the nptII gene varied among the ten lines, while within ten replicates of the same line, nptII expression appeared to be consistent, with the exception of one line, K3. Likewise, the level of GUS activity varied among transgenic lines, but was relatively consistent in plants derived from the same tissue, except for two lines, G4 and G5. Moreover, similar absolute values and pattern of gene expression of uidA was observed in the transgenic plants, for two consecutive years. Plantlets from eight lines survived a spray treatment with the equivalent of 2 kg/ha and 4 kg/ha of the commercial formulation Buster, whereas non-transformed controls died. Southern hybridisation analysis of embryogenic tissue and green needle tissue from putative transgenic lines demonstrated a relatively low number of gene insertions (from one to nine) of both the bar and nptII genes in the nine transgenic lines tested.     

去除转基因植物标记基因的研究进展

得到转基因植物以后 ,标记基因就失去了筛选的作用。但它的存在引起公众对转基因植物的安全性以及环境效应的担心 ,所以在目的基因转入后 ,要去除标记基因。该文主要就利用共转化、转座子、同源重组、位点特异重组酶等去除标记基因的方法进行了总结 ,并对各种方法的优缺点进行了比较 ,对该技术未来的发展趋势也进行了展望。     

Characterization of the potato MADS-box gene STMADS16 and expression analysis in tobacco transgenic plants

A new MADS-box gene, STMADS16, has been cloned in Solanum tuberosum L. that is expressed in all vegetative tissues of the plant, mainly in the stem, but not in flower organs. STMADS16 expression is established early during vegetative development and is not regulated by light. Sequence similarity besides the spatial and temporal expression patterns allow to define a novel MADS-box subfamily comprising STMADS16 and the gene STMADS11. Expression of the STMADS16 sense cDNA under the control of the 35S cauliflower mosaic virus promoter modifies the inflorescence structure by increasing both internode length and flower proliferation of the inflorescence meristems, and confers vegetative features to the flower. Moreover, STMADS16 ectopic expression overcomes the increase in flowering time and node number produced under short-day photoperiod, while the flowering time is not affected in long-day conditions. These results are discussed in terms of a possible role for STMADS16 in promoting vegetative development.     

The sequence of a pea vicilin gene and its expression in transgenic tobacco plants

A 5.5 kb Eco RI fragment containing a vicilin gene was selected from a Pisum sativum genomic library, and the protein-coding region and adjacent 5 and 3 regions were sequenced. A DNA construction comprising this 5.5 kb fragment together with a gene for neomycin phosphotransferase II was stably introduced into tobacco using an Agrobacterium tumefaciens binary vector, and the fidelity of expression of the pea vicilin gene in its new host was studied. The seeds of eight transgenic tobacco plants showed a sixteen-fold range in the level of accumulated pea vicilin. The level of accumulation of vicilin protein and mRNA correlated with the number of integrated copies of the vicilin gene. Pea vicilin was confined to the seeds of transgenic tobacco. Using immunogold labelling, vicilin was detected in protein bodies of eight out of ten embryos (axes plus cotyledons) and, at a much lower level, in two out of eleven endosperms. Pea vicilin was synthesized early in tobacco seed development; some molecules were cleaved as is the case in pea seeds, yielding a major parental component of M _r50000 together with a range of smaller polypeptides.     

A/T-rich sequences act as quantitative enhancers of gene expression in transgenic tobacco and potato plants

The role of an A/T-rich positive regulatory region (P268, -444 to -177 from the translation start site) of the pea plastocyanin gene (PetE) promoter has been investigated in transgenic plants containing chimeric promoters fused to the -glucuronidase (GUS) reporter gene. This region enhanced GUS expression in leaves of transgenic tobacco plants when fused in either orientation to a minimal pea PetE promoter (-176 to +4) and in roots when fused in either orientation upstream or downstream of a minimal cauliflower mosaic virus 35S promoter (-90 to +5). The region was also able to enhance GUS expression in microtubers of transgenic potato plants when placed in either orientation upstream of a minimal class I patatin promoter (-332 to +14). Dissection of P268 revealed that cis elements responsible for enhancing GUS expression from the minimal PetE promoter were distributed throughout P268. Multiple copies of a 31 bp A/T-rich sequence from within P268 and of a 26 bp random A/T sequence were able to enhance GUS expression from the minimal PetE promoter, indicating that A/T-rich sequences are able to act as quantitative, non-tissue-specific enhancer elements in higher plants. Abbreviations: CaMV, cauliflower mosaic virus; GUS, -glucuronidase; HMG, high-mobility group; MAR, matrix-associated region; MU, methylumbelliferone; SAR, scaffold-associated region.     

Evaluating the effect of codon optimization on expression of bar gene in transgenic tobacco plants

Journal of Plant Biochemistry and Biotechnology - Information of codon usage bias has been used for modifying genes for improved expression in heterologous systems. Codon modifications are carried...     

转基因植物中卡那霉素抗体（Ｋan^r）标记基因的生物安全性

卡那霉素抗性（Kan^r)基因是转基因植物中广泛使用的一类标记基因,其生物安全性受到普遍关注,本文详细讲座了转基因植物中Kan^r基因的漂流及其对自然生态环境的影响,并对Kan^r基因及编码蛋白ＡＰＨ（３′）－Ⅱ的人畜食用安全性进行了综述分析。     

Inducible expression of bacterio-opsin in transgenic tobacco and tomato plants

The development of new strategies to enhance resistance of plants to pathogens is instrumental in preventing agricultural losses. Lesion mimic, the spontaneous formation of lesions resembling hypersensitive response lesions in the absence of a pathogen, is a dramatic phenotype occasionally induced upon expression of certain transgenes in plants. These transgenes simulate the presence of a pathogen and, therefore, activate the plant anti-pathogen defense mechanisms and induce a state of systemic resistance. Lesion mimic genes have been successfully used to enhance the resistance of a number of different plants to pathogen attack. However, constitutive expression of these genes in plants is associated with the spontaneous formation of lesions on leaves and stems, reduced growth, and lower yield. We tested the possibility of using a wound-inducible promoter to control the expression of bacterio-opsin (bO), a transgene that confers a lesion mimic phenotype in tobacco and tomato plants when constitutively expressed. We found that plants with inducible expression of bO did not develop spontaneous lesions. Nevertheless, under controlled laboratory conditions, they were found to be resistant to infection by pathogens. The activation of defense mechanisms by the bO gene was not constitutive, and occurred in response to wounding or pathogen infection. Furthermore, wounding of transgenic tobacco plants resulted in the induction of systemic resistance to pathogen attack within 48 h. Our findings provide a promising initial assessment for the use of wound-inducible promoters as a new strategy to enhance pathogen resistance in transgenic crops by means of lesion mimic genes.     

Organ regulated expression of the Parasponia andersonii haemoglobin gene in transgenic tobacco plants

Summary Plant haemoglobin genes are known to occur in legume and non-legume families and in both nodulating (e.g. Parasponia andersonii) and non-nodulating species (e.g. Trema tomentosa). Their presence in non-nodulating plants raises the possibility that haemoglobins might serve a function in non-symbiotic tissues distinct from their role in the nitrogen-fixing root nodules induced by micro-organisms. We report here that a P. andersonii haemoglobin promoter can regulate expression of either the P. andersonii haemoglobin gene, or a hybrid construct with the bacterial chloramphenicol acetyltransferase gene (cat), in the nonsymbiotic plant, Nicotiana tabacum. Expression is predominantly in the roots, implying that haemoglobins might have a function in roots of non-nodulated plants. We have also observed a low level of haemoglobin protein in non-nodulated P. andersonii roots, but not leaves, supporting this assertion. The expression in transgenic plants will allow further characterization of the promoter sequences essential for the organ-specific expression of haemoglobins in nonsymbiotic tissues.