Marker_Free: a Novel Tendency of Transgenic Plants

Marker free is a rapidly developed strategy that offers a new approach for the elimination of public concerns caused by the selectable marker genes conferring antibiotic or herbicide resistance and so on. Furthermore, marker_free transgenic plants (MFTPs) have a number of special advantages, such as decreasing the concerns about safety of selectable marker and stacking transgenes progressively into transgenic plants, which significantly owns potential application value. Major approaches developed recently for obtaining MFTPs were reviewed in this paper.

Aphid-resistant Transgenic Tobacco Plants Expressing Modified gna Gene

A gene sequence coding for the precursor of Galanthus nivalis agglutinin (GNA) was modified by site-directed mutagenesis to change very low usage bias codons to higher usage bias ones for improvement of the gene expression in transgenic tobacco (Nicotiana tabacum L.) plants. Results from Western blot analysis of some of the transgenic tobacco plants showed that the expression level of GNA in plants transformed with the modified gene GNA34m reached 0.25% of total soluble proteins, while that of the GNA34 gene transgenic plants was 0.17%. Since the GNA expression level increased, the aphid resistance of GNA34m transgenic plants were also enhanced significantly as judged by a 71.0% aphid population inhibition in insect bioassay of GNA34m transformed plants and 63.7% for the plants transformed with the natural GNA34 gene.

Genetic and agronomic traits stability of marker-free transgenic wheat plants generated from Agrobacterium-mediated co-transformation in T2 and T3 generations

Genetically modified wheat has not been commercially utilized in agriculture largely due to regulatory hurdles associated with traditional transformation methods. Development of marker-free transgenic wheat plants will help to facilitate biosafety evaluation and the eventual environmental release of transgenic wheat varieties. In this study, the marker-free transgenic wheat plants previously obtained by Agrobacterium-mediated co-transformation of double T-DNAs vector were identified by fluorescence in situ hybridization(FISH) in the T1 generation, and their genetic stability and agronomic traits were analyzed in T2 and T3 generations. FISH analysis indicated that the transgene often integrated into a position at the distal region of wheat chromosomes. Furthermore, we show that the GUS transgene was stably inherited in the marker-free transgenic plants in T1 to T3 generations. No significant differences in agronomic traits or grain characteristics were observed in T3 generation, with the exception of a small variation in spike length and grains per spike in a few lines. The selection marker of bar gene was not found in the transgenic plants through T1 to T3 generations. The results from this investigation lay a solid foundation for the potential application of the marker-free transgenic wheat plants achieved through the co-transformation of double T-DNAs vector by Agrobacterium in agriculture after biosafty evaluation.

Features,Mechanisms and Applies of Post-transcriptional Gene Silencing in Transgenic Plants

Since transgene silencing was found in transgenic plants,many scholars have studied it extensively and considered that it has three functional mechanisms:post dependent gene silencing,transcriptional gene silencing,post transcriptional gene silencing.At the moment,people have mainly focused on the study of post transcriptional gene silencing and found its features:extensivity,conduction and peculiarity,also put forward some hypothesis for its mechanisms,for example,RNA threshold model,aberrant RNA model,inter or intra molecular base pairing model and so on.Furthermore,post transcriptional gene silencing is being applied in gene engineering of plants.Recently the people have found that post transcriptional gene silencing has bearing on capacity plants resisting virus.Many researchers have studied post transcriptional gene silencing,but there are some questions which need be solved in the future.This article summarizes progresses in features,mechanisms,applies of post transcriptional gene silencing about transgenic plants.

Photosynthetic Characteristics of Transgenic Rice Plants Overexpressing Maize Phosphoenopyruvate Carboxylase

用转PEPC、PPDK、NADP_ME、PEPC +PPDK 双基因水稻 (OryzasativaL .)及原种为材料 ,以光合酶活性、饱和光合速率及PSⅡ光化学效率 (Fv/Fm)为指标 ,研究了转PEPC基因水稻的光合生理特征。结果如下 :转PEPC基因水稻PEPC活性比原种提高 2 0倍 ;饱和光合速率比原种高 5 5 % ;用高光强或人工光氧化剂甲基紫精 (MV)处理后 ,与原种相比 ,转PEPC基因水稻光化学效率下降较少 ,证明其耐光抑制、耐光氧化能力增强 ,测定其光合日变化看出 :在 1d中不同时间 ,转PEPC基因水稻的光合速率均高于原种 ,且与PEPC活性的日变化有相似的趋势。上述结果为转PEPC基因水稻的生理机制和育种研究提供了依据和途径。

Transfer of Lysine-rich Protein Gene into Rice and Production of Fertile Transgenic Plants

Lysine-rich protein gene (lys) was cloned from Psophocarpus tetragonolobus (L.) DC. A plant expression plasmid was constructed and lys gene was under the control of maize ubiquitin promoter which is the highest efficient monocotyledon promoter. The plasmid was introduced into rice embryogenic calli by microprojectile bombardment. The regenerated fertile plants were obtained by effective selection for hygromycin B resistance. Genomic PCR and Southern blotting analyses showed that the lys gene has been integrated into rice genome. Simultaneously, the results of GUS histochemical assay demonstrated that gus report gene is also expressed in leaves, stems and roots of the transgenic rice plants. Data analysis showed that lysine content in most of the 11 transgenic plants is differently improved, and in one of them increased by 16.04%.

Alterations of Root and Fiber in Transgenic Cotton Plants with Chimeric Ph/P-ipt Gene Expression

The seed_specific phaseolin promoter (Ph/P) was fused to an ipt gene, then was cloned to a plant expression vector containing a gus gene driven by a 35S promoter. Cotton (Gossypium hirsutum L.) plants were transformed through pollen tube pathway methods. After seed germination, histochemical staining of the roots demonstrated that 32 GUS positive plants were obtained and three of which contained the chimeric Ph/P_ ipt transgene as confirmed by PCR analysis. An immunosorbent assay showed that two of the three transgenic cotton lines contained higher levels of zeatin equivalents in seeds than the control. Seedling development of these two transgenic lines differed from the control in a reduction of the shoot growth, showing a stunted phenotype as expected, but a surprisingly developed root system with a 3-4 fold fast_growing lateral roots. In addition, fibers (seed_hairs) of the two transgenic cotton lines were considerably shorter than those of the control. These results indicate that genetic engineering may be used to manipulate the development of cotton plants, particularly cotton fibers.

Anther culture of Transgenic rice plants

Anther culture is widely used in rice improve-ment and genome research.It is useful forgene transformation to stabilize foreign gene(s)and estimate the integrated copy number.We used two transgenic plants JB-3 andJB-4 as donors for anther culture.Their originwas a japonica cultivar Jingyin 119,whose im-mature embryos were transformed by particlebombardment with plasmid pCB1(a cecropin B

Leafy head formation of the progenies of transgenic plants of Chinese cabbage with exogenous auxin genes

The experiment was performed to evaluate the progenies of plant lines transgenic for auxin synthesis genes derived from Ri T-DNA. Four lines of the transgenic plants were selfcrossed and the foreign auxin genes in plants of T5 generation were confirmed by Southern hybridization. Two lines, D1232 and D1653, showed earlier folding of expanding leaves than untransformed line and therefore had early initiation of leaf y head. Leaf cuttings derived from plant of transgenic line D1653 produced more adventitious roots than the control whereas the cuttings from folding leaves had much more roots than rosette leaves at folding stage, and the cuttings from head leaves had more roots than rosette leaves at heading stage. It is demonstrated that early folding of transgenic leaf may be caused by the relatively higher concentration of auxin. These plant lines with auxin transgenes can be used for the study of hormonal regulation in differentiation and development of plant organs nd for the breeding of new varietywith rapid growth trait.

Maize Transformation of cry1Ac3 Gene and Insect Resistance of Their Transgenic Plants

The success for genetic transformation of maize (Zea mays L.) is highly related to genotype of target material. A few model varieties can be induced into type Ⅱ callus, which can be easily transformed with high regeneration frequency. However, most of cultivars could be only induced into type Ⅰ callus, which is difficult to be transformed with low regeneration. Thus, studying on the conditions of induction and transformation for type Ⅰ callus will show great importance for improving elite of maize directly with genetic engineering. Bacillus thuringiensis toxin protein (cry1Ac3) gene was successfully delivered into type Ⅰ calli of two elite inbred lines of maize, 340 and E28, via particle bombardment in this work. Fertile transgenic corn plants were obtained through phosphinothricin (PPT) or hygromycin B (HygB) selection, and the results of PCR, Southern blot assay and ELISA showed that foreign genes had been integrated into maize genome and expressed. In the meantime, strong resistance of some transgenic plants to corn borer was showed through bioassay. In addition, the comparison of selective effect between PPT and HygB showed that PPT, as a selective agent, was better than HygB for the growth and regeneration of resistant calli.

Increasing Accumulation Level of Foreign Protein in Transgenic Plants Through Protein Targeting

Targeting of the synthesized polypeptide in the cells is an important research field in modern cell biology. Cowpea trypsin inhibitor (cpti) gene has been modified and a fusion protein gene (sck) was produced by fusing a signal peptide sequence at cpti 5' end and an endoplasm reticulum (ER) retention signal peptide at cpti3' end respectively. The signal peptide can direct the newly synthesized polypeptide into ER, while ER retention signal can make the protein retained in the ER and its derivative protein body. ELISA test indicated that the accumulation level of foreign CpTI protein in sck transgenic tobacco (Nicotiana tabacum L.) was two times higher than cpti transgenic tobaccos and some individuals were four times higher. At the same time, sck transgenic tobacco has a high resistance to Lepidoptera pest due to the increased accumulation level of foreign CpTI protein. The strategy of foreign protein targeting can be used to increase the accumulation level of foreign protein in transgenic plants and can be widely applied to other related research field in plant genetic engineering.

Construction of Plant Expression Vector Carrying Two Insecticidal Genes and Obtain Insect resistant Transgenic Tobacco Plants

A plant expression vector carrying both pea lectin gene and Soybean trypsin inhibitor gene has been constructed and transferred into tobacco via Agrobacterium mediated transformation. Transgenic plants are further confirmed by ELISA, PCR and PCR Southern assays. Results of bioassays show that transgenic plants display notably inhibitory effects to larvae development and survival of Heliothis armigera Hubner.

Cloning of Glutamate Dehydrogenase cDNA from Chlorella sorokiniana and Analysis of Transgenic Tobacco Plants

A full_length cDNA has been cloned encoding nicotinamide adenine dinucleotide phosphate_specific glutamate dehydrogenase (NADP_GDH) from Chlorella sorokiniana with the RT_PCR method. The complete nucleotide sequence of NADP_GDH gene had 94% homology to the previously reported one . The NADP_GDH gene was constructed into a vector highly expressed in plants. The specific activity of NADP_GDH in transformants was detected, but not in the control plants. All transformed shoots on MS medium containing lower concentration of nitrogen and the transformed seedlings grown in lower concentration of nitrogen vermiculite had higher growth rate and more leaves than the control plants. Transformed leaf discs cultured on MS medium containing different nitrogen concentrations had more chlorophyll contents compared to the controls. These results suggested that exogenous NADP_GDH may enhance the absorption and utilization to ammonium in plants. The increased weight of transformed leaf discs cultured on medium supplemented with different concentrations of phosphinothricin (PPT) was more than that of control discs. 0.5 μg/mL PPT could be used as a selecting drug instead of kanamycin to develop the transformants. These results suggested that the NADP_GDH gene might be used as a new selecting gene in the future research of plant gene engineering.

Copper-Controllable, Site-Specific DMA Excision in Transgenic Plants

A copper-inducible, Cre-loxP recombination-mediated DNA excision system has been developed in transgenic tobacco plants. The copper inducible system derived from yeast was used for the control of the expression of the Cre recombinase. Upon copper induction, the GVS reporter gene expression unit flanked by two direct lox sites was excised from the transgenic tobacco genome. Quantitative fluorometric GUS assays, Northern blot and PCR analyses showed a high-efficient, copper-dependent and Cre-loxP mediated DNA recombination in all the tested transgenic lines. The copper inducible foreign gene excision might be of great potential in genetic control of transgenic crops.

Expression of Fusion Lytic Peptides Promotes Fungal Disease Resistance in Transgenic Plants

Many organisms produce small proteins which exhibit antimicrobial activities. In recent decades, the biological role of antimicrobial peptides (AMP) has been recognized as the main factor in the defense mechanisms against a broad range of pathogenic microbes. The increased worldwide incidence of microbial resistance to antibiotics makes AMPs promising alternative for the control of microbial disease. Exploring the potential of AMPs in transgenic crops could lead to the development of new and improved cultivars which are resistant to various economically important diseases. In the present study, two fusion lytic peptide gene constructs coding for antimicrobial peptides were expressed in Nicotiana benthamiana tobacco plants and tested against three fungal pathogens, Sclerotinia sclerotiorum, Rhizoctonia solani, and Pythium sp. Detached-leaf bioassay was employed for the transgenic plants carrying the fusion lytic peptide constructs (ORF13 and RSA1), transgenic vector only control plants (1234), and wild-type control plants (WT) against the three fungal pathogens. Symptom area of each leaf was measured with high accuracy and data were recorded and processed by statistical analyses. The results showed that transgenic plant lines ORF13 and RSL1 have substantial resistance to Sclerotinia sclerotiorum infection, producing significantly smaller lesion areas compared to vector only plant line 1234 and wild type plants. These transgenic lines also provided resistance against Rhizoctonia solani, however, these lines were not effective against the other fungal pathogen Pythium sp.

Cloning of Promoter of Chinese Bean GRP 1.8 Gene and Characterization of Its Function in Transgenic Tobacco Plants

In order to learn the expression pattern of GRP1 8(glycine rich protein) gene promoter in transgenic plants and to explore its potential application in plant genetic engineering for vascular specific expression of interested genes, GRP 1 8 promoter was amplified by PCR from Chinese bean genomic DNA. The intermediate vector was constructed by inserting vascular specific expression promoter of GRP 1 8 gene in vector pBI 101. The regenerated tobacco plants obtained were analyzed by PCR to select the putative transgenic plants. The histochemical localization of GUS( β D glucosidase) activity indicates that as for that of GRP 1 8 promoter we can confer the vascular specific expression of GUS gene.

Generating Marker-Free Transgenic Tobacco Plants by Agrobacteriummediated Transformation with Double T-DNA Binary Vector

We have developed a 'double T-DNA' binary vector system for generating selectable marker-free transgenic plants by Agrobacterium-mediated transformation. The 'double T-DNA' binary vector pDLBRBbarm which carried two independent T-DNAs, one containing a selectable marker neomycin phosphotransferase (nptII) gene and the other a bargene, was constructed. Transgenic tobacco (Nicotiana tabacum L.) plants were then produced by Agrobacterium-mediated transformation with this vector. Frequency of the primary transformants co-integrated with npt II gene and bar gene was 59.2%. Segregation of two T-DNA regions was found in 3 out of 4 T-1 lines from co-transformed T-0 plants with nptII and bar PPT-resistant and kanamycin-sensitive plants were in approximate 19.5% of the T-1 plants. The result indicated that this 'double T-DNA' vector system could be a workable approach to generate transgenic plants free from selectable marker genes. Co-transformation of nptII gene and bar gene to plants with mixtures of Agrobacterium tumefaciens strains containing single T-DNA vectors was also tested. Frequency of co-transformed plants was 20.0%-47.7% and relatively low as compared with that of 'double T-DNA' vector system.

Effects of External Chemical Regulation on Bt Transgenic Cotton Plants under Combined Stress of High Temperature and Water Deficit

[Objective] The study aimed to find a possible way to combat or alleviate the negative effects caused by high temperature and water deficit at the growth stage of peak boll-setting.[Method] With Bt transgenic cotton GK22 as the test cultivar,a potted experiment was carried out to investigate the effects of the regulation of external substances(the water solutions of pix,urea and their mixture) on the physiological parameters,insecticidal protein content,yield and yield component of cotton plants in artificial climate chambers treated with high temperature and water deficit.[Result] The application of external pix,urea or their mixture was effective in stabilizing the physiological parameters of cotton plants,insecticidal protein content,yield and yield components.Compared with the exclusive application of pix and urea,the mixture of pix and urea played the most effective role in stabilizing the content of chlorophyll,soluble sugar and insecticidal protein,alleviating the increase of the content of free amino acids and proline,and increasing boll number per plant,boll weight and seed cotton yield.[Conclusion] The water solutions of pix,urea or their mixtures can be used to combat or alleviate the stress of high temperature and water deficit if they are sprayed onto cotton plants prior to stress occurrence.

Risk Assessment of Synergism and Recombination on the Transgenic Plants Containing Viral Movement Protein and Replicase Genes

The transgenic tobacco plants transformed with movement protein gene of Tomato mosaic virus (ToMV) or Tobacco mosaic virus (TMV) and partial replicase gene of Cucumber mosaic virus (CMV) P1 isolate (CMV-P1), were inoculated with Potato virus X, Potato virus Y, TMV and CMV isolate RB (CMV-RB), respectively. Symptom observation showed there were no symptom differences in transgenic tobacco plants as compared with those in non-transgenie tobacco plants. ELISA also illustrated that the virus concentrations in the transgenic plants were similar to those in non-transgenic plants, indicating that no synergism is found in these plants. The transgenic tobacco plants expressing movement protein gene of ToMV or partial replicase gene of CMV-P1 were inoculated with TMV and CMV-RB, respectively. The local or systemic infected leaves were then used for elucidation of the possible virus recombination in transgenic plants with biological infectivity test, ELISA and immuno-capture RT-PCR. Within 16 months, no recombination was found between transformed genes and inoculated virus genomes. The research provides fundamental data for understanding of the possible risk of the transgenic plants expressing viral sequences.

Enhanced Stem Nematode Resistance of Transgenic Sweetpotato Plants Expressing Oryzacystatin-I Gene

Enhanced stem nematode resistance of transgenic sweetpotato （cv. Lizixiang） was achieved using Oryzacystatin-I （OCI） gene with Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 harbors a binary vector pCAMBIA1301 with OCI gene, gusA gene and hptII gene. Selection culture was conducted using 25 mg L-1 hygromycin. A total of 1 715 plants were produced from the inoculated 1 450 cell aggregates of Lizixiang via somatic embryogenesis. GUS assay and PCR analysis of the putative transgenic plants randomly sampled showed that 90.54% of them were transgenic plants. Transgenic plants exhibited significantly enhanced resistance to stem nematodes compared to the untransformed control plants by the field evaluation with stem nematodes. Stable integration of the OCI gene into the genome of resistant transgenic plants was confirmed by Southern blot analysis, and the copy number of integrated OCI gene ranged from 1 to 4. Transgene overexpression in stem nematode-resistant plants was demonstrated by quantitative real-time PCR analysis. This study provides a way for improving stem nematode resistance in sweetpotato.