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

１．ＩｎｔｒｏｄｕｃｔｉｏｎＣｒｏｐｐｌａｎｔｓａｒｅｓｕｓｃｅｐｔｉｂｌｅｔｏａｔａｃｋｂｙａｗｉｄｅｒａｎｇｅｏｆｈｅｒｂｉｖｏｒｏｕｓｉｎｓｅｃｔｓ．Ｉｔｉｓｅｓｔｉｍａｔｅｄｔｈａｔａｒｏｕｎｄ１３％ｏｆｔｈｅｔｏｔａｌｗｏｒｌｄｗｉｄｅ...

Fast,simple,efficient Agrobacterium rhizogenes-mediated transformation system to non-heading Chinese cabbage with transgenic roots

Non-heading Chinese cabbage, a variety of Brassica campestris, is an important vegetable crop in the Yangtze River Basin of China. However,the immaturity of its stable transformation system and its low transformation efficiency limit gene function research on non-heading Chinese cabbage. Agrobacterium rhizogenes-mediated(ARM) transgenic technology is a rapid and effective transformation method that has not yet been established for non-heading Chinese cabbage plants. Here, we optimized conventional ARM approaches(one-step and two-step transformation methods) suitable for living non-heading Chinese cabbage plants in nonsterile environments. Transgenic roots in composite non-heading Chinese cabbage plants were identified using phenotypic detection, fluorescence observation, and PCR analysis. The transformation efficiency of a two-step method on four five-day-old non-heading Chinese cabbage seedlings(Suzhouqing, Huangmeigui, Wuyueman, and Sijiu Caixin) was 43.33%-51.09%, whereas using the stout hypocotyl resulted in a transformation efficiency of 54.88% for the 30-day-old Sijiu Caixin.The one-step method outperformed the two-step method;the transformation efficiency of different varieties was above 60%, and both methods can be used to obtain transgenic roots for functional studies within one month. Finally, optimized ARM transformation methods can easily,quickly, and effectively produce composite non-heading Chinese cabbage plants with transgenic roots, providing a reliable foundation for gene function research and non-heading Chinese cabbage genetic improvement breeding.

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.

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.

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.

Rabbit Defensin(NP-1) Gene Transformation of Wheat and In Vitro Microbicidal Activity Assay of Transgenic Plants

ＩｎｔｒｏｄｕｃｔｉｏｎＳｉｎｃｅＶａｓｉｌｅｔａｌ．（１９９２）ｆｉｒｓｔｒｅｐｏｒｔｅｄｔｈｅｏｂｔａｉｎｍｅｎｔｏｆｔｒａｎｓｇｅｎｉｃｗｈｅａｔｐｌａｎｔｓ［１］，ｍｕｃｈｐｒｏｇｒｅｓｓｈａｓｂｅｅｎａｃｈｉｅｖｅｄｏｎｇｅｎｅｔｉｃｔｒａ...

Copper—Controllable,Site—Specific DNA 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.

The Application of TDZ in Enhancing Regeneration of Transgenic Plants

At present, transgenic plants are globally grown. Availability of a reliable regeneration system predominantly from a single transformed cell is the prerequisites for gene transfer, but regeneration is still a key

Identification of Bt-transgenic Rice Plants for Resistance to Stripe Stem Borer (Chilo suppressalis) and Genetic Analysis of the Transgenes

Resistance to herbicide Basta was used to identify Bt-transgenic rice plants and the progenies of crosses between the Bt-transgenic rice and the rice varieties cultivated in the Huang Huai area of China. The results demonstrated that the Basta-positive rice plants were highly resistant to stripe stem borer (Chilo sup-pressalis) both in the laboratory and field tests. Both cryIA (b) and bar genes were expressed and co-inherited in both self ing and crossing progenies. Mendelian segregation of the marker gene bar was observed in F2 and BC1 progenies. The results implicates that it is possible to transfer cryIA (b) gene into other cultivated varieties through crosses and back crosses.

Science Letters:Expression of a begomoviral DNAβ gene in transgenic Nicotiana plants induced abnormal cell division

An increasing number of monopartite begomoviruses are being identified that a satellite molecule (DNAβ) is required to induce typical symptoms in host plants. DNAβ encodes a single gene (termed βC1) encoded in the complementary-sense. We have produced transgenic Nicotiana benthamiana and N. tabacum plants expressing theβC1 gene of a DNAβ associated with Tomato yellow leaf curl China virus (TYLCCNV), under the control of the Cauliflower mosaic virus 35S promoter. Transgenic plants expressing βC1 showed severe developmental abnormalities in both species. Microscopic analysis of sections of both transgenic and non-transgenic N. tabacum leaves showed abnormal outgrowths of transgenic N. tabacum to be due to disorganized cell division (hyperplasia) of spongy and palisade parenchyma. Immuno-gold labeling of sections with a polyclonal antibody against the βC1 protein showed that the βC1 protein accumulated in the nuclei of cells. The possible biological function of the βC1 protein was discussed.

Expression of E. coli heat-labile enterotoxin B subunit in transgenic tobacco plants

Objective: To construct plant transformation vector containing Escherichia coli heat-labile enterotoxin B subunit(LT-B) gene and generate LT-B transgenic tobacco plants. Methods: The LT-B coding sequence was amplified from pMMB68 by PCR, subcloned into middle vector pUCmT and binary vector pBI121 to obtain plant expression vector pBI-LTB, in which LT-B expression was controlled under the Cauliflower mosaic virus (CaMV) 35S promoter. The tobacco plants (Nicotiana tobacum L.Cuttivar Xanthi) were transformed by co-cultivating leaf discs method via Agrobacterium tumefaciens LBA4404 harboring the plant expression vector. The regenerated transgenic tobacco plants were selected by kanamycin and confirmed by PCR, Southern blot, Western blot and ELISA. Results:LT-B gene integrated in the tobacco genomic DNA and were expressed in 9 strains of transgenic tobacco plants. The yield was varied from 3.36-10.56 ng/mg total soluble tobacco leaf protein. Conclusion: The plant binary expression vector pBI-LTB was constructed successfully, and transgenic LT-B tobacco plants was generated, and confirmed by Southern blot. The protein LT-B expressed by engineered plants was identified by Western blot analysis and had the expected molecular weight of LT-B pentamer protein. This result is an important step close to developing an edible vaccine and supplying a mucasal immunoajuvant, which will contribute to the prevention of mucosa-route evading pathogen.

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

PEG－mediated Gene Transfer into Orychophragmus Violaceus Cotyledon Protoplast and Regeneration of Transgenic Plants

ＰＥＧ－ｍｅｄｉａｔｅｄＧｅｎｅＴｒａｎｓｆｅｒｉｎｔｏＯｒｙｃｈｏｐｈｒａｇｍｕｓＶｉｏｌａｃｅｕｓＣｏｔｙｌｅｄｏｎＰｒｏｔｏｐｌａｓｔａｎｄＲｅｇｅｎｅｒａｔｉｏｎｏｆＴｒａｎｓｇｅｎｉｃＰｌａｎｔｓＺＨＯＵＪｉ－ｍｉｎｇ（周冀明），ＷＥＩＺｈ...

Transformation of Orychophragmus violaceus Using Agrobacterium tumefaciens And Regeneration of Transgenic Plants

ＴｒａｎｓｆｏｒｍａｔｉｏｎｏｆＯｒｙｃｈｏｐｈｒａｇｍｕｓｖｉｏｌａｃｅｕｓＵｓｉｎｇＡｇｒｏｂａｃｔｅｒｉｕｍｔｕｍｅｆａｃｉｅｎｓＡｎｄＲｅｇｅｎｅｒａｔｉｏｎｏｆＴｒａｎｓｇｅｎｉｃＰｌａｎｔｓａ￥ＺＨＯＵＪｉ－ｍｉｎｇ（周冀明）；ＷＥＩＺｈ...

Field Evaluation of the Asian Corn Borer Control in Hybrid of Transgenic Maize Event MON 810

In this study, a transgenic Bt maize hybrid (event MON 810 from Monsanto Company) expressing Cry1Ab protein derived from Bacillus thuringiensis (Bt) and its negative isoline hybrid were evaluated for control of the Asian corn borer, Ostrinia furnacalis (Guenée) (Lepidoptera: Pyralidae), in a field trial. Maize plants were artificially infested with neonate larvae of Asian corn borer at the mid-whorl (first-generation), pre-tassel (first- and/or second-generation), and silk (second-generation) growth stages. The transgenic Bt maize hybrid sustained significantly less leaf feeding damage (rating 1.0±0.0) than its negative isoline control (rating 7.3±0.1). With the Bt maize, 1.36.8% of plants were damaged by corn borer tunneling with <0.5 cm tunneling per stalk under different levels of infestation, compared with 100% of plants damaged with 9.325.0 cm tunneling per stalk for the negative isoline control. On average, transgenic Bt maize hybrids had only 0.010.05 tunnels per stalk and no stems were broken. In contrast, the negative isoline control had 3.118.36 tunnels per stalk and 31.273.9% of stems broken. Yields were significantly higher in transgenic Bt maize than in the control. These results demonstrate that transgenic Bt maize can significantly minimize yield losses caused by the Asian corn borer through resistance to the first- and second-generation larvae.

Rapid Generation of Selectable Marker-Free Transgenic Rice with Three Target Genes by Co-Transformation and Anther Culture

The ＇double T-DNA＇ binary vector p13HSR which harbored two independent T-DNAs, containing hygromycin phosphotransferase gene （hpf） in one T-DNA region and three target genes （hLF, SB401, RZ10） in another T-DNA region, was used to generate selectable marker-free transgenic rice by Agrobacterium-mediated transformation. The regenerated plants with both the three target genes and the selectable marker gene hpt were selected for anther culture. RT-PCR analysis indicated that target genes were inserted in rice genomic DNA and successfully transcribed. It took only one year to obtain double haploid selectable marker-free transgenic plants containing the three target genes with co-transformation followed by anther culture technique, and the efficiency was 12.2%. It was also noted that one or two target genes derived from the binary vector were lost in some transgenic rice plants.

Temporal and spatial changes in Bt toxin expression in Bt-transgenic poplar and insect resistance in field tests

Extensive planting of Bacillus thuringiensis （Bt）-transgenic plants economically benefits society; how-ever, the potential risk they pose is receiving increasing attention. This study used enzyme-linked immunosorbent assay and fluorescence quantitative PCR （RT-PCR） to monitor the temporal and spatial dynamics of the expression of Bt toxic protein in a forest of 6- to 8-year-old trees of transgenic insect-resistant poplar 741 for three consec- utive years. The enrichment, distribution, and degradation of Bt toxic protein and the influence of transgenic poplars on the targeted insect population, Hyphantria cunea, were investigated. The content of CrylAc toxic protein dynamically changed in transgenic poplar. During the annual growth cycle, the content initially increased, then decreased in the long and the short branches of the crown and in the root system, peaking in August. During the study, the protein did not accumulate overtime. The mRNA transcription of gene CrylAc was almost consistent with the level of the protein, but transcription peaked in July. In the transgenic and control forestland, microscale levels of the CrylAc toxic protein were detected from the soil, but increased accumulation was not observed with the planting year of transgenic poplar. Meanwhile, Bt was isolated and detected molecularly from the soil in the experimental forestland. A systematic investigation of the density of H. cunea in the experimental transgenic poplar forest indi- cated that transgenic Pb29 poplar could resist insects to a certain degree. At peak occurrence of the targeted insects, the density of H. cunea in the experimental forest was significantly lower than in the nontransgenic poplar forest.