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At least 343 records · Page 19Linked to original sources

[Expression characteristics of MtPAP1 and its exotic expression in Arabidopsis affecting organic phosphorus absorption of plants].

The MtPAP1 encodes 465 amino acids (Fig.1). The transcript of MtPAP1 was detected mainly in leaf under high-phosphate conditions (Fig.2). While under low-phosphate condition, the transcript level was low in leaves and very high in roots, with the strongest hybridization signal detected in roots (Fig.2). Chimeric MtPAP1 binary vector was constructed and the transcript levels of MtPAP1 in roots of transgenic T(3) Arabidopsis lines were analyzed (Fig.3). Under the condition in which phytate was the sole source of phosphorus, transgenic Arabidopsis plants over-expressing MtPAP1 showed acid phosphatase activities in root apoplast increasing markedly than that of the control plant (Fig.4). The results of analysis of organic phosphorus degradation in liquid culture by HPLC indicated that the APase secreted by the transgenic plants could quickly degrade the phytate (Fig.5). Significant increase in biomass production, Pi and total phosphorus content of plants achieved in the transgenic lines when phytate, an organic phosphorus compound, was supplied as the sole source of phosphorus (Fig.6,7).

Amino Acid Sequence↗

[The import of DNA topoisomerase type II from Drosophila melanogaster into the chloroplasts of transgenic Nicotiana tabacum plants].

The construct containing the structural gene of the type II DNA topoisomerase of Drosophila melanogaster fused to the sequence of the chloroplast transit peptide directed by the 35 S promoter was produced on the basis of the binary vector Bin 19. The construct was introduced into Nicotiana tabacum plants by Agrobacterium-mediated transformation. The integration of the construct into genomic DNA was demonstrated by PCR and Southern blot hybridization. Expression of the chimeric gene at the transcription level was studied by Northern blotting. The protein produce of the expression was identified in chloroplasts by Western blot hybridization with polyclonal anti-type II Drosophila topoisomerase antibodies.

Animals↗

cDNA cloning and sequence analysis of NIb gene of soybean mosaic virus.

cDNA of soybean mosaic virus (Beijing isolate, SMV-BJ) has been synthesized, using viral genomic RNA as a template and random hexanucleotides as primers. Based on the sequences of SMV-BJ coat protein (CP) gene as well as SMV- and WMV-II-related regions, oligonucleotides were made as primers for polymerase chain reaction (PCR). NIb gene of SMV-BJ was amplified by PCR, and cloned into pBluescript SK. The complete sequence was determined. The comparison of NIb genes between SMV-BJ and WMV-II (USA) shows that similarities for nucleotide sequence reach 80.3%; and the deduced amino acid sequence, 91.3%. In consideration of the high identities in between the CP gene and the 3'-non-coding region between them, WMV-II might be considered as a watermelon strain of SMV. Besides, some unexpected sequences were found in the 3'-region of 2 NIb gene clones. Following modification and splicing, a binary vector of NIb gene has been constructed for its expression in higher plant for the purpose of studying the possible replicase-mediated resistance in polyviruses.

Amino Acid Sequence↗

Cloning of polygalacturonase (PG) cDNA and inhibition effects of its antisense RNA on the expression of PG gene in transgenic tomato plants.

We have amplified a 1.5-kb PG cDNA including its complete open reading frame, using two primers specific to PG gene and PCR technique. Its fidelity and correctness were confirmed by restriction mapping and partial DNA sequencing. The PG gene was then inserted into a binary vector, pBin437, in an inverted orientation between the 35S promoter with duplicated enhancers and the Nos3' transcriptional termination sequence, and the resulting plasmid pBPG was introduced into LBA 4404. The A. tumefaciens harboring pBPG was used to transform tomato plants. Sixty kanamycin-resistant transformants were selected. PCR detection showed two-third of the transformed plants tested (30 plants) contain the exogenous PG gene. The PG activity assay and SDS-PAGE analysis of proteins from ripening fruit cell wall showed that the PG activity in transgenic tomato plants were inhibited in different extent. One of them, plant 3, gave only 7% PG activity compared with the control (100%). These results suggest that the expression of the anti-PG gene has effectively inhibited the expression of endogenous PG gene. The effect of low-level PG activity on tomato fruit ripening and storage is being investigated.

Base Sequence↗

Insect tolerance of transgenic Populus nigra plants transformed with Bacillus thuringiensis toxin gene.

Leaves and stem segments of Populus nigra were transformed with A. tumefaciens LBA4404 harboring a binary vector containing chimeric genes of NPT and 35S-omega-B.t. toxin-Nos. Nineteen regenerated kanamycin resistant plants were analyzed by DNA hybridization, out of which 10 were shown to be the candidates of transgenic plants. Insect tolerance tests showed that the transgenic plants were toxic to two lepidopteran pests, Lymantria dispar L. and Apocheimia cinerarius Erschoff. Based on the results of Southern blot of PCR products and the cluster analysis of their growth and insect resistance, three independent transgenic plants were selected, which were then propagated in the nursery field. This field test is currently in progress.

Agrobacterium tumefaciens↗

Expression of human hepatitis B virus surface antigen gene in transgenic tobacco.

Expression of Human hepatitis B virus surface antigen (HBsAg) gene in plant was reported for the first time. The recombinant plasmid pRoKII-HBsAg was constructed by inserting HBsAg gene into the downstream of CaMV 35S promoter of binary vector pRoKII and then introduced into Agrobacterium tumefaciens LBA4404. The kanamycin-resistant plants were obtained by Agrobacterium-mediated transformation system. It was shown that HBsAg gene was expressed in transgenic tobacco plants and their progenies by ELISA. The spherical particles of psi 22 nm is the leaf extract of transgenic tobacco were observed by immunosorbent electron microscopy.

Gene Expression↗

[Transgenic Brassica napus resistant to turnip mosaic virus].

A system for obtaining regenerated plantlets of "double low" Brassica napus by using cotyledonary petioles as material was established. The turnip mosaic virus coat protein (TuMV-CP) gene inserted in the binary vector pBTu in the Agrobacterium tumefaciens LBA 4404 was integrated into Brassica napus through co-culture of cotyledonary petioles with LBA4404, and the material in co-culture was selected under the stress of Kanamycin. Regenerated plantlets were obtained, and the specific TuMV-CP gene was proved to be integrated into the genomic DNA of the regenerated plants by the specific PCR amplification, dot hybridization and Southern blot. All these transgenic plants were proved to be resistant to virulent TuMV by virus challenge in different degree.

Brassica↗

Expression of the spinach betaine aldehyde dehydrogenase (BADH) gene in transgenic tobacco plants.

Plasmid pLS9 contains a 1.5-kb of spinach cDNA including its complete open reading frame. The 1.5-kb BADH cDNA was cut from pLS9 using restriction enzyme and was inserted into the expression cassette of plasmid pYH between the CaMV 35S promoter and polyA signal sequence. The 35S-BADH cDNA-polyA fragment of pYH was cloned into a polylinker cloning site of the binary vector pBin19. The resulting plasmid pBinBADH-S was transferred to Agrobacterium tumefacies LBA4404. The tobacco plants were transformed with strain LBA4404 containing pBinBADH-S, and more than ninety kanamycin-resistant transformants were selected. Polymerase chain reaction (PCR) detection showed that more than 60% of the transformed tobacco plants contained the foreign BADH gene. The Western blot analysis, BADH enzymatic assay, specific stain for BADH activity, and the test for salt tolerance showed that BADH gene was normally expressed in the transgenic tobacco plants. The BADH enzymes also presented in chloroplasts and cytosol of the transgenic plants. The transgenic tobacco plants having strong expression of BADH gene had strong ability to tolerate high salt stress.

Aldehyde Dehydrogenase↗

Inhibition effect of transgenic tobacco plants expressing snowdrop lectin on the population development of Myzus persicae.

The cDNAs of snowdrop lectin mature protein and its precursor protein, GNA12 and GNA34, were inserted downstream of a 35S promoter with a double enhancer and a "omega" fragment of TMV-RNA cDNA in the binary vector pBin438, or the phloem-specific CoYMV promoter in the vector pBcop1, respectively, resulting in the construction of four plant expression vectors pBGna12, pBGna34, pBCGna12, and pBCGna34. Leaf stripes of Nicotiana tabacum var. K326 were transformed with A. tumefaciens LBA4404 harboring the above expression vectors, respectively. PCR and Southern blot analysis showed that foreign GNA genes were inserted into the genome of transformed tobacco plants. Western blot analysis indicated that GNA could be expressed efficiently up to 0.08-0.15% of total soluble proteins in transgenic tobacco plants with pBGna34 or pBCGna34, while in those with pBGna12 and pBCGna12 GNA were hardly detected by immunoassay. The results from insect bioassay with a peach aphid (Myzus persicae) demonstrated that the transgenic plants of pBGna34 and pBCGna34 were aphid-resistant as shown by a 45-60% reduction in insect population density, with some individual transgenic lines being reduced by over 90%. In addition, it was evident that the 35S promoter with a double enhancer and CoYMV promoter had similar abilities to direct the GNA gene to express in transgenic tobacco plants, but because the CoYMV promoter drives the foreign gene in a phloem-specific expression manner, the transgenic plants of pBCGna34 showed higher aphid resistance.

Animals↗

Cadmium resistance in transgenic tobacco plants expressing the Nicotiana glutinosa L. metallothionein-like gene.

To understand the function of metallothioneins (MTs) in plants, we introduced the Nicotiana glutinosa MT gene into tobacco (N. tabacum) plants via an Agrobacterium mediated transformation. Full-length MT cDNA was fused between the cauliflower mosaic virus 35S (CaMV 35S) promoter and the nopaline synthase (nos) terminator of the pMBP1 binary vector in sense orientation. Tobacco leaf discs which were cocultivated with Agrobacterium carrying the chimeric MT gene, formed kanamycin-resistant shoots on medium containing kanamycin. The kanamycin-resistant shoots were subsequently rooted on medium containing 200 microM CdSO4. Approximately 30% of individual transgenic plants developed normally. Nontransgenic plants promptly underwent leaf chlorosis, and their growth and development were inhibited on MS medium containing 50 microM CdSO4. Genomic Southern blot analysis showed that the MT gene was stably integrated into the nuclear genome of transgenic tobacco plants. The expression level of MT transcripts was analyzed by RNA gel blot analysis. Self-pollinated seeds obtained from transgenic tobacco plants showing cadmium tolerance were germinated on a medium containing 100 microM CdSO4. PCR analysis from sensitive and stably resistant T2 seedlings for cadmium sulfate confirmed a high correlation between the phenotypic expression of the MT gene and the transgenic genotype, indicating that the MT gene is inherited in the next generation.

Cadmium↗

Amplified Muc1-specific gene expression in colon cancer cells utilizing a binary system in adenoviral vectors.

Mucin-1 is expressed in a variety of colon carcinomas and Muc-1/DF3 promoters have been utilized to reduce systemic toxicity through specific gene expression. To overcome weak expression, which is much lower than the widely used cytomegalovirus-promoter (CMV), new adenoviral vectors containing a binary system of transgene amplification have been developed. The Muc-1/DF3 promoter was used to control the expression of a Gal4VP16 fusion protein. This vector also contained Gal4 binding sites enabling the fusion protein to act as a transactivator, inducing transgene expression within the same construct. Mucin-1 expression was analyzed in a variety of colon cancer cell lines. After infection with recombinant adenoviruses, transgene expression was quantified using the luciferase system. Integration of the Gal4VP16-binary resulted in an up to 250-fold increase of Muc-1/DF3-specific gene expression. In mucin-positive cell lines utilizing this amplified Muc-1/DF3 promoter, expression was up to 590-fold higher as compared to the CMV-promoter. Western blot detected the presence of Gal4VP16 in infected muc-1-positive but not-negative cell lines. These new adenoviral vectors combing highly efficient and specific transgene expression and will contribute to the safety and efficacy of experimental approaches in cancer gene therapy.

Adenoviridae↗

Multiclass cancer classification by using fuzzy support vector machine and binary decision tree with gene selection.

We investigate the problems of multiclass cancer classification with gene selection from gene expression data. Two different constructed multiclass classifiers with gene selection are proposed, which are fuzzy support vector machine (FSVM) with gene selection and binary classification tree based on SVM with gene selection. Using F test and recursive feature elimination based on SVM as gene selection methods, binary classification tree based on SVM with F test, binary classification tree based on SVM with recursive feature elimination based on SVM, and FSVM with recursive feature elimination based on SVM are tested in our experiments. To accelerate computation, preselecting the strongest genes is also used. The proposed techniques are applied to analyze breast cancer data, small round blue-cell tumors, and acute leukemia data. Compared to existing multiclass cancer classifiers and binary classification tree based on SVM with F test or binary classification tree based on SVM with recursive feature elimination based on SVM mentioned in this paper, FSVM based on recursive feature elimination based on SVM can find most important genes that affect certain types of cancer with high recognition accuracy.

Journal Article↗

High efficiency transgene segregation in co-transformed maize plants using an Agrobacterium tumefaciens 2 T-DNA binary system.

For regulatory issues and research purposes it would be desirable to have the ability to segregate transgenes in co-transformed maize. We have developed a highly efficient system to segregate transgenes in maize that was co-transformed using an Agrobacterium tumefaciens 2 T-DNA binary system. Three vector treatments were compared in this study; (1) a 2 T-DNA vector, where the selectable marker gene bar (confers resistance to bialaphos) and the beta-glucuronidase (GUS) reporter gene are on two separate T-DNA's contained on a single binary vector; (2) a mixed strain treatment, where bar and GUS are contained on single T-DNA vectors in two separate Agrobacterium strains; (3) and a single T-DNA binary vector containing both bar and GUS as control treatment. Bialaphos resistant calli were generated from 52 to 59% of inoculated immature embryos depending on treatment. A total of 93.4% of the bialaphos selected calli from the 2 T-DNA vector treatment exhibited GUS activity compared to 11.7% for the mixed strain treatment and 98.2% for the cis control vector treatment. For the 2 T-DNA vector treatment, 86.7% of the bialaphos resistant/GUS active calli produced R0 plants exhibiting both transgenic phenotypes compared to 10% for the mixed strain treatment and 99% for the single T-DNA control vector treatment. A total of 87 Liberty herbicide (contains bialaphos as the active ingredient) resistant/GUS active R0 events from the 2 T-DNA binary vector treatment were evaluated for phenotypic segregation of these traits in the R1 generation. Of these R0 events, 71.4% exhibited segregation of Liberty resistance and GUS activity in the R1 generation. A total of 64.4% of the R0 2 T-DNA vector events produced Liberty sensitive/GUS active (indicating selectable-marker-free) R1 progeny. A high frequency of phenotypic segregation was also observed using the mixed strain approach, but a low frequency of calli producing R0 plants displaying both transgenic phenotypes makes this method less efficient. Molecular analyses were then used to confirm that the observed segregation of R1 phenotypes were highly correlated to genetic segregation of the bar and GUS genes. A high efficiency system to segregate transgenes in co-transformed maize plants has now been demonstrated.

Agrobacterium tumefaciens↗

A binary-BAC system for plant transformation with high-molecular-weight DNA.

A binary-BAC (BIBAC) vector suitable for Agrobacterium-mediated plant transformation with high-molecular-weight DNA was constructed. A BIBAC vector is based on the bacterial artificial chromosome (BAC) library vector and is also a binary vector for Agrobacterium-mediated plant transformation. The BIBAC vector has the minimal origin region of the Escherichia coli F plasmid and the minimal origin of replication of the Agrobacterium rhizogenes Ri plasmid, and thus replicates as a single-copy plasmid in both E. coli and in A. tumefaciens. The T-DNA of the BIBAC vector can be transferred into the plant nuclear genome. As examples, a 30-kb yeast genomic DNA fragment and a 150-kb human genomic DNA fragment were inserted into the BIBAC vector; these constructs were maintained in both E. coli and A. tumefaciens. In order to increase the efficiency of transfer of unusually large BIBAC T-DNAs, helper plasmids that carry additional copies of A. tumefaciens virulence genes virG and virE were constructed. These helper plasmids are compatible with, and can be present in addition to, the BIBAC vector in the A. tumefaciens host. This report details the components of the BIBAC system, providing information essential to the general understanding and the application of this new technology.

Bacterial Proteins↗

Expression of DNA coding for diphtheria toxin chain a is toxic to plant cells.

DNA coding for the enzymatically active subunit A of diphtheria toxin was placed under the control of the cauliflower mosaic virus 35S promoter and the Agrobacterium left transfer-DNA gene 7 polyadenylation signal. Agrobacteria carrying a binary plant vector with the chimeric diphtheria toxin A gene had very low transforming activity in tobacco (Nicotiana tabacum L.), and greatly diminished the recovery of stable transformants when mixed together with agrobacteria which alone transformed plant cells well. The introduction of this chimeric molecule into tobacco cells by electroporation lowered the level of the transient expression of the coelectroporated chloramphenicol acetyltransferase reporter gene indicating that expression of diphtheria toxin chain A in plant cells is toxic. We have developed a binary vector pGA987 which can be used for probing a variety of plant promoters.

Journal Article↗

Development of transformation system of rice based on binary bacterial artificial chromosome (BIBAC) vector.

An Agrobacterium-mediated transformation protocol using binary bacterial artificial chromosome (BIBAC) vector system in rice (Oryza sativa L.) was developed. Calli derived from mature embryos of japonica rice cv. H1493 were used as target tissues. Various aspects in transformation and regeneration processes including callus induction and culture, Agrobacterium concentration and duration of co-cultivation, bacterial elimination and transformant selection were examined in order to improve the transformation efficiency. An optimized transformation conditions was established including: using an Agrobacterium strain, LBA4404(HP4404), which carries a super-virulent helper plasmid pCH32, for the infection; a modified N6 medium system for callus induction and culture; pH 5.6 for media in pre-cultivation and co-cultivation; Agrobacterium concentration at OD600 = 1.0 for 3 days co-cultivation and 7 days for a resting period of the infected calli. Based on PCR and Southern blot analysis, it was demonstrated that insert DNA and marker genes carried by BIBAC2 were integrated into the rice genome.

Chromosomes, Artificial, Bacterial↗

A functional cloning strategy, based on a binary PVX-expression vector, to isolate HR-inducing cDNAs of plant pathogens.

We have devised a novel, high-throughput functional cloning method to isolate cDNAs from plant pathogens of which the products elicit a hypersensitive response (HR) in plants. Copy DNA, made from RNA isolated from the tomato pathogen Cladosporium fulvum grown under nutrient-limiting conditions in vitro, was cloned into a binary, potato virus X (PVX)-based expression vector and transformed to Agrobacterium tumefaciens. 9600 colonies were individually toothpick-inoculated onto leaflets of tomato plants resistant to C. fulvum. Four cDNAs were identified whose expression induced formation of a necrotic lesion around the inoculation site. One of these clones, specifically inducing HR on tomato plants carrying the Cf-4 resistance gene, encodes race-specific elicitor AVR4. The other three cDNAs, inducing a non-genotype-specific HR, encode a protein highly homologous to bZIP, basic transcription factors. To determine whether this approach has general applicability, part of the library was also inoculated onto Nicotiana tabacum var. Samsun NN, which is not a host for C. fulvum. Four independent HR-inducing cDNAs were identified which all encode ECP2, an extracellular protein of C. fulvum known to induce necrosis in certain Nicotiana species. These observations confirm that this functional screening method is a versatile strategy to identify cDNAs of pathogens that encode (race-specific) elicitors and other HR-inducing proteins.

Agrobacterium tumefaciens↗