We have reported that cDNA derived from entire coat protein (CP) gene of potato virus Y (PVY) could induce resistance to PVY infection in transgenic tobacco plants, and the resistance was further demonstrated to be RN...We have reported that cDNA derived from entire coat protein (CP) gene of potato virus Y (PVY) could induce resistance to PVY infection in transgenic tobacco plants, and the resistance was further demonstrated to be RNA-mediated rather than coat protein-mediated. In this study, we cloned cDNA fragments of 202 bp, 417 bp, and 603 bp in length derived from the 3′ end of the PVY CP gene, and the cDNA fragments were introduced into tobacco (var. NC89) plants via Agrobacterium-mediated transformation system. The results of resistance assay showed that the CP cDNA fragments of 417 bp and 603 bp could confer resistance of the trans-genic plants to PVY infection, but the fragment of 202 bp in length could not. Molecular analysis revealed that the resistance was RNA-mediated, which is believed to be a result of post-transcriptional gene silencing. The results indicate that the length of cDNA fragments needed for resistance induction was located somewhere between 202 bp and 417 bp from the 3′ end of PVY CP gene.展开更多
Insecticidal protein gene CryIA(c)from Bacillus thuringiensis HD-1(B.t.toxin gene)with 5’-end modified and 3’-end deleted to 4 different lengths were inserted downstream of 35S promoterwith double enhancer and"...Insecticidal protein gene CryIA(c)from Bacillus thuringiensis HD-1(B.t.toxin gene)with 5’-end modified and 3’-end deleted to 4 different lengths were inserted downstream of 35S promoterwith double enhancer and"Ω’"fragment of TMV-RNA cDNA in the binary vector pBin438 to constructthe chimeric expression vector of B.t.toxin gene.Leave stripes of tobacco plant var.NC89 widelygrown in China were transformed with A.tumefaciens LBA4404 harbouring the above expression vectorsrespectively,and kanamycin resistant tobacco plants were regenerated.Insect test with tobacco budwormH.assulta showed that insect-resistant transform.ants could be obtained from the regenerated plantstransformed with B.t.genes of different lengths though highest percentage(～50%)of plants with ahigh morality(90%-100%)to the testing insects is among those transformed with 1.8-kb toxin gene.Genetic,molecular and biological analyses of T1 and T2 progenies of plants with high efficient insect re-sistance showed that B.t.toxin gene and the character of insect resistance have been inherited in the pro-genies.Insect-resistant homozygotes D8-14 and D19-8 have been selected for small-scale field tests.展开更多
An intermediate expressing vector carrying the tobacco mosaic virus (TMV, Chinese common strain) coat protein (CP) gene was constructed by recombinant DNA techniques. The TMV-CP gene was transferred into the tobacco g...An intermediate expressing vector carrying the tobacco mosaic virus (TMV, Chinese common strain) coat protein (CP) gene was constructed by recombinant DNA techniques. The TMV-CP gene was transferred into the tobacco genome via Ti plasmid and a large number of regenerated plants, including both systemic and local lesion hosts for TMV, were obtained. Southern blot analysis revealed that 1-5 copies of the CP gene were integrated into the tobacco genome. RNA and protein analysis demonstrated that the TMV-CP gene was correctly expressed in the transgenic plants. The abundance of TMV-CP mRNA in total leaf RNA accounted for 0.005-0.01%, while the amount of coat proteins reached 0.05-0.2% of the total leaf soluble proteins. Virus challenge experiments showed that the symptom development of virus infection was markedly delayed and the replication as well as the spread of the virus was significantly inhibited in the transgenic plants expressing the TMV-CP gene. Three of these plants were completely protected after inoculation and they went to flowering and seeding normally. The possible mechanism of this kind of virus resistance could be the inhibition of the uncoating of the invading virus particles at the early stage of infection in the presence of the viral coat protein in plant cells, thus blocking the virus replication cycle.展开更多
To investigate the effects of 5’and 3’ untranslated region (UTR) from tobacco mosaicvirus (TMV) on expression of foreign genes, four plant-expression vectors pBG437, pBG438, pBG440 and pBG440ANOS containing expressi...To investigate the effects of 5’and 3’ untranslated region (UTR) from tobacco mosaicvirus (TMV) on expression of foreign genes, four plant-expression vectors pBG437, pBG438, pBG440 and pBG440ANOS containing expression cassettes of GUS-NOS; Ω-GUS-NOS, Ω-GUS-3’ UTR-NOS and Ω-GUS-3’ UTR downstream of CaMV 35S promoter with double enhancer sequences respectively have been constructed. Results from a large number of transgenic tobacco plants show that the GUS activity of pBG440 transformed plants is the highest, being 5-fold that of pBG437 and 1.6-fold that of pBG438. Similar results have been obtained in transient expression by injection of Agrobacterium tumefaciens harbouring these constructs into N. benthamiana leaves. These results obtained at the whole plant level confirm the conclusion drawn from the transient expression studies in protoplast system that TMV- Ω fragment can be a translation enhancer and the 3’ UTR has a coordinate effect with ft fragment to enhance foreign gene expression, but展开更多
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...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.展开更多
基金This work was supported by the National Natural Science Foundation of China(Grant No.30270875)Shandong Province Natural Science Foundation(Grant No.Z2000D02)Shandong Province Science and Technology Development Project.
文摘We have reported that cDNA derived from entire coat protein (CP) gene of potato virus Y (PVY) could induce resistance to PVY infection in transgenic tobacco plants, and the resistance was further demonstrated to be RNA-mediated rather than coat protein-mediated. In this study, we cloned cDNA fragments of 202 bp, 417 bp, and 603 bp in length derived from the 3′ end of the PVY CP gene, and the cDNA fragments were introduced into tobacco (var. NC89) plants via Agrobacterium-mediated transformation system. The results of resistance assay showed that the CP cDNA fragments of 417 bp and 603 bp could confer resistance of the trans-genic plants to PVY infection, but the fragment of 202 bp in length could not. Molecular analysis revealed that the resistance was RNA-mediated, which is believed to be a result of post-transcriptional gene silencing. The results indicate that the length of cDNA fragments needed for resistance induction was located somewhere between 202 bp and 417 bp from the 3′ end of PVY CP gene.
基金the Key Programme of the 7th Five-Year Plan,State Planning Commission of ChinaInternational Centre of Science and Culture (ICSC),World Laboratory,Lausanne,Switzerland
文摘Insecticidal protein gene CryIA(c)from Bacillus thuringiensis HD-1(B.t.toxin gene)with 5’-end modified and 3’-end deleted to 4 different lengths were inserted downstream of 35S promoterwith double enhancer and"Ω’"fragment of TMV-RNA cDNA in the binary vector pBin438 to constructthe chimeric expression vector of B.t.toxin gene.Leave stripes of tobacco plant var.NC89 widelygrown in China were transformed with A.tumefaciens LBA4404 harbouring the above expression vectorsrespectively,and kanamycin resistant tobacco plants were regenerated.Insect test with tobacco budwormH.assulta showed that insect-resistant transform.ants could be obtained from the regenerated plantstransformed with B.t.genes of different lengths though highest percentage(～50%)of plants with ahigh morality(90%-100%)to the testing insects is among those transformed with 1.8-kb toxin gene.Genetic,molecular and biological analyses of T1 and T2 progenies of plants with high efficient insect re-sistance showed that B.t.toxin gene and the character of insect resistance have been inherited in the pro-genies.Insect-resistant homozygotes D8-14 and D19-8 have been selected for small-scale field tests.
基金Project supported by the Chinese National Science Committee and the World Laboratory of International Center for Science and Culture, Geneva, Switzerland.
文摘An intermediate expressing vector carrying the tobacco mosaic virus (TMV, Chinese common strain) coat protein (CP) gene was constructed by recombinant DNA techniques. The TMV-CP gene was transferred into the tobacco genome via Ti plasmid and a large number of regenerated plants, including both systemic and local lesion hosts for TMV, were obtained. Southern blot analysis revealed that 1-5 copies of the CP gene were integrated into the tobacco genome. RNA and protein analysis demonstrated that the TMV-CP gene was correctly expressed in the transgenic plants. The abundance of TMV-CP mRNA in total leaf RNA accounted for 0.005-0.01%, while the amount of coat proteins reached 0.05-0.2% of the total leaf soluble proteins. Virus challenge experiments showed that the symptom development of virus infection was markedly delayed and the replication as well as the spread of the virus was significantly inhibited in the transgenic plants expressing the TMV-CP gene. Three of these plants were completely protected after inoculation and they went to flowering and seeding normally. The possible mechanism of this kind of virus resistance could be the inhibition of the uncoating of the invading virus particles at the early stage of infection in the presence of the viral coat protein in plant cells, thus blocking the virus replication cycle.
文摘To investigate the effects of 5’and 3’ untranslated region (UTR) from tobacco mosaicvirus (TMV) on expression of foreign genes, four plant-expression vectors pBG437, pBG438, pBG440 and pBG440ANOS containing expression cassettes of GUS-NOS; Ω-GUS-NOS, Ω-GUS-3’ UTR-NOS and Ω-GUS-3’ UTR downstream of CaMV 35S promoter with double enhancer sequences respectively have been constructed. Results from a large number of transgenic tobacco plants show that the GUS activity of pBG440 transformed plants is the highest, being 5-fold that of pBG437 and 1.6-fold that of pBG438. Similar results have been obtained in transient expression by injection of Agrobacterium tumefaciens harbouring these constructs into N. benthamiana leaves. These results obtained at the whole plant level confirm the conclusion drawn from the transient expression studies in protoplast system that TMV- Ω fragment can be a translation enhancer and the 3’ UTR has a coordinate effect with ft fragment to enhance foreign gene expression, but
基金Supported by the National Natural Science Foundation of China(No.39730 35 0 ) .
文摘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.
基金the scientific and technological program of Fujian Science and Technology Department(2004N026)the Natural Science Foundation of Fujian Province(B0410009)the scientific and technological program of undergraduate students in Fujian Normal university(BKL2007062)
