A chimeric gene, Bt29K, composed of coding sequences of activated Cry1Ac insecticidal protein and an endoplasm reticulum-retarding signal peptide, was synthesized. A plant expression vector containing two expression c...A chimeric gene, Bt29K, composed of coding sequences of activated Cry1Ac insecticidal protein and an endoplasm reticulum-retarding signal peptide, was synthesized. A plant expression vector containing two expression cassettes for the Bt29K and API-B genes was constructed. These two insect-resistant genes were transferred into two cotton ( Gossypium hirsutum L.) varieties ( or lines) via Agrobacterium-mediated transformation and nine homozygous transgenic cotton lines showing a mortality of 90.0% - 99.7% to cotton ballworm (Heliothis armigera) larvae and good agronomic traits were selected through six generations. Molecular biology analysis revealed that one or two copies of the insecticidal protein genes were integrated into the transgenic cotton genome and activated Cry1Ac and API-B protein expression was at a level of 0.17% and 0.09% of the total soluble protein in the transgenic cotton leaves, respectively. Comparison of the insect-resistance of the homozygous lines expressing the activated chimeric Cry1Ac and API-B with that expressing Cry1Ac only revealed that the insect-resistance of the former is apparently higher than the latter. These results also indicate that the strategy to construct a plant expression vector expressing two different insect-resistant genes reported here is reasonable.展开更多
In recent years, great progress has been made in. producing plants resistant to insect attack by genetic engineering of B. t δ-endotoxin gene or plant proteinase inhibitor genes, and how to avoid the emergence of the...In recent years, great progress has been made in. producing plants resistant to insect attack by genetic engineering of B. t δ-endotoxin gene or plant proteinase inhibitor genes, and how to avoid the emergence of the insect resistance to B. t δ-endotoxin produced by transgenic plants has been paid attention to. To combine one B. t δ-endotoxin gene with an incompatible B. t δ-endotoxin gene or with other genes such as proteinase inhibitor gene might alleviate the insect resistance problem. In this note, we study the展开更多
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 co...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.展开更多
Total RNA was extracted from rice immature seeds harvested 2 weeks after flowering; then mRNA was purified. cDNA with NotI and SaiI cohesive ends was synthesized and inserted into λgt22A. After packaged in vitno, the...Total RNA was extracted from rice immature seeds harvested 2 weeks after flowering; then mRNA was purified. cDNA with NotI and SaiI cohesive ends was synthesized and inserted into λgt22A. After packaged in vitno, the cDNA library was constructed with 1.5×106pfu. A 21-mer oligodeoxynucleotide was synthesized according to the 5’-end conserved coding sequence of oryzacystatin (a thiol proteinase inhibitor) and labeled as a probe. From 2.1 × 104 pfu, 9 positive dones have been isolated, 8 of which contain the entire coding region of oryzacystatin. λOC1 has the longest cDNA insert, which contains an open reading frame of 309 bp coding sequence, 84 bp 5’-end non-coding region and a poly(A) signal AATAAA at the 3’-end followed by 31 Nt of poly(A). The coding sequence is the same compared with oryzacystatin genomic DNA sequence, while there are some obvious differences such as insertion and variation in the non-coding region, especially lots of nonsucoessive insertion in the 3’ region after poly(A) signal.展开更多
A cDNA library was constructed with 1.5×10~6 pfu from rice immature seeds,fromwhich a cDNA clone for rice thiol proteinase inhibitor,oryzacystatin(OC),was isolated byscreening with synthesized oligodeoxynucleotid...A cDNA library was constructed with 1.5×10~6 pfu from rice immature seeds,fromwhich a cDNA clone for rice thiol proteinase inhibitor,oryzacystatin(OC),was isolated byscreening with synthesized oligodeoxynucleotide probe,which contained a 309bp open read-ing frame,84bp 5′-end noncoding region and a poly(A)signal AATAAA at the 3′-end fol-lowed by 31Nt poly(A).Then the coding region of OC was amplified and inserted into thedownstream of λP_RP_L promoter for thermal-inducible expression in E.coli.Shifting the cul-ture temperature from 30℃ to 42℃ led to a high level expression of OC,which exhibited adistinct band of 12.0 kDa and accounted for at least 10% of the total soluble proteins fromSDS-PAGE.The papain-inhibitory activity of the expressed OC was further confirmed.展开更多
文摘A chimeric gene, Bt29K, composed of coding sequences of activated Cry1Ac insecticidal protein and an endoplasm reticulum-retarding signal peptide, was synthesized. A plant expression vector containing two expression cassettes for the Bt29K and API-B genes was constructed. These two insect-resistant genes were transferred into two cotton ( Gossypium hirsutum L.) varieties ( or lines) via Agrobacterium-mediated transformation and nine homozygous transgenic cotton lines showing a mortality of 90.0% - 99.7% to cotton ballworm (Heliothis armigera) larvae and good agronomic traits were selected through six generations. Molecular biology analysis revealed that one or two copies of the insecticidal protein genes were integrated into the transgenic cotton genome and activated Cry1Ac and API-B protein expression was at a level of 0.17% and 0.09% of the total soluble protein in the transgenic cotton leaves, respectively. Comparison of the insect-resistance of the homozygous lines expressing the activated chimeric Cry1Ac and API-B with that expressing Cry1Ac only revealed that the insect-resistance of the former is apparently higher than the latter. These results also indicate that the strategy to construct a plant expression vector expressing two different insect-resistant genes reported here is reasonable.
文摘In recent years, great progress has been made in. producing plants resistant to insect attack by genetic engineering of B. t δ-endotoxin gene or plant proteinase inhibitor genes, and how to avoid the emergence of the insect resistance to B. t δ-endotoxin produced by transgenic plants has been paid attention to. To combine one B. t δ-endotoxin gene with an incompatible B. t δ-endotoxin gene or with other genes such as proteinase inhibitor gene might alleviate the insect resistance problem. In this note, we study the
文摘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.
基金Project supported by Prime Minister Foundation,863 Program Foundation and Rockefeller Foundation.
文摘Total RNA was extracted from rice immature seeds harvested 2 weeks after flowering; then mRNA was purified. cDNA with NotI and SaiI cohesive ends was synthesized and inserted into λgt22A. After packaged in vitno, the cDNA library was constructed with 1.5×106pfu. A 21-mer oligodeoxynucleotide was synthesized according to the 5’-end conserved coding sequence of oryzacystatin (a thiol proteinase inhibitor) and labeled as a probe. From 2.1 × 104 pfu, 9 positive dones have been isolated, 8 of which contain the entire coding region of oryzacystatin. λOC1 has the longest cDNA insert, which contains an open reading frame of 309 bp coding sequence, 84 bp 5’-end non-coding region and a poly(A) signal AATAAA at the 3’-end followed by 31 Nt of poly(A). The coding sequence is the same compared with oryzacystatin genomic DNA sequence, while there are some obvious differences such as insertion and variation in the non-coding region, especially lots of nonsucoessive insertion in the 3’ region after poly(A) signal.
基金Supported by Prime Minister FouNdationthe High Techriology Research and Development Programme of ChinaRockefeller Foun-dation.
文摘A cDNA library was constructed with 1.5×10~6 pfu from rice immature seeds,fromwhich a cDNA clone for rice thiol proteinase inhibitor,oryzacystatin(OC),was isolated byscreening with synthesized oligodeoxynucleotide probe,which contained a 309bp open read-ing frame,84bp 5′-end noncoding region and a poly(A)signal AATAAA at the 3′-end fol-lowed by 31Nt poly(A).Then the coding region of OC was amplified and inserted into thedownstream of λP_RP_L promoter for thermal-inducible expression in E.coli.Shifting the cul-ture temperature from 30℃ to 42℃ led to a high level expression of OC,which exhibited adistinct band of 12.0 kDa and accounted for at least 10% of the total soluble proteins fromSDS-PAGE.The papain-inhibitory activity of the expressed OC was further confirmed.
