The shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPs) is the target of nonselective herbicide glyphosate. A partial rice epsps cDNA was generated by RT-PCR with primers designed according to...The shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPs) is the target of nonselective herbicide glyphosate. A partial rice epsps cDNA was generated by RT-PCR with primers designed according to EST sequence in GenBank and used as probe for rice genomic library screening. In a screen of approximately 8.0×104 clones from the rice genomic library, sixteen positive clones were obtained, which strongly hybridized to the probe. One clone, E11, was selected for further analysis and the full-length 3661 bp rice epsps genomic sequence was obtained. Sequence analysis and homologous comparison revealed that epsps gene is composed of 8 exons and 7 introns. Analysis by restriction fragment length polymorphism with the probe of rice epsps cDNA fragment confirmed that rice epsps is located on chromosome 6 with an indica-japonica (ZYQ8-JX17) double-haploid (DH) population. This is the first report on the EPSP synthase from monocotyledons.展开更多
The DNA-binding protein TGA1a of tobacco can specially interact with the enhancer sequence as-1 (- 83 to - 63) of CaMV35S promoter and show the function of transcriptional acti-vation. In order to study the expression...The DNA-binding protein TGA1a of tobacco can specially interact with the enhancer sequence as-1 (- 83 to - 63) of CaMV35S promoter and show the function of transcriptional acti-vation. In order to study the expression of exogenous gene affected by TGA1a, a trans-acting regulation system was formed by tandem connecting tga1a under the control of the phloem- specific promoter rolC with reporter gene under the control of CaMV35S. Then, the system above was utilized to construct a plant expression vector. Moreover, two plant expression vectors were constructed with the report gene controlled by CaMV35S and rolC promoter respectively as posi-tive controls. Tobacco leaf disc transformed by Agrobacterium-mediated method and transgenic plants were regenerated. It was proved that the reporter gene existed in the genome of transgenic plants by Southern hybridization. The results of GUS activity indicated that the expression of tga1a controlled by rolC remarkably increased the expression of the reporter gene controlled by CaMV35S. GUS activity of transgenic plants containing trans-acting regulation system was higher than that of transgenic plants containing the reporter gene under the control of CaMV35S and rolC respectively, with the highest GUS activity of about tenfolds of two positive controls. Histochemical method demonstrated that GUS staining amassed mainly in phloem tissue of transgenic plants containing the trans-acting regulation system. A new model for arising the expression level and tissue-specific expression of exogenous gene in transgenic plant was established in this study.展开更多
A DNA fragment containing consensus sequence of matrix attachment region (MAR) has been isolated from pea genome. Compared with original DNA sequence, one 115 bp-long repeat sequence is deleted in the obtained DNA seq...A DNA fragment containing consensus sequence of matrix attachment region (MAR) has been isolated from pea genome. Compared with original DNA sequence, one 115 bp-long repeat sequence is deleted in the obtained DNA sequence. DNA fragments located upstream and down-stream of repeat DNA sequence respectively share 84% and 93% homology to the corresponding original sequence, and contain A-box or T-box and TATAA sequence, which is characteristics short sequence of MARs. To test the function of the DNA sequence, the plant expression vectors in which b-glucuronidase gene (GUS, uidA) was used as reporter gene were constructed and transferred into tobaccos via Agrobacterium-mediated transformation procedure. Quantitative GUS assay showed that the average level of uidA expression was increased twofold for the presence of MAR, and the highest level of GUS activity of transgenic plants could be increased six times. The results cited above suggest that the isolated DNA sequence contains consensus sequence of MARs and has capability to increase expression level of gene in transgenic plants.展开更多
基金This work was supported by the National High-Tech Program (863), National Natural Science Foundation of China (Grant Nos. 39989001, 39580012 & 39880023) National Special Program for Research and Industrialization of Transgenic Plants, and Rockefeller F
文摘The shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPs) is the target of nonselective herbicide glyphosate. A partial rice epsps cDNA was generated by RT-PCR with primers designed according to EST sequence in GenBank and used as probe for rice genomic library screening. In a screen of approximately 8.0×104 clones from the rice genomic library, sixteen positive clones were obtained, which strongly hybridized to the probe. One clone, E11, was selected for further analysis and the full-length 3661 bp rice epsps genomic sequence was obtained. Sequence analysis and homologous comparison revealed that epsps gene is composed of 8 exons and 7 introns. Analysis by restriction fragment length polymorphism with the probe of rice epsps cDNA fragment confirmed that rice epsps is located on chromosome 6 with an indica-japonica (ZYQ8-JX17) double-haploid (DH) population. This is the first report on the EPSP synthase from monocotyledons.
基金This work was supported by the National Natural Science Foundation of China (Grant Nos.3998002439880023&39989001)+2 种基金heHighTechnology Research and DevelopmentProgramofChina2001AA2120412001AA222251 101-06-01-06) and the National Key Fundamental Resear
文摘The DNA-binding protein TGA1a of tobacco can specially interact with the enhancer sequence as-1 (- 83 to - 63) of CaMV35S promoter and show the function of transcriptional acti-vation. In order to study the expression of exogenous gene affected by TGA1a, a trans-acting regulation system was formed by tandem connecting tga1a under the control of the phloem- specific promoter rolC with reporter gene under the control of CaMV35S. Then, the system above was utilized to construct a plant expression vector. Moreover, two plant expression vectors were constructed with the report gene controlled by CaMV35S and rolC promoter respectively as posi-tive controls. Tobacco leaf disc transformed by Agrobacterium-mediated method and transgenic plants were regenerated. It was proved that the reporter gene existed in the genome of transgenic plants by Southern hybridization. The results of GUS activity indicated that the expression of tga1a controlled by rolC remarkably increased the expression of the reporter gene controlled by CaMV35S. GUS activity of transgenic plants containing trans-acting regulation system was higher than that of transgenic plants containing the reporter gene under the control of CaMV35S and rolC respectively, with the highest GUS activity of about tenfolds of two positive controls. Histochemical method demonstrated that GUS staining amassed mainly in phloem tissue of transgenic plants containing the trans-acting regulation system. A new model for arising the expression level and tissue-specific expression of exogenous gene in transgenic plant was established in this study.
基金the National High Science and Technology Program, National NaturalScience Foundation of China (Grant Nos. 39989001 & 39880023), and National Special Program for Research and Industrialization of Transgenic Plants.
文摘A DNA fragment containing consensus sequence of matrix attachment region (MAR) has been isolated from pea genome. Compared with original DNA sequence, one 115 bp-long repeat sequence is deleted in the obtained DNA sequence. DNA fragments located upstream and down-stream of repeat DNA sequence respectively share 84% and 93% homology to the corresponding original sequence, and contain A-box or T-box and TATAA sequence, which is characteristics short sequence of MARs. To test the function of the DNA sequence, the plant expression vectors in which b-glucuronidase gene (GUS, uidA) was used as reporter gene were constructed and transferred into tobaccos via Agrobacterium-mediated transformation procedure. Quantitative GUS assay showed that the average level of uidA expression was increased twofold for the presence of MAR, and the highest level of GUS activity of transgenic plants could be increased six times. The results cited above suggest that the isolated DNA sequence contains consensus sequence of MARs and has capability to increase expression level of gene in transgenic plants.
