The constitutive promoter of cauliflower mosaic virus 35S （CaMV 35S） is a polar unidirectional promoter and is widely used in plant genetic engineering. In the present study, the unidirectional CaMV 35S promoter has...The constitutive promoter of cauliflower mosaic virus 35S （CaMV 35S） is a polar unidirectional promoter and is widely used in plant genetic engineering. In the present study, the unidirectional CaMV 35S promoter has been modified to a bi-directional promoter by fusing its minimal promoter element to the 5＇ end of CaMV 35S promoter in the opposite orientation. To qualitatively and quantitatively estimate its bi-directional transcriptional function and activity, two visible reporter genes, gusA （13-glucuronidase, GUS） and gfp （green fluorescent protein, GFP）, were fused to the two ends of the promoter in bi-orientations ending with NOS terminator sequences, respectively. Stable expression of gusA and gfp genes in transgenic tobacco （Nicotiana tabacum L.） was visulized by histochemically staining for GUS and fluorescence microscopic observation under UV for GFP in transgenic plants. The expression of two reporter genes showed that the constructed bi-directional promoter did have the bi-directional transcriptional function in both expected orientations. The quantitative estimation of GUS and GFP were determined on a HITACHI F1000 Fluorescence Spectrophotometer with various wavelengths of excitation and emission. The GUS activity varied from g to 250 pmol 4-MU/min/mg protein and the GFP content varied from 0.9 to 1.8 μg/ mg protein in various lines of transgenic tobacco plants. Higher GUS activity generally coupled with lower GFP content, and vice versa.展开更多
A method for transient gene expression was developed for western white pine(WWP,Pinus monticola Dougl.ex D.Don)using reporter gene uidA encodingβ-glucuronidase(GUS).GUS was transiently expressed in cross sections of ...A method for transient gene expression was developed for western white pine(WWP,Pinus monticola Dougl.ex D.Don)using reporter gene uidA encodingβ-glucuronidase(GUS).GUS was transiently expressed in cross sections of primary and secondary needles,cotyledons,and current and second year stems of WWP via vacuum-infiltration with Agrobacterium tumefaciens.Histochemical assays of cross sections of secondary needles showed stronger blue color indicating GUS expression at day 1 and 2 than on other days post agroinfiltration(dpa).GUS activity expressed inside WWP cells was confirmed using light microscopy.In fluorometric assays,GUS expression was high at 1 dpa and lasted until 4 dpa in detached secondary needles,while similarly high expression levels only lasted until 2 dpa in attached secondary needles then dropped significantly.Although the length of GUS-staining zones varied among different WWP organs and between growth and dormant seasons,all tested WWP tissues using the protocol had high levels of transient GUS expression.Thus,heterologous candidate genes or endogenous silencing can be expressed in various WWP tissues or organs using this agroinfiltration approach.The current protocol for efficient transient gene expression will aid functional genomics study of WWP and its pathogens and related conifer species.展开更多
Agrobacterium tumefaciens strain LBA 4404 carrying pBI121 plasmid was used to transform mature zygotic embryos of three genotypes (E-Hb, E-Ma, and E-Mc) of loblolly pine. The results demonstrated that the expression f...Agrobacterium tumefaciens strain LBA 4404 carrying pBI121 plasmid was used to transform mature zygotic embryos of three genotypes (E-Hb, E-Ma, and E-Mc) of loblolly pine. The results demonstrated that the expression frequency of (-glucuronidase reporter gene (GUS) varied among genotypes after mature zygotic embryos were infected with Agrobacterium tumefaciens cultures. The highest frequency (27.8%) of GUS expressing embryos was obtained from genotype E-Mc with mean number of 21.9 blue GUS spots per embryo. Expression of (-glucuronidase reporter gene was observed on cotyledons, hypocotyls, and radicles of transformed mature zygotic embryos, as well as on organogenic callus and regenerated shoots derived from co-cultivated mature zygotic embryos. Nineteen regenerated transgenic plants were obtained from GUS expression and kanamycin resistant calli. The presence and integration of the GUS gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. These results suggested that an efficient Agrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine has been developed and that this transformation system could be useful for the future studies on transferring economically important genes to loblolly pine.展开更多
In mammals, myeloid progenitors infiltrate the developing central nervous system (CNS), through the immature blood-brain barrier (BBB), the ventricular layer or the pial surface migrate and give rise to resident micro...In mammals, myeloid progenitors infiltrate the developing central nervous system (CNS), through the immature blood-brain barrier (BBB), the ventricular layer or the pial surface migrate and give rise to resident microglia. In the mature brain, however, the BBB hampers such recruitment from the blood-stream and long-term establishment of blood borne myeloid cells in the CNS thus appears at best limited. Hematopoietic stem cell-derived microglia, nevertheless, represents a promising tool for the correction of genetic deficits in the brain. We thus investigated the fate of primary human monocytes, and monocyte-derived macrophages, following transplantation into the adult mouse brain overpassing the BBB. Furthermore, we documented the ability of such cells to deliver a lysosomal enzyme into the brain following genetic modification with a recombinant adenoviral vector carrying the human β-glucuronidase cDNA. When implanted into the mouse striatum, the engineered primary cells survived and expressed the transgene for as much as 8 months. Moreover, the donor cells could migrate out of the grafting site and settle along blood vessels or myelin tracts although at limited distance. Migrating donor cells down-regulated the expression of CD14 andHLA DR, suggesting the adoption of a deactivated microglia-like phenotype. Our observations establish the ability of circulating mononuclear phagocytes to integrate into the brain after transplantation and express a transgene on the long term. These cells might thus be employed for autologous transplantation for the delivery of secreted therapeutic proteins in the context of a wide range of brain affections.展开更多
对乳酸杆菌pPG质粒表达系统理想表达条件进行了探索。以干酪乳杆菌Lactobacillus casei 393为宿主菌,β-葡萄糖苷酸酶(gusA)基因作为表达的报告基因,根据干酪乳杆菌的生长特性对细胞表面表达载体pPG表达过程中各个关键因素的影响进行了...对乳酸杆菌pPG质粒表达系统理想表达条件进行了探索。以干酪乳杆菌Lactobacillus casei 393为宿主菌,β-葡萄糖苷酸酶(gusA)基因作为表达的报告基因,根据干酪乳杆菌的生长特性对细胞表面表达载体pPG表达过程中各个关键因素的影响进行了探索。得出最佳表达条件:将其培养于质量浓度为1%乳糖,初始pH值在6.0～6.5之间的MRS培养基中,30～32℃,静止培养至A590OD达0.5,培养时间约8 h,表达效率最高,外源蛋白的表达量可占菌体总蛋白的14%。展开更多
To obtain a suitable condition for electroporation transformation in indica rice, the 10-day-old immature embryos were selected for optimization experiments. The results showed that one pulse at 850 V/cm, 950μF capac...To obtain a suitable condition for electroporation transformation in indica rice, the 10-day-old immature embryos were selected for optimization experiments. The results showed that one pulse at 850 V/cm, 950μF capacitance, 200 μL electroporation buffer with 70 mmol/L sodium glutamate, 100 μg/mL plasmid, 50μg/mL carrier DNA, 20 embryos per cuvette, 0℃ treatment and CC medium were the best parameters, which not only improved the transformation efficiency to 30.89%, but also ameliorated the embryo survival ratio to 95.92%. To further verify the practicability of this condition, the embryos from another indica rice variety and a rice type Ⅱ metallothionein-like gene （OsMT2bL） promotec：mgfp5：：gusA construct were tested, and specific GUS expression on the embryos was visualized by histochemical staining. The results showed that the GUS expression on the embryos activated by the OsMT2bL promoter was mainly concentrated on the apical point of the plumule whereas the expression driven by CaMV35S promoter was distributed on nearly all areas of the electroporated tissues. These results indicated that the optimized embryo electroporation conditions could be used not only in genetic transformation of indica rice but also in assay of gene regulation on embryos.展开更多
Artemisia annua L. produces small amounts of the sesquiterpenoid artemisinin, which is used for treatment of malaria. A worldwide shortage of the drug has led to intense research to increase the yield of artemisinin i...Artemisia annua L. produces small amounts of the sesquiterpenoid artemisinin, which is used for treatment of malaria. A worldwide shortage of the drug has led to intense research to increase the yield of artemisinin in the plant. In order to study the regulation of expression of a key enzyme of artemisinin biosynthesis, the promoter region of the key enzyme amorpha-4,11-diene synthase (ADS) was cloned and fused with the β-glucuronidase (GUS) reporter gene. Transgenic plants of A. annua expressing this fusion were generated and studied. Transgenic plants expressing the GUS gene were used to establish the activity of the cloned promoter by a GUS activity staining procedure. GUS under the control of the ADS promoter showed specific expression in glandular trichomes. The activity of the ADS promoter varies temporally and in old tissues essentially no GUS staining could be observed. The expression pattern of GUS and ADS in aerial parts of the transgenic plant was essentially the same indicating that the cis-elements controlling glandular trichome specific expression are included in the cloned promoter. However, some cis-element(s) that control expression in root and old leaf appears to be missing in the cloned promoter. Furthermore, qPCR was used to compare the activity of the wild-type ADS promoter with that of the cloned ADS promoter. The latter promoter showed a considerably lower activity than the wild-type promoter as judged from the levels of GUS and ADS transcripts, respectively, which may be due to the removal of an enhancing cis-element from the ADS promoter. The ADS gene is specifically expressed in stalk and secretory cells of glandular trichomes of A. annua.展开更多
Two plasmids, p13GUS and p13GUS2, were constructed to create a gene trap system containing the promotsriess β-glucuronidase （GUS） reporter gene in the T-DNA region. Transformation of these two plasmids into the ric...Two plasmids, p13GUS and p13GUS2, were constructed to create a gene trap system containing the promotsriess β-glucuronidase （GUS） reporter gene in the T-DNA region. Transformation of these two plasmids into the rice variety Zhonghua 11 （Oryza sativa ssp. japonica cv.）, mediated by Agrobacterium tumefaciens, resulted in 942 independent transgenic lines. Histochemical GUS assays revealed that 31 To plants had various patterns of the reporter gene expression, including expression in only one tissue, and simultaneously in two or more tissues. Hygromycin-resistsnt （hygr） homozygotes were screened and the copy number of the T-DNA inserts was determined in the GUS-positive transgenic plants. The flanking sequences of the T-DNA were isolated by inverse-polymerase chain reaction and the insert positions on the rice genome of T-DNA were determined by a basic local alignment search tool in the GUS-positive transgenic plants transformed with plasmid p13GUS. Moreover, calli induced from the seeds of the T1 generation of 911 GUS-negative transgenic lines were subjected to stress and hormone treatments. Histochemical GUS assays were carried out on the calli before and after treatment. The results revealed that calli from 21 lines displayed differential GUS expression after treatment. All of these data demonstrated that this trap system is suitable for identifying rice genes, including those that are sensitive to induction.展开更多
The dehydration-responsive element-binding factor 2C （DREB2C） is a member of the CBF/DREB subfamily of proteins, which contains a single APETALA2/Ethylene responsive element-binding factor （AP2/ERF） domain. To ide...The dehydration-responsive element-binding factor 2C （DREB2C） is a member of the CBF/DREB subfamily of proteins, which contains a single APETALA2/Ethylene responsive element-binding factor （AP2/ERF） domain. To identify the expression pattern of the DREB2C gene, which contains multiple transcription cis-regulatory elements in its promoter, an approximately 1.4 kb upstream DREB2C sequence was fused to the β-glucuronidase reporter gene （GUS） and the recombinant p1244 construct was transformed into Arabidopsis thaliana （L.） Heynh. The promoter of the gene directed prominent GUS activity in the vasculature in diverse young dividing tissues. Upon applying heat stress （HS）, GUS staining was also enhanced in the vasculature of the growing tissues. Analysis of a series of 5＇-deletions of the DREB2C promoter revealed that a proximal upstream sequence sufficient for the tissue-specific spatial and temporal induction of GUS expression by HS is localized in the promoter region between -204 and -34 bps relative to the transcriptional start site. Furthermore, electrophoretic mobility shift assay （EMSA） demonstrated that nuclear protein binding activities specific to a -120 to -32 bp promoter fragment increased after HS. These results indicate that the TATA-proximal region and some latent trans-acting factors may cooperate in HS-induced activation of the Arabidopsis DREB2C promoter.展开更多
Inclusion of MARs in transgene cassettes enhances their expression and reduces position-effect variations in the transgenic host. Four new MARs (TM2, TM3, AM1 and AM2) were isolated from tobacco and Arabidopsis by PCR...Inclusion of MARs in transgene cassettes enhances their expression and reduces position-effect variations in the transgenic host. Four new MARs (TM2, TM3, AM1 and AM2) were isolated from tobacco and Arabidopsis by PCR method. The nuclei isolated from suspension-cultured cells of rice were used to prepare nuclear matrix. With a characterized MAR (TM1) as a positive control, the Matrix-MAR interactions were tested by an in vitro binding assay to identify the DNA sequences as MARs and their binding strength to nuclear matrix in vitro was compared. The results showed that TM2 and TM3 had stronger binding strength than TM1. To determine the functions of the four new MARs in vivo, binary vectors pBI121 carrying a uidA GUS reporter gene were modified with direct repeat MARs inserted on both sides of the reporter gene cassette and were transferred into tobaccos via Agrobacterium-mediated transformation procedure. Quantitative GUS assays of the transgenic tobaccos showed that when flanking a GUS reporter gene展开更多
文摘The constitutive promoter of cauliflower mosaic virus 35S （CaMV 35S） is a polar unidirectional promoter and is widely used in plant genetic engineering. In the present study, the unidirectional CaMV 35S promoter has been modified to a bi-directional promoter by fusing its minimal promoter element to the 5＇ end of CaMV 35S promoter in the opposite orientation. To qualitatively and quantitatively estimate its bi-directional transcriptional function and activity, two visible reporter genes, gusA （13-glucuronidase, GUS） and gfp （green fluorescent protein, GFP）, were fused to the two ends of the promoter in bi-orientations ending with NOS terminator sequences, respectively. Stable expression of gusA and gfp genes in transgenic tobacco （Nicotiana tabacum L.） was visulized by histochemically staining for GUS and fluorescence microscopic observation under UV for GFP in transgenic plants. The expression of two reporter genes showed that the constructed bi-directional promoter did have the bi-directional transcriptional function in both expected orientations. The quantitative estimation of GUS and GFP were determined on a HITACHI F1000 Fluorescence Spectrophotometer with various wavelengths of excitation and emission. The GUS activity varied from g to 250 pmol 4-MU/min/mg protein and the GFP content varied from 0.9 to 1.8 μg/ mg protein in various lines of transgenic tobacco plants. Higher GUS activity generally coupled with lower GFP content, and vice versa.
文摘A method for transient gene expression was developed for western white pine(WWP,Pinus monticola Dougl.ex D.Don)using reporter gene uidA encodingβ-glucuronidase(GUS).GUS was transiently expressed in cross sections of primary and secondary needles,cotyledons,and current and second year stems of WWP via vacuum-infiltration with Agrobacterium tumefaciens.Histochemical assays of cross sections of secondary needles showed stronger blue color indicating GUS expression at day 1 and 2 than on other days post agroinfiltration(dpa).GUS activity expressed inside WWP cells was confirmed using light microscopy.In fluorometric assays,GUS expression was high at 1 dpa and lasted until 4 dpa in detached secondary needles,while similarly high expression levels only lasted until 2 dpa in attached secondary needles then dropped significantly.Although the length of GUS-staining zones varied among different WWP organs and between growth and dormant seasons,all tested WWP tissues using the protocol had high levels of transient GUS expression.Thus,heterologous candidate genes or endogenous silencing can be expressed in various WWP tissues or organs using this agroinfiltration approach.The current protocol for efficient transient gene expression will aid functional genomics study of WWP and its pathogens and related conifer species.
文摘Agrobacterium tumefaciens strain LBA 4404 carrying pBI121 plasmid was used to transform mature zygotic embryos of three genotypes (E-Hb, E-Ma, and E-Mc) of loblolly pine. The results demonstrated that the expression frequency of (-glucuronidase reporter gene (GUS) varied among genotypes after mature zygotic embryos were infected with Agrobacterium tumefaciens cultures. The highest frequency (27.8%) of GUS expressing embryos was obtained from genotype E-Mc with mean number of 21.9 blue GUS spots per embryo. Expression of (-glucuronidase reporter gene was observed on cotyledons, hypocotyls, and radicles of transformed mature zygotic embryos, as well as on organogenic callus and regenerated shoots derived from co-cultivated mature zygotic embryos. Nineteen regenerated transgenic plants were obtained from GUS expression and kanamycin resistant calli. The presence and integration of the GUS gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. These results suggested that an efficient Agrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine has been developed and that this transformation system could be useful for the future studies on transferring economically important genes to loblolly pine.
基金funded by the CNRS,The INSERM and Vaincre les Maladie Lysosomiales(vml-asso.org).
文摘In mammals, myeloid progenitors infiltrate the developing central nervous system (CNS), through the immature blood-brain barrier (BBB), the ventricular layer or the pial surface migrate and give rise to resident microglia. In the mature brain, however, the BBB hampers such recruitment from the blood-stream and long-term establishment of blood borne myeloid cells in the CNS thus appears at best limited. Hematopoietic stem cell-derived microglia, nevertheless, represents a promising tool for the correction of genetic deficits in the brain. We thus investigated the fate of primary human monocytes, and monocyte-derived macrophages, following transplantation into the adult mouse brain overpassing the BBB. Furthermore, we documented the ability of such cells to deliver a lysosomal enzyme into the brain following genetic modification with a recombinant adenoviral vector carrying the human β-glucuronidase cDNA. When implanted into the mouse striatum, the engineered primary cells survived and expressed the transgene for as much as 8 months. Moreover, the donor cells could migrate out of the grafting site and settle along blood vessels or myelin tracts although at limited distance. Migrating donor cells down-regulated the expression of CD14 andHLA DR, suggesting the adoption of a deactivated microglia-like phenotype. Our observations establish the ability of circulating mononuclear phagocytes to integrate into the brain after transplantation and express a transgene on the long term. These cells might thus be employed for autologous transplantation for the delivery of secreted therapeutic proteins in the context of a wide range of brain affections.
文摘对乳酸杆菌pPG质粒表达系统理想表达条件进行了探索。以干酪乳杆菌Lactobacillus casei 393为宿主菌,β-葡萄糖苷酸酶(gusA)基因作为表达的报告基因,根据干酪乳杆菌的生长特性对细胞表面表达载体pPG表达过程中各个关键因素的影响进行了探索。得出最佳表达条件:将其培养于质量浓度为1%乳糖,初始pH值在6.0～6.5之间的MRS培养基中,30～32℃,静止培养至A590OD达0.5,培养时间约8 h,表达效率最高,外源蛋白的表达量可占菌体总蛋白的14%。
基金This project was supported by the National Natural Science Foundation of China(Grant No.30570103)the Key Grant Project of Chinese Ministry of Education(Grant No.307018).
文摘To obtain a suitable condition for electroporation transformation in indica rice, the 10-day-old immature embryos were selected for optimization experiments. The results showed that one pulse at 850 V/cm, 950μF capacitance, 200 μL electroporation buffer with 70 mmol/L sodium glutamate, 100 μg/mL plasmid, 50μg/mL carrier DNA, 20 embryos per cuvette, 0℃ treatment and CC medium were the best parameters, which not only improved the transformation efficiency to 30.89%, but also ameliorated the embryo survival ratio to 95.92%. To further verify the practicability of this condition, the embryos from another indica rice variety and a rice type Ⅱ metallothionein-like gene （OsMT2bL） promotec：mgfp5：：gusA construct were tested, and specific GUS expression on the embryos was visualized by histochemical staining. The results showed that the GUS expression on the embryos activated by the OsMT2bL promoter was mainly concentrated on the apical point of the plumule whereas the expression driven by CaMV35S promoter was distributed on nearly all areas of the electroporated tissues. These results indicated that the optimized embryo electroporation conditions could be used not only in genetic transformation of indica rice but also in assay of gene regulation on embryos.
文摘Artemisia annua L. produces small amounts of the sesquiterpenoid artemisinin, which is used for treatment of malaria. A worldwide shortage of the drug has led to intense research to increase the yield of artemisinin in the plant. In order to study the regulation of expression of a key enzyme of artemisinin biosynthesis, the promoter region of the key enzyme amorpha-4,11-diene synthase (ADS) was cloned and fused with the β-glucuronidase (GUS) reporter gene. Transgenic plants of A. annua expressing this fusion were generated and studied. Transgenic plants expressing the GUS gene were used to establish the activity of the cloned promoter by a GUS activity staining procedure. GUS under the control of the ADS promoter showed specific expression in glandular trichomes. The activity of the ADS promoter varies temporally and in old tissues essentially no GUS staining could be observed. The expression pattern of GUS and ADS in aerial parts of the transgenic plant was essentially the same indicating that the cis-elements controlling glandular trichome specific expression are included in the cloned promoter. However, some cis-element(s) that control expression in root and old leaf appears to be missing in the cloned promoter. Furthermore, qPCR was used to compare the activity of the wild-type ADS promoter with that of the cloned ADS promoter. The latter promoter showed a considerably lower activity than the wild-type promoter as judged from the levels of GUS and ADS transcripts, respectively, which may be due to the removal of an enhancing cis-element from the ADS promoter. The ADS gene is specifically expressed in stalk and secretory cells of glandular trichomes of A. annua.
基金grants from the Ministry of Science and Technology of China(2002AA2Z1003 and 2005CB120803).
文摘Two plasmids, p13GUS and p13GUS2, were constructed to create a gene trap system containing the promotsriess β-glucuronidase （GUS） reporter gene in the T-DNA region. Transformation of these two plasmids into the rice variety Zhonghua 11 （Oryza sativa ssp. japonica cv.）, mediated by Agrobacterium tumefaciens, resulted in 942 independent transgenic lines. Histochemical GUS assays revealed that 31 To plants had various patterns of the reporter gene expression, including expression in only one tissue, and simultaneously in two or more tissues. Hygromycin-resistsnt （hygr） homozygotes were screened and the copy number of the T-DNA inserts was determined in the GUS-positive transgenic plants. The flanking sequences of the T-DNA were isolated by inverse-polymerase chain reaction and the insert positions on the rice genome of T-DNA were determined by a basic local alignment search tool in the GUS-positive transgenic plants transformed with plasmid p13GUS. Moreover, calli induced from the seeds of the T1 generation of 911 GUS-negative transgenic lines were subjected to stress and hormone treatments. Histochemical GUS assays were carried out on the calli before and after treatment. The results revealed that calli from 21 lines displayed differential GUS expression after treatment. All of these data demonstrated that this trap system is suitable for identifying rice genes, including those that are sensitive to induction.
基金supported by the Next-Generation BioGreen 21 Program (SSAC, PJ008173, Rural Development Administration,Republic of Korea)the Basic Science Research Program(20120002903, through the NRF funded by the Ministry of Education, Science and Technology, Republic of Korea)
文摘The dehydration-responsive element-binding factor 2C （DREB2C） is a member of the CBF/DREB subfamily of proteins, which contains a single APETALA2/Ethylene responsive element-binding factor （AP2/ERF） domain. To identify the expression pattern of the DREB2C gene, which contains multiple transcription cis-regulatory elements in its promoter, an approximately 1.4 kb upstream DREB2C sequence was fused to the β-glucuronidase reporter gene （GUS） and the recombinant p1244 construct was transformed into Arabidopsis thaliana （L.） Heynh. The promoter of the gene directed prominent GUS activity in the vasculature in diverse young dividing tissues. Upon applying heat stress （HS）, GUS staining was also enhanced in the vasculature of the growing tissues. Analysis of a series of 5＇-deletions of the DREB2C promoter revealed that a proximal upstream sequence sufficient for the tissue-specific spatial and temporal induction of GUS expression by HS is localized in the promoter region between -204 and -34 bps relative to the transcriptional start site. Furthermore, electrophoretic mobility shift assay （EMSA） demonstrated that nuclear protein binding activities specific to a -120 to -32 bp promoter fragment increased after HS. These results indicate that the TATA-proximal region and some latent trans-acting factors may cooperate in HS-induced activation of the Arabidopsis DREB2C promoter.
基金This work was supported by the National Special Program for Research and Industrialization of Transgenic Plants (Grant No. J99-A-038) the National Natural Science Foundation of China (Grant No. 39970075).
文摘Inclusion of MARs in transgene cassettes enhances their expression and reduces position-effect variations in the transgenic host. Four new MARs (TM2, TM3, AM1 and AM2) were isolated from tobacco and Arabidopsis by PCR method. The nuclei isolated from suspension-cultured cells of rice were used to prepare nuclear matrix. With a characterized MAR (TM1) as a positive control, the Matrix-MAR interactions were tested by an in vitro binding assay to identify the DNA sequences as MARs and their binding strength to nuclear matrix in vitro was compared. The results showed that TM2 and TM3 had stronger binding strength than TM1. To determine the functions of the four new MARs in vivo, binary vectors pBI121 carrying a uidA GUS reporter gene were modified with direct repeat MARs inserted on both sides of the reporter gene cassette and were transferred into tobaccos via Agrobacterium-mediated transformation procedure. Quantitative GUS assays of the transgenic tobaccos showed that when flanking a GUS reporter gene
