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
High-molecular-weight glutenin subunits （HMW-GSs）, one class of seed storage proteins in wheat, play an important role in determining bread-making quality of flour. More and more proves support that HMW-GS- 1Bx 14 a...High-molecular-weight glutenin subunits （HMW-GSs）, one class of seed storage proteins in wheat, play an important role in determining bread-making quality of flour. More and more proves support that HMW-GS- 1Bx 14 and - 1Bx 15 subunits are strongly positively associated with good bread-making and excellent noodle-making quality. The two subunits are encoded by two genes, Glu-1Bx14 and Glu-1Bx15, which are tightly linked and located on the 1BL. Protein assay by SDS-PAGE indicated that the expression of Glu-1Bx14 gene was always much stronger than that of Glu-1By15 in the same variety. But, variation of expression level for Glu-1By15 gene existed among varieties, such as in Xiaoyan 54, Xiaoyan 6, Yanzhan 1 and Shanyou 225. We also investigated the transcription difference of Glu-1By14 and Glu-1By15 genes in Xiaoyan 54 and Shanyou 225 by semi-quantitative RT-PCR method. The Glu-1By14 always transcripts much more than the Glu-1By15. This was basically consistent with the translation difference between the two genes. Promoters of 1Bx14, 1By15, 1By8, 1Dx2 and 1Dy12 were cloned from Xiaoyan 54, Chinese Spring and Aegilops tauschii. Sequence analysis indicated that the HMW-GS genes had high homology at their promoter regions. However, significant difference existed between sequence of 1Bx14 promoter and those of other HMW-GS genes. The transient expression experiment showed that the promoter of 1By15 has lower activity than that of 1Bx14, which was consistent with their transcription level of the two genes in varieties. In addition, transient expression of the gus driven by the promoter （P2） of HMW-GS 1Dx2 gene was higher than by other HMW-GS promoters. Therefore, we constructed 1By15 gene expression vector driven by the 1Dx2 promoter, and transformed the 1By15 gene into wheat commercial variety, Jimai 20 by pollen tube method. Of 45 independent transgenic lines identified by PCR, 3 were confirmed to contain the HMW-GS 1By15 gene via Southern hybridization. The delivered 1By15 gene expressed the expected HMW-GS protein in the seeds of transgenic plants.展开更多
Odontoglossum ringspot virus (ORSV) infects perennial orchids (Phalaenopsis amabilis) and causes a widespread viral disease. RNA-silencing of viral genes is a promising and effective way of controlling viral infection...Odontoglossum ringspot virus (ORSV) infects perennial orchids (Phalaenopsis amabilis) and causes a widespread viral disease. RNA-silencing of viral genes is a promising and effective way of controlling viral infection in plants. An inverted repeat (IR) fragment of the ORSV coat protein gene, cp, was inserted into the pXGY1 vector to generate the silencing construct, pXGY1-ORSV, which was introduced into Nicotiana benthamiana via Agrobacterium-mediated infiltration. A total of 15 homozygous pXGY1-ORSV transgenic N. benthamiana T1 plants were obtained from five transgenic lines, and ORSV cp gene multiplication was reduced by at least 75% - 95% in 12 T2 plants, demonstrating their increased resistance to ORSV. An infectious ORSV clone, pCAMBIA2300-ORSV, was generated to facilitate rigorous analyses of plant viral resistance. Semi-quantitative RT-PCR (sqRT-PCR) and northern-blot analyses revealed that levels of ORSV multiplication and ORSV coat protein were significantly reduced in pXGY1-ORSV transgenic N. benthamiana. Western-blot from pXGY1-ORSV inoculated leaves of ORSV infected P. amabilis also revealed the significant decrease and even degradation of ORSV-CP protein. Disease symptoms were not observed in transgenic plants. These results indicate a high level of ORSV-resistance in pXGY1-ORSV transgenic N. benthamiana.展开更多
RNAi with natural defence mechanism of homologous RNA degradation is widely used in research of antiviral plant.It is important to construct a highly efficent RNAi vector for transgenic plants of virus resis-tance.In ...RNAi with natural defence mechanism of homologous RNA degradation is widely used in research of antiviral plant.It is important to construct a highly efficent RNAi vector for transgenic plants of virus resis-tance.In this study,part fragments of coat protein gene of Potato virus Y(PVY)(451-750 bp) were inserted into the two expression vectors.Vector pROKY300 without intron and pHelY300 with PDK and CAT introns on the hpRNA stem were constructed.The silence efficiency of virus resistance of the two vectors was investigated as 88%(22/25)for pROKY300 and 92%(23/25) for pHelY300 through transient expression mediated by agroinfiltration.The results showed that both vectors were highly antiviral and elucidated the validity of RNAi-medicates resistance to virus.展开更多
To obtain a regulatory element for generating mammary bioreactors. a DNA fragment derived from bovine β-lactoglobulin (BLG) gene was cloned, which consisted of a 650-bp 5’ flanking sequence, exon Ⅰ, intron Ⅰ and e...To obtain a regulatory element for generating mammary bioreactors. a DNA fragment derived from bovine β-lactoglobulin (BLG) gene was cloned, which consisted of a 650-bp 5’ flanking sequence, exon Ⅰ, intron Ⅰ and exon Ⅱ. A 661-bp region of the cloned fragment, consisting of the 650-bp 5’ flanking sequence and a non-coding sequence of 11 bp downstream of the transcription initiation site, was used as a regulatory element to combine with human growth hormone (hGH) gene to generate a bovine BLG/hGH fusion construct, which was then introduced into cultured primary mammary epithelial cells of goat for transient expression of hGH gene. It was demonstrated that the hGH gene was able to express following hormone induction and the expressed product was able to be secreted into the medium. The bovine BLG/hGH fusion construct was also used to generate transgenic mice by microinjection. Subsequently, five transgenic mice were generated. The hGH in milk by one transgenic female mouse was 420μg/mL, while the展开更多
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High-molecular-weight glutenin subunits （HMW-GSs）, one class of seed storage proteins in wheat, play an important role in determining bread-making quality of flour. More and more proves support that HMW-GS- 1Bx 14 and - 1Bx 15 subunits are strongly positively associated with good bread-making and excellent noodle-making quality. The two subunits are encoded by two genes, Glu-1Bx14 and Glu-1Bx15, which are tightly linked and located on the 1BL. Protein assay by SDS-PAGE indicated that the expression of Glu-1Bx14 gene was always much stronger than that of Glu-1By15 in the same variety. But, variation of expression level for Glu-1By15 gene existed among varieties, such as in Xiaoyan 54, Xiaoyan 6, Yanzhan 1 and Shanyou 225. We also investigated the transcription difference of Glu-1By14 and Glu-1By15 genes in Xiaoyan 54 and Shanyou 225 by semi-quantitative RT-PCR method. The Glu-1By14 always transcripts much more than the Glu-1By15. This was basically consistent with the translation difference between the two genes. Promoters of 1Bx14, 1By15, 1By8, 1Dx2 and 1Dy12 were cloned from Xiaoyan 54, Chinese Spring and Aegilops tauschii. Sequence analysis indicated that the HMW-GS genes had high homology at their promoter regions. However, significant difference existed between sequence of 1Bx14 promoter and those of other HMW-GS genes. The transient expression experiment showed that the promoter of 1By15 has lower activity than that of 1Bx14, which was consistent with their transcription level of the two genes in varieties. In addition, transient expression of the gus driven by the promoter （P2） of HMW-GS 1Dx2 gene was higher than by other HMW-GS promoters. Therefore, we constructed 1By15 gene expression vector driven by the 1Dx2 promoter, and transformed the 1By15 gene into wheat commercial variety, Jimai 20 by pollen tube method. Of 45 independent transgenic lines identified by PCR, 3 were confirmed to contain the HMW-GS 1By15 gene via Southern hybridization. The delivered 1By15 gene expressed the expected HMW-GS protein in the seeds of transgenic plants.
文摘Odontoglossum ringspot virus (ORSV) infects perennial orchids (Phalaenopsis amabilis) and causes a widespread viral disease. RNA-silencing of viral genes is a promising and effective way of controlling viral infection in plants. An inverted repeat (IR) fragment of the ORSV coat protein gene, cp, was inserted into the pXGY1 vector to generate the silencing construct, pXGY1-ORSV, which was introduced into Nicotiana benthamiana via Agrobacterium-mediated infiltration. A total of 15 homozygous pXGY1-ORSV transgenic N. benthamiana T1 plants were obtained from five transgenic lines, and ORSV cp gene multiplication was reduced by at least 75% - 95% in 12 T2 plants, demonstrating their increased resistance to ORSV. An infectious ORSV clone, pCAMBIA2300-ORSV, was generated to facilitate rigorous analyses of plant viral resistance. Semi-quantitative RT-PCR (sqRT-PCR) and northern-blot analyses revealed that levels of ORSV multiplication and ORSV coat protein were significantly reduced in pXGY1-ORSV transgenic N. benthamiana. Western-blot from pXGY1-ORSV inoculated leaves of ORSV infected P. amabilis also revealed the significant decrease and even degradation of ORSV-CP protein. Disease symptoms were not observed in transgenic plants. These results indicate a high level of ORSV-resistance in pXGY1-ORSV transgenic N. benthamiana.
文摘RNAi with natural defence mechanism of homologous RNA degradation is widely used in research of antiviral plant.It is important to construct a highly efficent RNAi vector for transgenic plants of virus resis-tance.In this study,part fragments of coat protein gene of Potato virus Y(PVY)(451-750 bp) were inserted into the two expression vectors.Vector pROKY300 without intron and pHelY300 with PDK and CAT introns on the hpRNA stem were constructed.The silence efficiency of virus resistance of the two vectors was investigated as 88%(22/25)for pROKY300 and 92%(23/25) for pHelY300 through transient expression mediated by agroinfiltration.The results showed that both vectors were highly antiviral and elucidated the validity of RNAi-medicates resistance to virus.
文摘To obtain a regulatory element for generating mammary bioreactors. a DNA fragment derived from bovine β-lactoglobulin (BLG) gene was cloned, which consisted of a 650-bp 5’ flanking sequence, exon Ⅰ, intron Ⅰ and exon Ⅱ. A 661-bp region of the cloned fragment, consisting of the 650-bp 5’ flanking sequence and a non-coding sequence of 11 bp downstream of the transcription initiation site, was used as a regulatory element to combine with human growth hormone (hGH) gene to generate a bovine BLG/hGH fusion construct, which was then introduced into cultured primary mammary epithelial cells of goat for transient expression of hGH gene. It was demonstrated that the hGH gene was able to express following hormone induction and the expressed product was able to be secreted into the medium. The bovine BLG/hGH fusion construct was also used to generate transgenic mice by microinjection. Subsequently, five transgenic mice were generated. The hGH in milk by one transgenic female mouse was 420μg/mL, while the
