[Objective] The aim was to construct the fusion expression vector of polyphosphate kinase（PPK） and green fluorescent protein（GFP） genes.[Method] In this study,the primers were designed based on PPK gene sequence（...[Objective] The aim was to construct the fusion expression vector of polyphosphate kinase（PPK） and green fluorescent protein（GFP） genes.[Method] In this study,the primers were designed based on PPK gene sequence（L03719） of E.coli DH5α in Genbank.Genomic DNA of E.coli DH 5α was extracted as template for the amplification of PPK gene by PCR method.By using In-Fusion@ HD Cloning Kit,the PPK gene was directionally cloned into NcoI site of the pCAMBIA1302 vector.[Result] Sequencing results showed that the 2.0 kb long fragment of PPK gene was inserted into the plant-based expression vector pCAMBIA1302 in front of GFP gene.[Conclusion] The fusion expression vector of PPK and GFP genes were successfully constructed.展开更多
[ Objective] The study is to generate pharmaceutical protein via plant transgenic technique. [Methed] Using the cotyledons with petiole as transformation receptor, the fusion gene of rapeseed oil-body gene and bFGF wa...[ Objective] The study is to generate pharmaceutical protein via plant transgenic technique. [Methed] Using the cotyledons with petiole as transformation receptor, the fusion gene of rapeseed oil-body gene and bFGF was introduced into the rapeseed （ Brassica campestris L. ） by Agrobacterium tumefaciens-mediated transformation; meanwhile regeneration conditions of rapeseed were also optimized, and the regenerated resistant plantlets were detected by PCR and Southern blot. [ Result] This fusion gene had been integrated into rapeseed genome successfully, and the optimized conditions of transformation and regeneration were as follows： explants pre-culture for 2 d, co-culture for 3 d, bacteria solution OD600 for 0.3 and infection time for 5 min. [ Conclusion] The results laid a solid foundation for extraction, isolation and purification of protein in transgenic plant seeds.展开更多
[Objective] The aim was to isolate the CBL-interacting protein kinases(CIPK)from maize(Zea mays L.)and construct the fusion gene expression vector which consisted the ZmCIPK8 and GFP.[Method] The ZmCIPK8 cDNA was succ...[Objective] The aim was to isolate the CBL-interacting protein kinases(CIPK)from maize(Zea mays L.)and construct the fusion gene expression vector which consisted the ZmCIPK8 and GFP.[Method] The ZmCIPK8 cDNA was successfully cloned by using RT-PCR method.And then,it was connected to the pBlueScript SK(pSK)plasmid,which contained the GFP gene.So that the fusion gene vector pSK-CIPK-GFP was obtained.Then,the fusion gene was connected into the efficient plant expression vector PBI121 to construct the fusion gene expression vector PBI-CIPK-GFP.At last,the recombined expression vector was transformed to Agrobacterium tumefaciems LBA4404 to produce the engineering strain LBA4404-PBI-CIPK-GFP.[Result] The fusion gene expression vector which consisted of GFP and ZmCIPK8 gene and engineering strain LBA4404-PBI-CIPK-GFP were successfully constructed.[Conclusion] The results lays a foundation for further study of subcellular localization of ZmCIPK8,which can help to clarify the molecular mechanism of regulation serious stresses,and also provides an important basis for the research on resistance stress engineering of maize.展开更多
A pair of primers was designed according to the reported conserved sequence of the defective in anther dehiscencel （DAD1） gene ofArabidopsis thaliana and Brassica rapa. A 558 bp long fragment was amplified from geno...A pair of primers was designed according to the reported conserved sequence of the defective in anther dehiscencel （DAD1） gene ofArabidopsis thaliana and Brassica rapa. A 558 bp long fragment was amplified from genomic DNA of Chinese kale, showing more than 88% identity with the known DAD1 nucleotide sequence and no intron. The reverse of the amplified fragment was ligated to the downstream of the CaMV35S promoter in the plant expression vector pBIl21. Antisense expression vector pBII21-DAD1F was constructed with DAD1 fragment of Chinese kale, and was transferred into Agrobacterium tumefaciens, which will be used in the transformation to create male sterile materials of Chinese kale.展开更多
基金Supported by National Natural Science Foundation of China(31070451)Qianjiang Talent Project of Zhejiang Province(2009R10016)Zhejiang Provincial Natural Science Foundation of China(Y5110067)~~
文摘[Objective] The aim was to construct the fusion expression vector of polyphosphate kinase（PPK） and green fluorescent protein（GFP） genes.[Method] In this study,the primers were designed based on PPK gene sequence（L03719） of E.coli DH5α in Genbank.Genomic DNA of E.coli DH 5α was extracted as template for the amplification of PPK gene by PCR method.By using In-Fusion@ HD Cloning Kit,the PPK gene was directionally cloned into NcoI site of the pCAMBIA1302 vector.[Result] Sequencing results showed that the 2.0 kb long fragment of PPK gene was inserted into the plant-based expression vector pCAMBIA1302 in front of GFP gene.[Conclusion] The fusion expression vector of PPK and GFP genes were successfully constructed.
基金Supported by Bioreactor Important Special Item of 863-Program inthe "Eleventh Five-Year" Plan (No. 2007AA100503)Science and Technology Development Key Plan of Jilin Province( No.20070922)+1 种基金Cultivation Fund of Scientific and Technical Innovation Project Major Program of Higher Education Institutions ( No.70S018)Science and Technology Plan of Changchun City (No.06GG150)~~
文摘[ Objective] The study is to generate pharmaceutical protein via plant transgenic technique. [Methed] Using the cotyledons with petiole as transformation receptor, the fusion gene of rapeseed oil-body gene and bFGF was introduced into the rapeseed （ Brassica campestris L. ） by Agrobacterium tumefaciens-mediated transformation; meanwhile regeneration conditions of rapeseed were also optimized, and the regenerated resistant plantlets were detected by PCR and Southern blot. [ Result] This fusion gene had been integrated into rapeseed genome successfully, and the optimized conditions of transformation and regeneration were as follows： explants pre-culture for 2 d, co-culture for 3 d, bacteria solution OD600 for 0.3 and infection time for 5 min. [ Conclusion] The results laid a solid foundation for extraction, isolation and purification of protein in transgenic plant seeds.
文摘[Objective] The aim was to isolate the CBL-interacting protein kinases(CIPK)from maize(Zea mays L.)and construct the fusion gene expression vector which consisted the ZmCIPK8 and GFP.[Method] The ZmCIPK8 cDNA was successfully cloned by using RT-PCR method.And then,it was connected to the pBlueScript SK(pSK)plasmid,which contained the GFP gene.So that the fusion gene vector pSK-CIPK-GFP was obtained.Then,the fusion gene was connected into the efficient plant expression vector PBI121 to construct the fusion gene expression vector PBI-CIPK-GFP.At last,the recombined expression vector was transformed to Agrobacterium tumefaciems LBA4404 to produce the engineering strain LBA4404-PBI-CIPK-GFP.[Result] The fusion gene expression vector which consisted of GFP and ZmCIPK8 gene and engineering strain LBA4404-PBI-CIPK-GFP were successfully constructed.[Conclusion] The results lays a foundation for further study of subcellular localization of ZmCIPK8,which can help to clarify the molecular mechanism of regulation serious stresses,and also provides an important basis for the research on resistance stress engineering of maize.
文摘A pair of primers was designed according to the reported conserved sequence of the defective in anther dehiscencel （DAD1） gene ofArabidopsis thaliana and Brassica rapa. A 558 bp long fragment was amplified from genomic DNA of Chinese kale, showing more than 88% identity with the known DAD1 nucleotide sequence and no intron. The reverse of the amplified fragment was ligated to the downstream of the CaMV35S promoter in the plant expression vector pBIl21. Antisense expression vector pBII21-DAD1F was constructed with DAD1 fragment of Chinese kale, and was transferred into Agrobacterium tumefaciens, which will be used in the transformation to create male sterile materials of Chinese kale.
