supported by grants from the National Natural Science Foundation of China （30671178）;the Shanxi Province Science Foundation for Youths, China （2014021029-2）
Our previous studies have revealed that the Th CAP gene plays a vital role in transgenic Populus(P.davidiana 9 P.bolleana) in response to cold stress.However,the regulatory mechanism of Th CAP gene expression has been...Our previous studies have revealed that the Th CAP gene plays a vital role in transgenic Populus(P.davidiana 9 P.bolleana) in response to cold stress.However,the regulatory mechanism of Th CAP gene expression has been unclear.In this study,the 50 flanking region of the Th CAP promoter(PTh CAP) was cloned using a genomewalking method.By analyzing cis-acting regulatory elements of PTh CAP,a DRE motif and MYC and MYB elements were found to be located in the promoter.To identify the regulatory elements that control the expression of the Th CAP gene promoter,a series of deletion derivatives ofPTh CAP,P1–P5,from the translation start code(-1538,-1190,-900,-718 and-375 bp),were fused to the GUS reporter gene,and then each deletion was stably introduced into Arabidopsis thaliana plants.Deletion analysis of the promoter suggested that only the P2 fragment had strong GUS expression in leaves and roots of A.thaliana exposed to low temperature stress.These results suggest that this290-bp region(-1190 to-900 bp),as an important part in PTh CAP,was associated with cold tolerance of A.thaliana.Our results provide evidence for the regulatory mechanism of Th CAP gene involved in the response to cold stress,and that the gene is promising candidate gene for genetic improvement of crops.展开更多
A rapid, efficient and high-performance transformation protocol employing Agrobacterium rhizogenes was developed for the common bean Phaseolus vulgaris. In this study, we examined competencies of various protocols to ...A rapid, efficient and high-performance transformation protocol employing Agrobacterium rhizogenes was developed for the common bean Phaseolus vulgaris. In this study, we examined competencies of various protocols to induce and explants that respond to hairy root transformation in bean plants. Utilizing young seedlings with severed radicles/hypocotyls, we developed a highly efficient procedure for achieving hairy root transformation frequencies as high as 100% as visualized by GUS reporter gene expression system. Transgenic hairy roots in these young composite plants were susceptible to nodulation by rhizobia, and form an excellent system for high throughput genomic analysis to study root biology and endosymbiosis in common bean.展开更多
A promoter of the PNZIP （Pharbitis nil leucine zipper） gene （1.459 kb） was cloned from Pharbitis nil and fused to the GUS（^-glucuronidase） and Bacillus thuringiensis endotoxin （Cry9C） genes. Several transgenic...A promoter of the PNZIP （Pharbitis nil leucine zipper） gene （1.459 kb） was cloned from Pharbitis nil and fused to the GUS（^-glucuronidase） and Bacillus thuringiensis endotoxin （Cry9C） genes. Several transgenic PNZIP：：GUS and PNZIP：：Cry9C cotton lines were developed by Agrobacterium-mediated transformation. Strong GUS staining was detected in the green tissues of the transgenic PNZIP：：GUS cotton plants. In contrast, GUS staining in the reproductive structures such as petals, anther, and immature seeds of PNZIP：：GUS cotton was very faint. Two transgenic PNZIP：：Cry9C lines and one trans- genic cauliflower mosaic virus （CaMV） 35S：：Cry9C line were selected for enzyme-linked immunosorbent assay （ELISA） and insect bioassays. Expression of the Cry9C protein in the 35S：：Cry9C line maintained a high level in most tissues ranging from 24.6 to 45.5 ~tg g-I fresh weight. In green tissues such as the leaves, boll rinds, and bracts of the PNZIP：：Cry9C line, the Cry9C protein accumulated up to 50.2, 39.7, and 48.3 jag g-a fresh weight respectively. In contrast, seeds of the PNZIP：：Cry9C line （PZ1.3） accumulated only 0.26 ~ag g-~ fresh weight of the Cry9C protein, which was 100 times lower than that recorded for the seeds of the CaMV 35S：：Cry9C line. The insect bioassay showed that the transgenic PNZIP：：Cry9C cotton plant exhibited strong resistance to both the cotton bollworm and the pink bollworm. The PNZIP promoter could effectively drive Bt toxin ex- pression in green tissues of cotton and lower accumulated levels of the Bt protein in seeds. These features should allay public concerns about the safety of transgenic foods. We propose the future utility of PNZIP as an economical, environmentally friendly promoter in cotton biotechnology.展开更多
In this study, an efficient plant regeneration protocol in vitro and transformation by Agrobacterium-mediated method of Camellia sinensis was achieved, which would lay the foundation for genetic improvement of tea pla...In this study, an efficient plant regeneration protocol in vitro and transformation by Agrobacterium-mediated method of Camellia sinensis was achieved, which would lay the foundation for genetic improvement of tea plant by genetic engineering technology. The cotyledon callus of C.sinensis were used as the receptors for transformation by Agrobacterium tumefaciens EHA105 containing PS1aG-3. Some factors which affected the result of Agrobacterium-mediated transformation of C. sinensis were studied on the basis of GUS transient expression system. The optimum system of Agrobacterium-mediated transformation was that the cotyledon callus were pre-cultured for 3 d, and then infected by EHA105 for 15 min followed by 3 d co-culture in the dark on the YEB medium containing 150 μmol·L^(-1) acetosyringone(AS). The transient expression rate of GUS gene was 62.6%. After being delayed selective culture for 3 d, infected callus were transferred into the differentiation medium and the root induction medium both of which were supplemented with 100 mg·L^(-1) spectinomycin, and then resistant seedlings of C. sinensis were obtained. The conversion rate was 3.6%.展开更多
基金supported by grants from the National Natural Science Foundation of China (30671178)the Shanxi Province Science Foundation for Youths, China (2014021029-2)
文摘supported by grants from the National Natural Science Foundation of China （30671178）;the Shanxi Province Science Foundation for Youths, China （2014021029-2）
基金supported by the State Key Laboratory of Tree Genetics and Breeding(Northeast Forestry University)(201102)Natural Science Foundation of Heilongjiang Province(QC2012C057)+2 种基金China Postdoctoral Science Foundation(2013M531005)Foundation for the Postdoctoral of Heilongjiang Province(LBH-Z12007)National Natural Science Foundation of China(31200510)
文摘Our previous studies have revealed that the Th CAP gene plays a vital role in transgenic Populus(P.davidiana 9 P.bolleana) in response to cold stress.However,the regulatory mechanism of Th CAP gene expression has been unclear.In this study,the 50 flanking region of the Th CAP promoter(PTh CAP) was cloned using a genomewalking method.By analyzing cis-acting regulatory elements of PTh CAP,a DRE motif and MYC and MYB elements were found to be located in the promoter.To identify the regulatory elements that control the expression of the Th CAP gene promoter,a series of deletion derivatives ofPTh CAP,P1–P5,from the translation start code(-1538,-1190,-900,-718 and-375 bp),were fused to the GUS reporter gene,and then each deletion was stably introduced into Arabidopsis thaliana plants.Deletion analysis of the promoter suggested that only the P2 fragment had strong GUS expression in leaves and roots of A.thaliana exposed to low temperature stress.These results suggest that this290-bp region(-1190 to-900 bp),as an important part in PTh CAP,was associated with cold tolerance of A.thaliana.Our results provide evidence for the regulatory mechanism of Th CAP gene involved in the response to cold stress,and that the gene is promising candidate gene for genetic improvement of crops.
基金Centro de Ciencias de Genomicas,Universidad Autonoma de Mexico,Cuernavaca,Morelos,Mexico for financial and lab support
文摘A rapid, efficient and high-performance transformation protocol employing Agrobacterium rhizogenes was developed for the common bean Phaseolus vulgaris. In this study, we examined competencies of various protocols to induce and explants that respond to hairy root transformation in bean plants. Utilizing young seedlings with severed radicles/hypocotyls, we developed a highly efficient procedure for achieving hairy root transformation frequencies as high as 100% as visualized by GUS reporter gene expression system. Transgenic hairy roots in these young composite plants were susceptible to nodulation by rhizobia, and form an excellent system for high throughput genomic analysis to study root biology and endosymbiosis in common bean.
基金the National Natural Science Foundation of China (31171592, 31371673)Fundamental Research Funds for the Central Universities (2013PY064)
文摘A promoter of the PNZIP （Pharbitis nil leucine zipper） gene （1.459 kb） was cloned from Pharbitis nil and fused to the GUS（^-glucuronidase） and Bacillus thuringiensis endotoxin （Cry9C） genes. Several transgenic PNZIP：：GUS and PNZIP：：Cry9C cotton lines were developed by Agrobacterium-mediated transformation. Strong GUS staining was detected in the green tissues of the transgenic PNZIP：：GUS cotton plants. In contrast, GUS staining in the reproductive structures such as petals, anther, and immature seeds of PNZIP：：GUS cotton was very faint. Two transgenic PNZIP：：Cry9C lines and one trans- genic cauliflower mosaic virus （CaMV） 35S：：Cry9C line were selected for enzyme-linked immunosorbent assay （ELISA） and insect bioassays. Expression of the Cry9C protein in the 35S：：Cry9C line maintained a high level in most tissues ranging from 24.6 to 45.5 ~tg g-I fresh weight. In green tissues such as the leaves, boll rinds, and bracts of the PNZIP：：Cry9C line, the Cry9C protein accumulated up to 50.2, 39.7, and 48.3 jag g-a fresh weight respectively. In contrast, seeds of the PNZIP：：Cry9C line （PZ1.3） accumulated only 0.26 ~ag g-~ fresh weight of the Cry9C protein, which was 100 times lower than that recorded for the seeds of the CaMV 35S：：Cry9C line. The insect bioassay showed that the transgenic PNZIP：：Cry9C cotton plant exhibited strong resistance to both the cotton bollworm and the pink bollworm. The PNZIP promoter could effectively drive Bt toxin ex- pression in green tissues of cotton and lower accumulated levels of the Bt protein in seeds. These features should allay public concerns about the safety of transgenic foods. We propose the future utility of PNZIP as an economical, environmentally friendly promoter in cotton biotechnology.
基金supported financially by the China Earmarked Fund for Modern Agro-industry Technology Research System (CARS-23)Jiangsu Provincial Agricultural Project (SXGC[2015]018)+3 种基金Fudiyingcai Talent Project of Jurong (2014)the Priority Academic Program Development of Jiangsu Higher Education InstitutionsJiangsu Provincial Project of Research and Practice on the Teaching Reform of Postgraduate Education (JGLX15_111)the Teaching Research Project of College of Horticulture of Nanjing Agricultural University
文摘In this study, an efficient plant regeneration protocol in vitro and transformation by Agrobacterium-mediated method of Camellia sinensis was achieved, which would lay the foundation for genetic improvement of tea plant by genetic engineering technology. The cotyledon callus of C.sinensis were used as the receptors for transformation by Agrobacterium tumefaciens EHA105 containing PS1aG-3. Some factors which affected the result of Agrobacterium-mediated transformation of C. sinensis were studied on the basis of GUS transient expression system. The optimum system of Agrobacterium-mediated transformation was that the cotyledon callus were pre-cultured for 3 d, and then infected by EHA105 for 15 min followed by 3 d co-culture in the dark on the YEB medium containing 150 μmol·L^(-1) acetosyringone(AS). The transient expression rate of GUS gene was 62.6%. After being delayed selective culture for 3 d, infected callus were transferred into the differentiation medium and the root induction medium both of which were supplemented with 100 mg·L^(-1) spectinomycin, and then resistant seedlings of C. sinensis were obtained. The conversion rate was 3.6%.
