The results of the investigation on transgenic rice with maize C4-specific phosphoenolpyruvate carboxylase (pepc) gene showed that the transgenic rice plants with the maize pepc gene expressed at high level and the ma...The results of the investigation on transgenic rice with maize C4-specific phosphoenolpyruvate carboxylase (pepc) gene showed that the transgenic rice plants with the maize pepc gene expressed at high level and the maize PEPC expression was inherited in the progenies in a Mendelian manner. The transgenic plants had PEPC activity of more than 10-fold higher than untransformed plants. As compared with untransformed plants, the panicle per plant, spikelet per panicle, 1000-grain weight and grain-weight per plant for transgenic plants increased by 14.9 % , 5.7%, 1.3 % and 13.9 %, respectively. By crossing the maize pepc gene was incorporated into the parents of hybrid rice, which were the photo-sensitive genie male sterile (PGMS) lines of two-line hybrid rice such as Peiai64s, 7001s, 2302s, 2304s and 2306s-1, and the BT type of cytoplas-mic male sterile (CMS) line of three-line hybrid rice such as Shuangjiu A, and restorer lines 5129, Wanjing97 in the spring of 1998. The following progresses were made: (1) The inheritance of the high-level expression of the maize PEPC was stable in different genetic background of rice; (2) PEPC activity of hybrid was the mean of the two parents. Its saturated photosynthetic rate (Pn) rose to 50 % higher than that of the receptor parent. These results demonstrated that it is possible to increase the vigor of the rice plant by transgenic approach with maize pepc gene; (3) The activity of PEPC in leaf could be considered as the major physiological index because the correlation coefficient between PEPC activity and Pn was 0.6470* * ; (4) We have developed three rice lines with maize pepc gene; (5) The selection method of high photosynthetic efficiency rice has been established, which includes soaking seeds into solution of hygromycin phosphotransferase to germinate, tracing the pepc gene by PCR analysis, evaluating the performance of the rice plants in the field and examining PEPC activities and Pn of rice plants with maize pepc gene.展开更多
Since pepc gene encoding phosphoenolpyruvate carboxylase （PEPCase） has been cloned from Anabaena sp. PCC 7120 and other cyanobacteria, the effects of pepc gene expression on photosynthesis have not been reported yet...Since pepc gene encoding phosphoenolpyruvate carboxylase （PEPCase） has been cloned from Anabaena sp. PCC 7120 and other cyanobacteria, the effects of pepc gene expression on photosynthesis have not been reported yet. In this study, we constructed mutants containing either upregu-lated （forward） or downregulated （reverse） pepc gene in Anabaena sp. PCC 7120. Results from real‐time quantitative polymerase chain reaction （RT‐qPCR）, Western blot and enzymatic analysis showed that PEPCase activity was signifi-cantly reduced in the reverse mutant compared with the wild type, and that of the forward mutant was obviously increased. Interestingly, the net photosynthesis in both the reverse mutant and the forward mutant were higher than that of the wild type, but dark respiration was decreased only in the reverse mutant. The absorbance changes of P700 upon saturation pulse showed the photosystem I （PSI） activity was inhibited, as reflected by Y（I）, and Y（NA） was elevated, and＆amp;nbsp;dark reduction of P700t was stimulated, indicating enhanced cyclic electron flow （CEF） around PSI in the reverse mutant. Additional y, the reverse mutant photosynthesis was higher than that of the wild type in low temperature, low and high pH, and high salinity, and this implies increased tolerance in the reverse mutant through downregulated pepc gene.展开更多
Maize intact C4-pepc gene was amplified through LA-PCR and successfully sub-cloned into modified vector pGreen0029 to form a stable expression construct named as pBAC214 (12 kb), which contains CaMV 35S promoter drive...Maize intact C4-pepc gene was amplified through LA-PCR and successfully sub-cloned into modified vector pGreen0029 to form a stable expression construct named as pBAC214 (12 kb), which contains CaMV 35S promoter driven bar gene as selection marker. Comparing the cloned DNA sequences (6.7 kb) with published maize C4-pepc gene (GenBank accession E17154) sequences, the identity of DNA sequence alignment is 98.96%. There are only 49 differences between these two intact DNA sequences, of which 13 occur in the region of promoter, 18 in introns, and 18 in exons. The homology of mRNA sequence alignment is 99.38%, and the putative amino acids sequence identity is 99.38%. There are only 15 differences between these two mRNA, and these differences bring 4 sites mutant on the putative amino acids of PEPC protein. Through biolistic bombardment of PDS1000/He system, expression vector pBAC214 has been transformed into winter wheat. Southern blotting results show that the intact C4-pepc gene has been integrated into genome of winter wheat. SDS-PAGE analysis of leaf soluble protein in transgenic wheat showed that the intact C4-pepc gene was well transcribed, spliced and trans-lated as in maize. The enzyme activity of leaf PEPC in trans-genic wheat has been detected. The activities of leaf PEPC increased over 35 times in some transgenic plants. The data of photosynthesis rate and transpiration rate of trans-genic wheat flag leaves showed that the C4-pepc gene can increase the photosynthesis rate and transpiration rate of transgenic wheat.展开更多
文摘The results of the investigation on transgenic rice with maize C4-specific phosphoenolpyruvate carboxylase (pepc) gene showed that the transgenic rice plants with the maize pepc gene expressed at high level and the maize PEPC expression was inherited in the progenies in a Mendelian manner. The transgenic plants had PEPC activity of more than 10-fold higher than untransformed plants. As compared with untransformed plants, the panicle per plant, spikelet per panicle, 1000-grain weight and grain-weight per plant for transgenic plants increased by 14.9 % , 5.7%, 1.3 % and 13.9 %, respectively. By crossing the maize pepc gene was incorporated into the parents of hybrid rice, which were the photo-sensitive genie male sterile (PGMS) lines of two-line hybrid rice such as Peiai64s, 7001s, 2302s, 2304s and 2306s-1, and the BT type of cytoplas-mic male sterile (CMS) line of three-line hybrid rice such as Shuangjiu A, and restorer lines 5129, Wanjing97 in the spring of 1998. The following progresses were made: (1) The inheritance of the high-level expression of the maize PEPC was stable in different genetic background of rice; (2) PEPC activity of hybrid was the mean of the two parents. Its saturated photosynthetic rate (Pn) rose to 50 % higher than that of the receptor parent. These results demonstrated that it is possible to increase the vigor of the rice plant by transgenic approach with maize pepc gene; (3) The activity of PEPC in leaf could be considered as the major physiological index because the correlation coefficient between PEPC activity and Pn was 0.6470* * ; (4) We have developed three rice lines with maize pepc gene; (5) The selection method of high photosynthetic efficiency rice has been established, which includes soaking seeds into solution of hygromycin phosphotransferase to germinate, tracing the pepc gene by PCR analysis, evaluating the performance of the rice plants in the field and examining PEPC activities and Pn of rice plants with maize pepc gene.
基金supported by the National High-tech R&D Program (863 Program) (Nos.2009AA064401 and 2014AA093506)National Administration Project from Chinese Ministry of Environmental Protection (No.04)supported by Shanghai Universities First-class Disciplines Project of Fisheries
文摘Since pepc gene encoding phosphoenolpyruvate carboxylase （PEPCase） has been cloned from Anabaena sp. PCC 7120 and other cyanobacteria, the effects of pepc gene expression on photosynthesis have not been reported yet. In this study, we constructed mutants containing either upregu-lated （forward） or downregulated （reverse） pepc gene in Anabaena sp. PCC 7120. Results from real‐time quantitative polymerase chain reaction （RT‐qPCR）, Western blot and enzymatic analysis showed that PEPCase activity was signifi-cantly reduced in the reverse mutant compared with the wild type, and that of the forward mutant was obviously increased. Interestingly, the net photosynthesis in both the reverse mutant and the forward mutant were higher than that of the wild type, but dark respiration was decreased only in the reverse mutant. The absorbance changes of P700 upon saturation pulse showed the photosystem I （PSI） activity was inhibited, as reflected by Y（I）, and Y（NA） was elevated, and＆amp;nbsp;dark reduction of P700t was stimulated, indicating enhanced cyclic electron flow （CEF） around PSI in the reverse mutant. Additional y, the reverse mutant photosynthesis was higher than that of the wild type in low temperature, low and high pH, and high salinity, and this implies increased tolerance in the reverse mutant through downregulated pepc gene.
文摘Maize intact C4-pepc gene was amplified through LA-PCR and successfully sub-cloned into modified vector pGreen0029 to form a stable expression construct named as pBAC214 (12 kb), which contains CaMV 35S promoter driven bar gene as selection marker. Comparing the cloned DNA sequences (6.7 kb) with published maize C4-pepc gene (GenBank accession E17154) sequences, the identity of DNA sequence alignment is 98.96%. There are only 49 differences between these two intact DNA sequences, of which 13 occur in the region of promoter, 18 in introns, and 18 in exons. The homology of mRNA sequence alignment is 99.38%, and the putative amino acids sequence identity is 99.38%. There are only 15 differences between these two mRNA, and these differences bring 4 sites mutant on the putative amino acids of PEPC protein. Through biolistic bombardment of PDS1000/He system, expression vector pBAC214 has been transformed into winter wheat. Southern blotting results show that the intact C4-pepc gene has been integrated into genome of winter wheat. SDS-PAGE analysis of leaf soluble protein in transgenic wheat showed that the intact C4-pepc gene was well transcribed, spliced and trans-lated as in maize. The enzyme activity of leaf PEPC in trans-genic wheat has been detected. The activities of leaf PEPC increased over 35 times in some transgenic plants. The data of photosynthesis rate and transpiration rate of trans-genic wheat flag leaves showed that the C4-pepc gene can increase the photosynthesis rate and transpiration rate of transgenic wheat.
