Salt stress is one of the major factors affecting plant growth and yield in soybean under saline soil condition. Despite many studies on salinity tolerance of soybean during the past few decades, the detailed signalin...Salt stress is one of the major factors affecting plant growth and yield in soybean under saline soil condition. Despite many studies on salinity tolerance of soybean during the past few decades, the detailed signaling pathways and the signaling molecules for salinity tolerance regulation have not been clarified. In this study, a proteomic technology based on two-dimensional gel electrophoresis(2-DE) and mass spectrometry(MS) were used to identify proteins responsible for salinity tolerance in soybean plant. Real-time quantitative PCR(qRT-PCR) and Western blotting(WB) were used to verify the results of 2-DE/MS. Based on the results of 2-DE and MS, we selected glucosyltransferase(GsGT4), 4-coumarate, coenzyme A ligase(Gs4 CL1), mitogen-activated protein kinase 4(GsMAPK4), dehydration responsive element binding protein(GsDREB1), and soybean cold-regulated gene(GsSRC1) in the salinity tolerant soybean variety, and GsMAPK4 for subsequent research. We transformed soybean plants with mitogen-activated-protein kinase 4(GsMAPK4) and screened the resulting transgenics soybean plants using PCR and WB, which confirmed the expression of GsMAPK4 in transgenic soybean. GsMAPK4-overexpressed transgenic plants showed significantly increased tolerance to salt stress, suggesting that GsMAPK4 played a pivotal role in salinity tolerance. Our research will provide new insights for better understanding the salinity tolerance regulation at molecular level.展开更多
Methionine and lysine are restrictive essential amino acids of livestock, they are also the most attentive indexes in the feed production to carry out the quality control and quality evaluation. Their contents in feed...Methionine and lysine are restrictive essential amino acids of livestock, they are also the most attentive indexes in the feed production to carry out the quality control and quality evaluation. Their contents in feed directly affect livestock protein synthesis. Bacillus natto has excellent probiotic properties. In this experiment, we used the genetic engineering method, fusion PCR technique, to connect methionine-rich gene （zein） from maize endosperm protein with lysine-rich gene （Cflr） from the pepper anther, then the fusion gene was inserted into the expression vector pHT43, and the recombinant plasmid pHT43/zein-Cflr was constructed. The recombinant plasmid was transferred into Bacillus natto, and induced by IPTG for the expression of the fusion gene. We found an apparent band at 40 ku site for the recombinant strain by SDS-PAGE. The contents of methionine and lysine were individually detected with HPLC, the quantities of methionine and lysine in the recombinant strain increased by 18.37% and 24.68% than the wild one, respectively. We also verified the stability of the recombinant bacterium during passaging, and found the stability was 100%. This study provided research-basis for the application of the recombined Bacillus natto as feed additive.展开更多
基金supported by the Science and Technology Research Project of Department of Education of Heilongjiang Province, China (12541047)
文摘Salt stress is one of the major factors affecting plant growth and yield in soybean under saline soil condition. Despite many studies on salinity tolerance of soybean during the past few decades, the detailed signaling pathways and the signaling molecules for salinity tolerance regulation have not been clarified. In this study, a proteomic technology based on two-dimensional gel electrophoresis(2-DE) and mass spectrometry(MS) were used to identify proteins responsible for salinity tolerance in soybean plant. Real-time quantitative PCR(qRT-PCR) and Western blotting(WB) were used to verify the results of 2-DE/MS. Based on the results of 2-DE and MS, we selected glucosyltransferase(GsGT4), 4-coumarate, coenzyme A ligase(Gs4 CL1), mitogen-activated protein kinase 4(GsMAPK4), dehydration responsive element binding protein(GsDREB1), and soybean cold-regulated gene(GsSRC1) in the salinity tolerant soybean variety, and GsMAPK4 for subsequent research. We transformed soybean plants with mitogen-activated-protein kinase 4(GsMAPK4) and screened the resulting transgenics soybean plants using PCR and WB, which confirmed the expression of GsMAPK4 in transgenic soybean. GsMAPK4-overexpressed transgenic plants showed significantly increased tolerance to salt stress, suggesting that GsMAPK4 played a pivotal role in salinity tolerance. Our research will provide new insights for better understanding the salinity tolerance regulation at molecular level.
基金Supported by the Funding of High Technology Project of Ministry of Science and Technology of China(863 Project,2013AA102504-03)
文摘Methionine and lysine are restrictive essential amino acids of livestock, they are also the most attentive indexes in the feed production to carry out the quality control and quality evaluation. Their contents in feed directly affect livestock protein synthesis. Bacillus natto has excellent probiotic properties. In this experiment, we used the genetic engineering method, fusion PCR technique, to connect methionine-rich gene （zein） from maize endosperm protein with lysine-rich gene （Cflr） from the pepper anther, then the fusion gene was inserted into the expression vector pHT43, and the recombinant plasmid pHT43/zein-Cflr was constructed. The recombinant plasmid was transferred into Bacillus natto, and induced by IPTG for the expression of the fusion gene. We found an apparent band at 40 ku site for the recombinant strain by SDS-PAGE. The contents of methionine and lysine were individually detected with HPLC, the quantities of methionine and lysine in the recombinant strain increased by 18.37% and 24.68% than the wild one, respectively. We also verified the stability of the recombinant bacterium during passaging, and found the stability was 100%. This study provided research-basis for the application of the recombined Bacillus natto as feed additive.
