Nitrogen is one of the most needed elements by coffee plants. Utilization of biological nitrogen fixation by non symbiotic bacteria offers alternative to reduce the N fertilizer usage. This study was focused to obtain...Nitrogen is one of the most needed elements by coffee plants. Utilization of biological nitrogen fixation by non symbiotic bacteria offers alternative to reduce the N fertilizer usage. This study was focused to obtain aerobic non symbiotic nitrogen-fixing bacteria from coffee rhizosphere. The application of those bacteria was expected to enhance coffee seedling growth. Sixty four aerobic nitrogen-fixing bacterial isolates were isolated from coffee plants rhizosphere from Jember, East Java using several nitrogen free medium, such as Ashby, malate acid, and fahreus agar. The nitrogen fixation ability of the isolates was determined by measuring their ability in pellicle formation on semi solid medium and ammonium excretion on growth medium. Ab Kws.l, Asm E6s.3.a, Asm Bsl.1, and Asm E6s were the isolates which showed the best performance on nitrogen fixation with excreted ammonium concentration ranged from 129.6 up to 239.8 pM/mg dry weight cell. Acetylene reduction assay was used to detect nitrogenase activity. Ab Kws.1 was the isolate which had the highest nitrogenase activity （7.4 mmol N2 fixed/gram dry weight cell/hour）. Inoculation of the four best isolates onto Robusta coffee seedling positively enhanced the seedling growth in this green house experiment. Based on the results of Becton Dickinson＇s （BD） PhoenixTM Automated Microbiology System biochemical tests, Asm Bls.l isolates has similarities with Achromobacter sp., Asm E6s.l and Asm E6s.3.a had similarities with Stenotrophomonas maltophilia, while Ab Kws. 1 had similarities with Leifsonia aquatica.展开更多
Peroxisome proliferator-activated receptors （PPARs） are ligand-activated transcription factors known to play a pivotal role in regulations of metabolism. In order to yield soluble ligand binding domain of PPARδ （P...Peroxisome proliferator-activated receptors （PPARs） are ligand-activated transcription factors known to play a pivotal role in regulations of metabolism. In order to yield soluble ligand binding domain of PPARδ （PPARδLBD） for screening ligands, the cDNA was amplified using total RNA from HepG2 cells by RT-PCR. Then the enzyme-digested product was inserted . downstream of the malE gene in the vectorpMAL-p2x, which encoded maltose-binding protein （MBP）, resulting in the expression of an MBP-PPARδLBD fusion protein. The recombinant plasmid was transformed into E. coli TBI that was cultured shakily at 30 ℃, 200 r/min and induced by 0.4 mmol/L IPTG for 6 h. The cells were harvested by centrifugation and broken by sonication. The expressed fusion protein was soluble and accounted for 0.31 of the total protein in the supernatant. Western blot analysis showed that the expressed MBP-PPARδLBD could bind to anti-MBP-antibody. The MBP-PPARδLBD fusion protein of 77 kDa and the PPARδLBD protein of 34 kDa were obtained by amylose-resin affinity chromatography without or with digestion of Factor Xa. They were both homogeneity, judged by SDS-PAGE. The recombinant MBP-PPARδLBD and PPARδLBD protein with high purity is obtained, which provides the necessary material for screening and researching PPARδ ligands.展开更多
文摘Nitrogen is one of the most needed elements by coffee plants. Utilization of biological nitrogen fixation by non symbiotic bacteria offers alternative to reduce the N fertilizer usage. This study was focused to obtain aerobic non symbiotic nitrogen-fixing bacteria from coffee rhizosphere. The application of those bacteria was expected to enhance coffee seedling growth. Sixty four aerobic nitrogen-fixing bacterial isolates were isolated from coffee plants rhizosphere from Jember, East Java using several nitrogen free medium, such as Ashby, malate acid, and fahreus agar. The nitrogen fixation ability of the isolates was determined by measuring their ability in pellicle formation on semi solid medium and ammonium excretion on growth medium. Ab Kws.l, Asm E6s.3.a, Asm Bsl.1, and Asm E6s were the isolates which showed the best performance on nitrogen fixation with excreted ammonium concentration ranged from 129.6 up to 239.8 pM/mg dry weight cell. Acetylene reduction assay was used to detect nitrogenase activity. Ab Kws.1 was the isolate which had the highest nitrogenase activity （7.4 mmol N2 fixed/gram dry weight cell/hour）. Inoculation of the four best isolates onto Robusta coffee seedling positively enhanced the seedling growth in this green house experiment. Based on the results of Becton Dickinson＇s （BD） PhoenixTM Automated Microbiology System biochemical tests, Asm Bls.l isolates has similarities with Achromobacter sp., Asm E6s.l and Asm E6s.3.a had similarities with Stenotrophomonas maltophilia, while Ab Kws. 1 had similarities with Leifsonia aquatica.
基金Supported by the National Natural Science Foundation of China (30572353)
文摘Peroxisome proliferator-activated receptors （PPARs） are ligand-activated transcription factors known to play a pivotal role in regulations of metabolism. In order to yield soluble ligand binding domain of PPARδ （PPARδLBD） for screening ligands, the cDNA was amplified using total RNA from HepG2 cells by RT-PCR. Then the enzyme-digested product was inserted . downstream of the malE gene in the vectorpMAL-p2x, which encoded maltose-binding protein （MBP）, resulting in the expression of an MBP-PPARδLBD fusion protein. The recombinant plasmid was transformed into E. coli TBI that was cultured shakily at 30 ℃, 200 r/min and induced by 0.4 mmol/L IPTG for 6 h. The cells were harvested by centrifugation and broken by sonication. The expressed fusion protein was soluble and accounted for 0.31 of the total protein in the supernatant. Western blot analysis showed that the expressed MBP-PPARδLBD could bind to anti-MBP-antibody. The MBP-PPARδLBD fusion protein of 77 kDa and the PPARδLBD protein of 34 kDa were obtained by amylose-resin affinity chromatography without or with digestion of Factor Xa. They were both homogeneity, judged by SDS-PAGE. The recombinant MBP-PPARδLBD and PPARδLBD protein with high purity is obtained, which provides the necessary material for screening and researching PPARδ ligands.
