To study cold signaling, we screened for Arabidopsis mutants with altered cold-induced transcription of a firefly luciferase reporter gene driven by the CBF3 promoter （CBF3-LUC）. One mutant, chy1-10, displayed reduc...To study cold signaling, we screened for Arabidopsis mutants with altered cold-induced transcription of a firefly luciferase reporter gene driven by the CBF3 promoter （CBF3-LUC）. One mutant, chy1-10, displayed reduced cold-induction of CBF3-LUC luminescence. RNA gel blot analysis revealed that expression of endogenous CBFs also was reduced in the chyl mutant, chy1-10 mutant plants are more sensitive to freezing treatment than wild-type after cold acclimation. Both the wild-type and chyl mutant plants are sensitive to darkness-induced starvation at warm temperatures, although chyl plants are slightly more sensitive. This dark-sensitivity is suppressed by cold temperature in the wildtype but not in chyl. Constitutive CBF3 expression partially rescues the sensitivity of chyl-10 plants to dark treatment in the cold. The chyl mutant accumulates higher levels of reactive oxygen species, and application of hydrogen peroxide can reduce cold-induction of CBF3-LUC in wild-type. Map-based cloning of the gene defective in the mutant revealed a nonsense mutation in CHY1, which encodes a peroxisomal β-hydroxyisobutyryl （HIBYL）-CoA hydrolase needed for valine catabolism and fatty acid β-oxidation. Our results suggest a role for peroxisomal metabolism in cold stress signaling, and plant tolerance to cold stress and darkness-induced starvation.展开更多
We cloned and expressed bile salt hydrolase gene ofLactobacillus plantarum M1-UVS29 in Lactococcus lactis NZ9000 successfully. Gene-specific primers for amplification of L. plantarum bsh were designed by using sequenc...We cloned and expressed bile salt hydrolase gene ofLactobacillus plantarum M1-UVS29 in Lactococcus lactis NZ9000 successfully. Gene-specific primers for amplification of L. plantarum bsh were designed by using sequence which availabled from GenBank. The production of PCR amplicon was confirmed by sequencing and cloned into pMD18-T vector, and then recombined into expression vector pNZ8148 and yielding vector pNZ8148-BSH, pNZ8148-BSH was transferred into Lactococcus lactis NZ9000. Sequencing indicated that the cloned bsh fragment contained 995 nucleotides, and shared 99.3% sequence homology with bsh gene from L. plantarum MBUL10. Cloned bsh fragment was successfully transduced into NICE expression system and confirmed by PCR and restriction digest. Recombinant BSH protein was analyzed by SDS-PAGE. The molecular weight of BSH protein was approximately 37 ku. Activity of the expressed protein was 0.77 μmol· min^-1. The successfully expressed proteins by genetic engineering technology made the function of lactic acid bacteria be abundant and laid the foundation for further researches into cholesterol-lowering lactic acid bacterium food and probiotics.展开更多
文摘To study cold signaling, we screened for Arabidopsis mutants with altered cold-induced transcription of a firefly luciferase reporter gene driven by the CBF3 promoter （CBF3-LUC）. One mutant, chy1-10, displayed reduced cold-induction of CBF3-LUC luminescence. RNA gel blot analysis revealed that expression of endogenous CBFs also was reduced in the chyl mutant, chy1-10 mutant plants are more sensitive to freezing treatment than wild-type after cold acclimation. Both the wild-type and chyl mutant plants are sensitive to darkness-induced starvation at warm temperatures, although chyl plants are slightly more sensitive. This dark-sensitivity is suppressed by cold temperature in the wildtype but not in chyl. Constitutive CBF3 expression partially rescues the sensitivity of chyl-10 plants to dark treatment in the cold. The chyl mutant accumulates higher levels of reactive oxygen species, and application of hydrogen peroxide can reduce cold-induction of CBF3-LUC in wild-type. Map-based cloning of the gene defective in the mutant revealed a nonsense mutation in CHY1, which encodes a peroxisomal β-hydroxyisobutyryl （HIBYL）-CoA hydrolase needed for valine catabolism and fatty acid β-oxidation. Our results suggest a role for peroxisomal metabolism in cold stress signaling, and plant tolerance to cold stress and darkness-induced starvation.
基金Supported by the National Natural Science Fund Project(31171657)Heilongjiang Province Natural Fund Project(ZD201207)Heilongjiang Province Postdoctoral Special Funds(LBH-Q13133)
文摘We cloned and expressed bile salt hydrolase gene ofLactobacillus plantarum M1-UVS29 in Lactococcus lactis NZ9000 successfully. Gene-specific primers for amplification of L. plantarum bsh were designed by using sequence which availabled from GenBank. The production of PCR amplicon was confirmed by sequencing and cloned into pMD18-T vector, and then recombined into expression vector pNZ8148 and yielding vector pNZ8148-BSH, pNZ8148-BSH was transferred into Lactococcus lactis NZ9000. Sequencing indicated that the cloned bsh fragment contained 995 nucleotides, and shared 99.3% sequence homology with bsh gene from L. plantarum MBUL10. Cloned bsh fragment was successfully transduced into NICE expression system and confirmed by PCR and restriction digest. Recombinant BSH protein was analyzed by SDS-PAGE. The molecular weight of BSH protein was approximately 37 ku. Activity of the expressed protein was 0.77 μmol· min^-1. The successfully expressed proteins by genetic engineering technology made the function of lactic acid bacteria be abundant and laid the foundation for further researches into cholesterol-lowering lactic acid bacterium food and probiotics.
