[Objective] This study aimed to screen an Na＋/H＋ antiporter gene from the halophiles colonizing in the Dagong Ancient Brine Well in Zigong City, China, and then analyze the gene structure and properties of the prote...[Objective] This study aimed to screen an Na＋/H＋ antiporter gene from the halophiles colonizing in the Dagong Ancient Brine Well in Zigong City, China, and then analyze the gene structure and properties of the protein encoded by this gene. [Method] Metagenomic DNA libraries of halophiles from the Dagong Ancient Brine Well were used for screening genes with Na＋/H＋ antiporter activity in antiporter-defi- cient E. coil KNabc strain by functional complementation. Then the start codon, stop codon, ORF, -35 region, -10 region and SD sequence of Na~/H＋ antiporter gene, as well as the molecular weight, isoelectric point, hydrophobic region, transmembrane domain, phyletic evolution and salt resistance of protein encoded by the gene were investigated. [Result] A new Na＋/H＋ antiporter gene m-nha was obtained, which ,ren- dered the antiporter-negative mutant E. coil KNabc cells with both the resistance to Na＋ and the ability to grow under alkaline conditions. [Conclusion] The structure and amino acid sequence of M-Nha was different from the previously reported Na＋/H~ antiporters, and the m-nha gene disclosed from the Dagong Ancient Brine Well was identified as a novel Na＋/H＋ antiporter gene. This study was significant not only in helping us understand the salt tolerance of halophiles in ancient brine wells and develop and utilize the genes resource, but also in exploring new salt-tolerant genes.展开更多
Our previous study demonstrated that a chloroplast co-chaperonin 20(CPN20),one of the interaction partners of the magnesium-protoporphyrin IX chelatase H subunit(CHLH/ABAR),negatively regulates ABA signaling at the sa...Our previous study demonstrated that a chloroplast co-chaperonin 20(CPN20),one of the interaction partners of the magnesium-protoporphyrin IX chelatase H subunit(CHLH/ABAR),negatively regulates ABA signaling at the same node with ABAR but upstream of WRKY40 transcription repressor in Arabidopsis thaliana.In the present experiment,we showed that ABA directly inhibits the ABAR-CPN20 interaction,and also represses expression of CPN20,which depends on ABAR.CPN20 inhibits ABAR-WRKY40 interaction by competitively binding to ABAR.ABAR downregulates,but CPN20 upregulates,WRKY40 expression.The cpn20-1 mutation induces downregulation of WRKY40,and suppresses the upregulated level of WRKY40 due to the cch mutation in the ABAR gene.ABA-induced repressive effect of the WRKY40 gene is strengthened by downregulation of CPN20 but reduced by upregulation of CPN20.Together with our previously reported genetic data,we provide evidence that CPN20 functions through antagonizing the ABAR-WRKY40 coupled pathway,and ABA relieves this pathway of repression by inhibiting the ABAR-CPN20 interaction to activate ABAR-WRKY40 interaction.展开更多
基金Supported by Chunhui Plan of Ministry of Education(Z2010101)Open Fund of Food Biotechnology Key Laboratory of Sichuan Province(SZJJ2009-014)Scientific Research Foundation of Xihua University(000022)~~
文摘[Objective] This study aimed to screen an Na＋/H＋ antiporter gene from the halophiles colonizing in the Dagong Ancient Brine Well in Zigong City, China, and then analyze the gene structure and properties of the protein encoded by this gene. [Method] Metagenomic DNA libraries of halophiles from the Dagong Ancient Brine Well were used for screening genes with Na＋/H＋ antiporter activity in antiporter-defi- cient E. coil KNabc strain by functional complementation. Then the start codon, stop codon, ORF, -35 region, -10 region and SD sequence of Na~/H＋ antiporter gene, as well as the molecular weight, isoelectric point, hydrophobic region, transmembrane domain, phyletic evolution and salt resistance of protein encoded by the gene were investigated. [Result] A new Na＋/H＋ antiporter gene m-nha was obtained, which ,ren- dered the antiporter-negative mutant E. coil KNabc cells with both the resistance to Na＋ and the ability to grow under alkaline conditions. [Conclusion] The structure and amino acid sequence of M-Nha was different from the previously reported Na＋/H~ antiporters, and the m-nha gene disclosed from the Dagong Ancient Brine Well was identified as a novel Na＋/H＋ antiporter gene. This study was significant not only in helping us understand the salt tolerance of halophiles in ancient brine wells and develop and utilize the genes resource, but also in exploring new salt-tolerant genes.
基金supported by the National Key Basic Research Program of China(2012CB114302)National Natural Science Foundation of China(90817104 and 31170268)Ministry of Agriculture of China(2013ZX08009-003)
文摘Our previous study demonstrated that a chloroplast co-chaperonin 20(CPN20),one of the interaction partners of the magnesium-protoporphyrin IX chelatase H subunit(CHLH/ABAR),negatively regulates ABA signaling at the same node with ABAR but upstream of WRKY40 transcription repressor in Arabidopsis thaliana.In the present experiment,we showed that ABA directly inhibits the ABAR-CPN20 interaction,and also represses expression of CPN20,which depends on ABAR.CPN20 inhibits ABAR-WRKY40 interaction by competitively binding to ABAR.ABAR downregulates,but CPN20 upregulates,WRKY40 expression.The cpn20-1 mutation induces downregulation of WRKY40,and suppresses the upregulated level of WRKY40 due to the cch mutation in the ABAR gene.ABA-induced repressive effect of the WRKY40 gene is strengthened by downregulation of CPN20 but reduced by upregulation of CPN20.Together with our previously reported genetic data,we provide evidence that CPN20 functions through antagonizing the ABAR-WRKY40 coupled pathway,and ABA relieves this pathway of repression by inhibiting the ABAR-CPN20 interaction to activate ABAR-WRKY40 interaction.
