Diaphanes is the fourth largest genus in Lampyridae, but no luciferase gene from this genus has been reported. In this paper, by PCR amplification of the genomic DNA, the luciferase gene of Diaphanes pectinealis, whic...Diaphanes is the fourth largest genus in Lampyridae, but no luciferase gene from this genus has been reported. In this paper, by PCR amplification of the genomic DNA, the luciferase gene of Diaphanes pectinealis, which is the first case from Diaphanes, was identified and sequenced. The luciferase gene from D. pectinealis spans 1958 base pairs （bp） from the start to the stop codon, including seven exons separated by six introns, and encoding a 547-residuelong polypeptide. Its deduced amino acid sequence showed high protein similarity to those of the Lampyrini tribe （93 - 94% ） and the Cratomorphini tribe （92%）, while low similarity was found with the North American firefly Photinus pyralis （83%） of the Photinini tribe within the same subfamily Lampyrinae. The phylogenetic analysis performed with the deduced amino acid sequences of the luciferase gene further confirms that D. pectinealis, Pyrocoelia, Lampyris, Cratomorphus, and Photinus belong to the same subfamily Lampyrinae, and Diaphanes is closely related to Pyrocoelia, Lampyris, and Cratomorphus. Furthemore, the phylogenetic analysis based on the nucleotide sequences of the luciferase gene indicates Diaphanes is a sister to Lampyris. The phylogenetic analyses are partly consistent with morphological （Branham ＆ Wenzel, 2003） and mitochondrial DNA analyses （Li et al, 2006）.展开更多
The cDNA encoding the luciferase from lantern mRNA of one diurnal firefly Pyrocoelia pygidialis Pic, 1926 has been cloned, sequenced and functionally expressed. The cDNA sequence of P pygidialis luciferase is 1647 bas...The cDNA encoding the luciferase from lantern mRNA of one diurnal firefly Pyrocoelia pygidialis Pic, 1926 has been cloned, sequenced and functionally expressed. The cDNA sequence of P pygidialis luciferase is 1647 base pairs in length, coding a protein of 548 amino acid residues. Sequence analysis of the deduced amino acid sequence showed that this luciferase had 97.8% resemblance to luciferases from the fireflies Lampyris noctiluca, Lampyris turkestanicus and Nyctophila cf. caucasica. Phylogenetic analysis using deduced amino acid sequence showed that P pygidialis located at the base of Lampyris＋Nyctophila clade with robust support （BP=97%）; but did not show a monophyletic relationship with its congeneric species P pectoralis, P tufa and P miyako, all three are strong luminous and nocturnal species. The expression worked in recombinant Escherichia coli. Expression product had a 70kDa band and emitted yellow-green luminescence in the presence of luciferin. Five loops in the P pygidialis luciferase, L1 （NI98-G208）, L2 （T240-G247）, L3 （G317-K322）, L4 （L343-I350） and L5 （G522-D532）, were found from the structure modeling analysis in the cleft, where it was considered the active site for the substrate compound entering and binding. Different amino acid residues between the luciferases of P. pygidialis and the three other known strong luminous species can not explain the situation of weak or strong luminescence. Future study of these loops, residues or crystal structure analysis may be helpful in understanding the real differences between the luciferases between diurnal and nocturnal species.展开更多
Background:To study the effects of the main diterpene esters in Euphorbia factor L_(1),L_(2),L_(3),L_(7a),L_(7b)and L_(8)on the transcriptional activity and protein expression of liver X receptor(LXR).Methods:The effe...Background:To study the effects of the main diterpene esters in Euphorbia factor L_(1),L_(2),L_(3),L_(7a),L_(7b)and L_(8)on the transcriptional activity and protein expression of liver X receptor(LXR).Methods:The effect of the main diterpene ester components in Semen Euphorbiae on the viability of HEK293 cells were studied by MTT assay.The LXR-Luc plasmid vector was transfected into HEK293 cells and treated with Euphorbia factor L_(1),L_(2),L_(3),L_(7a),L_(7b)and L_(8)for 24 h.The effect of the main diterpene ester components of Semen Euphorbiae on LXR-Luc luciferase activity was investigated by dual luciferase reporter gene system,and the expression of LXRαprotein was detected by Western Blot.Results:Euphorbia factor L_(1),L_(2),L_(3),L_(7a),L_(7b)and L_(8)could significantly reduce the relative luciferase activity(RLU)of LXRα,and the expression level of LXRαprotein was significantly down-regulated.Conclusion:Euphorbia factor L_(1),L_(2),L_(3),L_(7a),L_(7b)and L_(8)can inhibit the expression of LXR protein level,which may be achieved by inhibiting the transcriptional activity of LXR.展开更多
文摘Diaphanes is the fourth largest genus in Lampyridae, but no luciferase gene from this genus has been reported. In this paper, by PCR amplification of the genomic DNA, the luciferase gene of Diaphanes pectinealis, which is the first case from Diaphanes, was identified and sequenced. The luciferase gene from D. pectinealis spans 1958 base pairs （bp） from the start to the stop codon, including seven exons separated by six introns, and encoding a 547-residuelong polypeptide. Its deduced amino acid sequence showed high protein similarity to those of the Lampyrini tribe （93 - 94% ） and the Cratomorphini tribe （92%）, while low similarity was found with the North American firefly Photinus pyralis （83%） of the Photinini tribe within the same subfamily Lampyrinae. The phylogenetic analysis performed with the deduced amino acid sequences of the luciferase gene further confirms that D. pectinealis, Pyrocoelia, Lampyris, Cratomorphus, and Photinus belong to the same subfamily Lampyrinae, and Diaphanes is closely related to Pyrocoelia, Lampyris, and Cratomorphus. Furthemore, the phylogenetic analysis based on the nucleotide sequences of the luciferase gene indicates Diaphanes is a sister to Lampyris. The phylogenetic analyses are partly consistent with morphological （Branham ＆ Wenzel, 2003） and mitochondrial DNA analyses （Li et al, 2006）.
基金the Natural Foundation of Sciences of Yunnan Province (2006C0046Q)Partly by the Chinese Academy of Sciences (O706551141)
文摘The cDNA encoding the luciferase from lantern mRNA of one diurnal firefly Pyrocoelia pygidialis Pic, 1926 has been cloned, sequenced and functionally expressed. The cDNA sequence of P pygidialis luciferase is 1647 base pairs in length, coding a protein of 548 amino acid residues. Sequence analysis of the deduced amino acid sequence showed that this luciferase had 97.8% resemblance to luciferases from the fireflies Lampyris noctiluca, Lampyris turkestanicus and Nyctophila cf. caucasica. Phylogenetic analysis using deduced amino acid sequence showed that P pygidialis located at the base of Lampyris＋Nyctophila clade with robust support （BP=97%）; but did not show a monophyletic relationship with its congeneric species P pectoralis, P tufa and P miyako, all three are strong luminous and nocturnal species. The expression worked in recombinant Escherichia coli. Expression product had a 70kDa band and emitted yellow-green luminescence in the presence of luciferin. Five loops in the P pygidialis luciferase, L1 （NI98-G208）, L2 （T240-G247）, L3 （G317-K322）, L4 （L343-I350） and L5 （G522-D532）, were found from the structure modeling analysis in the cleft, where it was considered the active site for the substrate compound entering and binding. Different amino acid residues between the luciferases of P. pygidialis and the three other known strong luminous species can not explain the situation of weak or strong luminescence. Future study of these loops, residues or crystal structure analysis may be helpful in understanding the real differences between the luciferases between diurnal and nocturnal species.
基金supported by National Natural Science Foundation of China(Grant No.82074021).
文摘Background:To study the effects of the main diterpene esters in Euphorbia factor L_(1),L_(2),L_(3),L_(7a),L_(7b)and L_(8)on the transcriptional activity and protein expression of liver X receptor(LXR).Methods:The effect of the main diterpene ester components in Semen Euphorbiae on the viability of HEK293 cells were studied by MTT assay.The LXR-Luc plasmid vector was transfected into HEK293 cells and treated with Euphorbia factor L_(1),L_(2),L_(3),L_(7a),L_(7b)and L_(8)for 24 h.The effect of the main diterpene ester components of Semen Euphorbiae on LXR-Luc luciferase activity was investigated by dual luciferase reporter gene system,and the expression of LXRαprotein was detected by Western Blot.Results:Euphorbia factor L_(1),L_(2),L_(3),L_(7a),L_(7b)and L_(8)could significantly reduce the relative luciferase activity(RLU)of LXRα,and the expression level of LXRαprotein was significantly down-regulated.Conclusion:Euphorbia factor L_(1),L_(2),L_(3),L_(7a),L_(7b)and L_(8)can inhibit the expression of LXR protein level,which may be achieved by inhibiting the transcriptional activity of LXR.
