萤火虫的荧光素酶基因克隆表达及发光行为的初步研究

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	萤火虫的荧光素酶基因克隆表达及发光行为的初步研究
	董平轩
学位类型	硕士
导师	梁醒财
	2008-06
学位授予单位	中国科学院研究生院
学位授予地点	北京
关键词	日行性萤火虫 P. P.gidialis 荧光素酶卵发光
其他摘要	本研究围绕萤火虫的荧光素酶克隆表达和发光行为两方面来进行。首先对萤火虫的发光体系和发光行为进行了概述，然后对萤火虫荧光素酶cDNA进行了克隆表达分析，探讨了日行性和夜行性萤火虫荧光素酶的差异。最后通过萤火虫的饲养和发光行为观察研究，对卵发光的意义进行了探讨。本研究克隆了三种云南萤火虫的荧光素酶基因，并成功表达出两种有活性的荧光素酶。其中日行性萤火虫云南窗萤(Pyrocoelia pygidialis)的荧光素酶基因完成了首次克隆，并在大肠杆菌中成功表达出有功能的荧光素酶。测序结果表明：P. pygidialis荧光素酶的cDNA序列有1647个碱基，编码548个氨基酸残基。系统发育分析表明云南窗萤与同属三种发光很亮的萤火虫不形成单系，在一定程度上印证了P. pygidialis以及同属微弱发光的日行性种类与持续发出很亮荧光的夜行性种类构成窗萤属两个分支的观点；并且从发光器官的形态差异初步推断这两个分支之间是独立进化的。荧光素酶的结构模拟分析表明，云南窗萤荧光素酶的N端和C端结构域之间的裂沟处存在着五个多肽环，这是已知荧光素酶推测的活性位点。日行性的云南窗萤和三种夜行性萤火虫的荧光素酶有13个不同氨基酸，位于模拟的分子结构表面。这些氨基酸的差异，有利于进一步分析日行性和夜行性萤火虫荧光素酶结构的不同。对栉角雪萤(Diaphanes pectinealis)的荧光素酶cDNA也完成了克隆表达。结果表明其cDNA序列有1647个碱基，编码548个氨基酸残基，比从基因组中获得基因序列多3个碱基。体外表达产物在底物存在时发出黄绿色光。对雌光萤科Rhagophthalmus sp.的荧光素酶基因进行了克隆和氨基酸序列比对。得出其荧光素酶基因与来自同一科的荧光素酶基因有最高的一致性为90.7％，和鞘翅目另外三个科的物种的一致性在65％以下，显示出雌光萤科独特的分类地位。通过对采自昆明郊区的云南窗萤和一种扁萤属萤火虫的饲养和发光行为的观察，得出萤火虫的卵和蛹阶段持续发微弱的光的结论，且首次发现了卵发光在发育过程中的变化。本文对卵发光行为进行了描述，并在此基础上对萤火虫卵和蛹两阶段发光的原因和意义进行了探讨。; This is the study focus on the firefly’s luciferase and egg glowing behavior. The development of firefly bioluminescence system and the glowing behavior were reviewed at first. Then the difference between the luciferases from diurnal and nocturnal fireflies was first discussed after cloning and expression the cDNA encoding P. pygidialis luciferase. In the end, through rearing and observation the egg glowing behavior of two firefly species from Kunming north suburb, the egg luminescence function was discussed for the first time. The cDNA encoding the luciferase in P. pygidialis was cloned and expressed functionally in Escherichia coli. The Open Reading Frame (ORF) of this cDNA consists of 1647 bp, encoding a polypeptide of 548 amino acid residues. Analysis of the deduced amino acid sequence demonstrated that this luciferase has 97.8% identity to luciferase from the firefly Nictophila cf. caucasica. Phylogenetic analysis showed that P. pygidialis didn’t form monophyly with its congeneric species Pyrocoelia pectoralis, P. rufa and P. miyako, all these three are strong luminous species. The analysis of 16S mitochondrial DNA sequence also supported P. pygidialis, together with other weakly luminous congeneric diurnal species, constituted a separate group from those strong luminous nocturnal taxa. It was deduced that these weak luminous species, with degenerate photic organs in morphology, evolve in a separated routine from those strongly luminous species. The expression product of P. pygidialis luciferase cDNA has been determined as a 70 kDa band in recombinant E. coli and emitted yellow-green luminescence in the presence of luciferin. Five loops in the P. pygidialis luciferase (L1 (N198-G208), L2 (T240-G247), L3 (G317-K322), L4 (L343-I350) and L5 (G522-D532)) were found from the structure modeling analysis in the cleft, which were deduced as the active site. 13 different residues of the P. pygidialis luciferase with other known luciferases of Pyrocoelia species were located on the surface of the luciferase molecule. The existence of these residues may be helpful for understanding difference between the luciferases from diurnal and nocturnal species. The luciferase gene of Diaphanes pectinealis was cloned and expressed also for the first time. The ORF of this cDNA consists of 1647 bp encoding a polypeptide of 548 amino acid residues, 3bp and 1 amino acid residue more than the luciferase gene from its genome. The expression product of D. pectinealis luciferase cDNA has been determined as a 70 kDa band and emitted yellow-green luminescence in the presence of luciferin, closely to the P. pygidialis luciferase. The luciferase gene from Rhagophthalmus sp. was also cloned and sequenced. The deduced amino acid sequence demonstrated that this luciferase has 90.7% identity with luciferase from the Rhagophthalmus ohbai, not more than 65% identity with the luciferase of Lampyridae, Phengodidae and Elateridae. It indicated that Rhagophthalmidae is special in luminous beetles. By rearing and observation two species firefly from Kunming north suburb, the glowing behavior of P. pygidialis and Lamprigera sp. was described. The result showed that the eggs and pupae of these two species glow weakly and continuously. It was deduced that the eggs and pupae luminescence have other biological function except for the sexual communication, defense behavior and predation. To discuss this function would help us understand the bioluminescent essence and origin of the firefly and other bioluminescence organism.
学科领域	动物学
语种	中文
文献类型	学位论文
条目标识符	http://ir.kiz.ac.cn/handle/152453/11015
专题	其他
作者单位	中国科学院昆明动物研究所
推荐引用方式 GB/T 7714	董平轩. 萤火虫的荧光素酶基因克隆表达及发光行为的初步研究[D]. 北京. 中国科学院研究生院,2008.

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