CAJ | 학술논문

从一种来自中国日行性萤火虫(云南窗萤)发光器官mRNA中克隆、测序并表达了有功能的荧光素酶.云南窗萤荧光素酶的cDNA序列有1647个碱基,编码548个氨基酸残基.从推测得到的氨基酸序列的比对分析得出:云南窗萤的荧光素酶与来自Lampyris noctiluca,L.turkestanicus和Nyctophila cf.caucasica三种萤火虫的荧光素酶有97.8%的序列一致性.从推测得出的氨基酸序列进行系统发育分析,其结果表明:云南窗萤和Lampyris+Nyctophila聚在一起,与同属的发光强夜行性的萤火虫不形成的单系.云南窗萤荧光素酶在大肠杆菌中表达的条带大约70kDa,并且在有荧光素存在时发出黄绿色荧光.对荧光素酶的结构模拟和分析表明,云南窗萤荧光素酶基因的氨基端和羧基端结构域之间的裂沟处存在这5个多肽环,这正是从其他荧光素酶推测得到的催化荧光反应时的底物结合位点.云南窗萤和窗萤属的其他3种萤火虫的荧光素酶卡目比,有13个不同氨基酸位点,位于模拟分子结构的表面.对于这些多肽环、不刚氨基酸残基和晶体结构的进一步研究有利于解释日行和夜行性萤火虫荧光素酶的差异.
종일충래자중국일행성형화충(운남창형)발광기관mRNA중극륭、측서병표체료유공능적형광소매.운남창형형광소매적cDNA서렬유1647개감기,편마548개안기산잔기.종추측득도적안기산서렬적비대분석득출:운남창형적형광소매여래자Lampyris noctiluca,L.turkestanicus화Nyctophila cf.caucasica삼충형화충적형광소매유97.8%적서렬일치성.종추측득출적안기산서렬진행계통발육분석,기결과표명:운남창형화Lampyris+Nyctophila취재일기,여동속적발광강야행성적형화충불형성적단계.운남창형형광소매재대장간균중표체적조대대약70kDa,병차재유형광소존재시발출황록색형광.대형광소매적결구모의화분석표명,운남창형형광소매기인적안기단화최기단결구역지간적렬구처존재저5개다태배,저정시종기타형광소매추측득도적최화형광반응시적저물결합위점.운남창형화창형속적기타3충형화충적형광소매잡목비,유13개불동안기산위점,위우모의분자결구적표면.대우저사다태배、불강안기산잔기화정체결구적진일보연구유리우해석일행화야행성형화충형광소매적차이.
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 rufa 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 lueiferin. Five loops in the P. pygidialis lueiferase, 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, 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.