基于转录组比较探讨萤火虫生物荧光体系的分子进化机制

Bioluminescence, the conversion of chemical energy into light in living organisms, is dependent on two principal components, an enzyme luciferase and the substrate luciferin. Fireflies, including the coleopteran family Lampyridae and to a lesser extent Rhagophthalmidae, make up the most widespread and abundant bioluminescent terrestrial organisms on the planet. Despite the facts that luciferin appears to be conserved in structures among firefly species, and that the amino acid sequences of luciferases determined from dozens of firefly species have been found to share high levels of identity, there exists diverse bioluminescent behaviors not only among phylogenetically close species but even among different development stages of a species. It is of a theoretical and practical significance to understand the molecular mechanisms of the different bioluminescent behaviors for both clarifying the evolutionary nature of bioluminescent systems and better applying it in the related field of biology. Unfortunately, the strong explanation could not be given for the phenomenon of differently bioluminescence only based on the data of enzymology, sequence and structure of luciferase per se, and further the lack knowledge of luiciferin biosynthetic pathway is possibly a bottleneck. Next generation sequencing technology affords new opportunities in ecological genetics, and various functional genomic tools can be achieved by comparing the transcriptomes of the organs with a phenotype of interest. In order to explore the molecular nature of different bioluminescence, we propose the current project to compare the transcriptomes of luminous organs among species and developmental stages of a species with different biluminescent systems. We aim to provide a complete transcriptomic picture of luminous organs, and clarify molecular evolution mechanisms of bioluminescent systems by the means of a comparatively transcriptomic technology and methodology. In addition, we plan to construct a new reporter system of combined luciferase and luciferin-regenerating enzyme together and evaluate its possible applications.

荧光酶和荧光素是萤火虫生物荧光体系的两个重要组分，并有广泛应用。虽然荧光酶自身的特征（如酶学、序列和结构等）已有很多报道，但对其表达调控的分子机制和对荧光素生物合成途径却知之甚少，这制约着对生物荧光体系起源进化本质的理解。尽管所有萤火虫具有相同的荧光素，荧光酶在序列、功能结构上也极相似，但我们野外观察发现不同种类和虫态的发光行为仍差异极大。了解其差异的分子进化机制对揭示生物荧光体系的起源进化本质和更好挖掘其应用价值有重要意义。非常遗憾的是，已知的荧光酶和荧光素知识无法解释这种差异。解析所关注表型器官的转录组是功能基因组学最经济有效的方法。为此，本项目将利用高通量二代测序技术对发光行为差异的萤火虫种类及虫态的发光器官进行转录组测序、比较和分析，旨在从转录组水平揭示生物荧光体系的遗传全貌；探讨不同种类和虫态发光行为的分子进化机制；并从全新视角探讨荧光素-荧光酶体系作为一个整体的应用潜力。

生物荧光是自然界存在的一类活体生物可发光的有趣而奇特的生命现象，荧光酶和荧光素是萤火虫生物荧光体系的两个重要组分，并有广泛应用。虽然荧光酶自身的特征（如酶学、序列和结构等）已有很多报道，但对其表达调控的分子机制和对荧光素生物合成途径却知之甚少，这制约着对生物荧光体系起源进化本质的理解。尽管所有萤火虫具有相同的荧光素，荧光酶在序列、功能结构上也极相似，但我们野外观察发现不同种类和虫态的发光行为仍差异极大，非常遗憾的是目前的研究还无法解释这种差异。为了解析这种差异的分子进化机制，揭示生物荧光体系的起源进化本质和更好挖掘其应用价值，本项目基于前期的研究，在系统发育的框架下，选取具有不同发光行为萤火虫作为研究对象，通过整合多组学数据（基因组、转录组、蛋白组）、行为学和形态学数据及CRISPR/Cas9遗传操作等技术，揭示了萤火虫生物荧光体系及其进化的遗传全貌，阐述了不同种类之间发光行为差异的遗传分子机制，为改善发光体系应用的可能途径和方式如荧光酶-荧光素再生酶体系的应用潜力提供了分子证据，对解析生物荧光的起源与进化具有重要的意义。此外，围绕着生物荧光及萤火虫系统发育进化的主题，我们也完成了其它一些如萤火虫种类与系统发育等相关研究。