家蚕i-lem突变基因的精细定位及其抑制lem幼虫黄体色的分子机理

Diversity of insect body color plays an important role in the reproduction of species and adaptation to environment. Synthesis, transportation and deposition of endogenous pigments to form body color of insects are controlled by many complicated molecular mechanisms. Although synthesis and metabolism networks of pigments have been studied to a certain extent in insect, there are many color patterns difficult to be well explained by far. As a lepidopteran model insect, the silkworm Bombyx mori has various body color mutants, which are good resources to recognize more genes, elements and pathways in the body color formation. i-lem is a larval body color mutant of B. mori, which can inhibit lem, a yellow body color mutant, and make yellow evidently weakened in lem. But there is no knowledge about i-lem gene itself and the inhibition mechanism remains unknown. In our previous study, we have identified that BmSpr, a key enzyme gene acting in the synthesis and metabolism pathway of pterin pigments, is responsible for the body color mutation of lem larvae. In this research, transcriptome sequencing will be performed and compared between i-lem and lem libraries to analyzing differencially expressed genes and pathways involving in the synthesis and metabolism of pigments. Also, we will finely map the i-lem mutant gene by positional cloning strategy, indentify the function of i-lem candidate and interpret the molecular basis for i-lem inhibiting lem. Together with the findings in morphological investigation and pigment deposition patterns, this research aims at revealing the co-acting mechanism how i-lem and BmSpr coordinate the formation of silkworm body color, especially in the regulation of pterin pigments pathway. We hope our achievements in this study could provide scientific basis for exploring i-lem mutant gene and utilizing its function in the future.

昆虫体色的多样性在适应环境和繁衍种群中扮演重要的生物学功能。构成昆虫体色内源性色素的合成、转运及贮存受到复杂的分子机制调控，目前仍有不少体色模式的形成机制难以阐释。鳞翅目模式昆虫家蚕的i-lem体色突变体对黄体色lem具有明显的抑制作用，但i-lem的分子实质及其与lem在家蚕幼虫体色形成过程的互作机制尚不知晓。前期研究结果表明，lem突变基因是蝶呤类色素合成代谢途径的关键酶基因之一BmSpr。本项目利用转录组测序技术比较i-lem与lem之间色素合成代谢途径及相关功能基因的转录表达差异；利用定位克隆技术对i-lem突变体进行精细定位，并对锁定的候选基因进行功能鉴定，解析i-lem对lem发生抑制作用的分子基础。结合体壁组织形态学和色素沉积模式的差异分析，阐明i-lem与BmSpr在家蚕幼虫体色模式形成中的相互作用机制，为进一步利用i-lem在蝶呤类色素合成调控网络中的功能提供科学依据。

昆虫体色模式的形成受到复杂的分子机制调控，目前仍有不少体色模式的形成机制难以阐释。鳞翅目模式昆虫家蚕（Bombyx mori）i-lem体色突变体对黄体色lem具有明显的抑制作用，但是i-lem的分子实质及其与lem在家蚕幼虫体色形成过程的互作机制尚不知晓。本项目针对这一科学问题，以l82 (i-lem)、ah09 (lem)和p50 (wt)为研究对象，主要开展了4个方面的研究工作：1）组织形态学观察及色素沉积模式鉴别；2）i-lem与转录组测序与差异分析；3）i-lem突变基因的定位克隆及其功能鉴定；4）i-lem基因与lem（BmSpr）在家蚕幼虫体色模式形成中的相互作用机制研究。主要研究结果如下：1）转录组数据显示28个表皮蛋白编码基因和GST等9个参与家蚕先天性免疫应答系统的DEGs及其参与的代谢通路，是i-lem与lem突变体之间存在的主要差异。这可能是由于四氢生物喋呤（BH4）的缺乏，i-lem和lem突变蚕体内存在较多的活性氧（ROS），而i-lem突变使解毒相关基因上调表达，部分缓解了ROS的有害损伤。2）i-lem突变体基因组中2段序列的缺失，造成BMgn009799基因缺失5个外显子，编码一个缩短型AKR（BMgn009799mutant）；转基因实验结果表明BMgn009799mutant的过表达抑制了蚕体内由于BmSpr缺乏引起的黄体色。3）当BmSPR发生功能障碍时，BMgn009799mutant替代BmSPR参与BH4合成代谢途径，减少黄色的墨蝶呤及其脱氨产物的生成量，提升了BH4的合成水平，从而使得i-lem幼虫体壁的黄色变淡。本项目通过阐明i-lem对lem发生抑制作用的分子基础，揭示了BmAkr 与BmSpr协同调控BH4合成代谢水平的互作机制，为今后将家蚕lem与i-lem突变体作为人类BH4缺陷型相关疾病模型的应用基础研究提供重要的理论依据。