家蚕BH4选择性合成途径的分子调控机制研究

Tetrahydrobiopterin (BH4) is a necessary cofactor for aromatic amino acid hydroxylases and nitric oxide synthase. Synthesis disorder of BH4 leads to various complicatedly metabolic syndromes or neuropsychological diseases in human. Biosynthetic pathways of BH4 in mammals have been widely focused on due to its important physiological functions. But the alternative synthesis branches, regulating mechanism of related genes and roles of different pathways remain unclear. By far, knowledge about BH4 synthesis and regulatory network is limited in insects. Our previous studies showed that the de novo synthesis pathway is similar between the silkworm Bombyx mori and higher animal. Therefore, achievements in the molecular regulating mechanism of BH4 biosynthesis pathways contribute to better understanding and clarifying the problems present in higher animal. In this research, using several B. mori mutant models, such as lem, which are differentially deficient in BH4 synthesis, genomic-wide screening of genes that probably take part in the BH4 synthesis pathways will be performed to analyze their special-time expression patterns. Next, shRNA mediated transgenic RNAi will be carried out to study the gene roles in B. mori BH4 synthesis network and to comprehend the whole molecular regulating mechanism. Especially, biological functions of salvage pathway and recycling pathway will be investigated and discussed. In theory, findings in this study count for using such silkworm mutants to set up invertebrate models for some human BH4 deficiency-dependent diseases, furthermore, provide scientific basis for screening new medicine candidates or targets to apply them in the accurate diagnosis and therapy in the future.

四氢生物蝶呤(BH4)是芳香族氨基酸羟化酶的重要辅酶，其合成不足可导致人罹患多种复杂的神经性代谢综合症。哺乳动物BH4的生物合成通路因其所具有的重要生理功能而研究较多，但至今BH4的选择性合成支路及其调控机理有待探明，不同合成途径的生理病理作用模式及功能地位尚不清楚。我们的前期研究表明，家蚕具有和高等动物类似的BH4从头合成途径，研究家蚕BH4合成代谢网络的分子调控机制有助于阐释高等动物的相关问题。本项目以lem等家蚕BH4合成缺陷型突变体为模型，充分发掘家蚕全基因组数据库中与BH4合成相关的基因，解明各基因的表达模式；利用shRNA介导的转基因RNAi技术，研究基因功能及其对不同合成途径的调控机制；着重探讨补救途径和回收途径在家蚕BH4合成调控网络的生物学功能。研究成果对于开发家蚕突变体作为人BH4缺陷型疾病的昆虫模型具有重要理论价值，为新型药物及靶标的筛选和应用提供科学依据。

四氢生物蝶呤(BH4)是芳香族氨基酸羟化酶的必需辅酶，也是一氧化氮合酶 (NOS) 的重要辅助因子，还能作为NOS的调控因子或直接作为自由基清除剂参与氧化应激作用，其合成不足可导致人罹患多种复杂的神经性代谢综合症。然而，哺乳动物BH4的选择性合成支路及其调控机理有待探明，不同合成途径的生理病理作用模式及功能地位尚不清楚。本项目针对这一科学问题，以模式昆虫家蚕（Bombyx mori）的lem (leml)、al、和i-lem等BH4合成缺陷型突变体为研究对象，主要开展了4个方面的研究工作：1）BH4合成相关基因的克隆、表达与鉴定；2）突变体中相关基因的表达互作模式；3）BmAKR、BmCBR调控BH4合成的功能；4）以转基因shRNAi研究BH4选择性合成途径的分子调控机理。主要研究结果如下：1）获得了与人的BH4合成途径中发挥重要作用的hCBR1、hCBR3亲缘关系最近的4个BmSDR成员，确定了BmSDR50为重要的参与家蚕BH4选择性合成途径的CBR；2）当从头合成途径受阻激发BmDHFR上调表达，可将BH2还原成BH4，从而提高BH4/BH2比值，因此BmDHFR介导的补救合成途径对于维持机体细胞的BH4水平和BH4/BH2比值具有重要的生理作用；3）BmSpr直接调控SP和BH4在脂肪体的合成，BH4主要由脂肪体优先向表皮、头部和生殖腺转运，而母体BH4的合成水平对子代胚胎发育具有母性影响的遗传效应，这在哺乳动物中尚未见报道。本项目为将家蚕BH4合成缺陷型突变体作为人类相关疾病模型的建模对象研究，奠定了必要的生物化学与分子生物学理论基础，所取得的研究成果对于阐释哺乳动物BH4合成代谢通路的生理病理作用机制，从分子水平明确BH4缺陷依赖性疾病的病因具有重要科学意义。