鳞翅目昆虫新发现的海藻糖酶基因在不同昆虫间的功能分化研究

Insect trehalase has a very important role in many aspects, such as insect growth and development, flight energy supply, chitin biosynthesis and regulation as well as the body’s resistance. In our previous study, we have identified a novel trehalase gene in lepidopteran insects, named as Tre-1B, which shows structural differences among insects. A preliminary study has been conducted on the gene expression and enzymatic activity of Tre-1B. However, the mechanism of evolution and functional differentiation is unknown. In the present study, we will compare the gene expression, enzymology and biochemical properties of two typical Tre-1B from Bombyx mori and Trilocha varians. To examine effects of key amino acid changes on enzymology characteristics of Tre-1B, site-directed mutagenesis will be performed. Wild-type and the mutant Tre-1B will be expressed in insect cell, purified and characterized. Furthermore, we will use the CRISPR/Cas9 system and embryonic RNAi to explore the biological function of Tre-1B of Bombyx mori and Trilocha varians. The results of this project will contribute to understand the mechanism of evolution and functional differentiation of Tre-1B in lepidopteran insects. These results can also demonstrate metabolic mechanism of trehalose and provide a solid theoretical basis for energy supply and carbon cycling in Lepidoptera. Moreover, our result will provide a fruitful foundation for developing strategies to control lepidopteran pest species.

昆虫海藻糖酶在昆虫生长发育，飞行能量供给，几丁质合成的调节以及机体抗逆性等方面具有重要作用。课题组前期研究发现鳞翅目昆虫存在一种新的海藻糖酶基因Tre-1B，且昆虫间基因结构存在差异。课题组对Tre-1B的表达方式和酶活性进行了初步研究，但它的进化和功能分化机制国内外尚无报道。本研究以家蚕和三角斑褐蚕蛾Tre-1B为研究对象，分析和比较鳞翅目昆虫两类典型Tre-1B基因的时空表达特征和酶学特性。通过点突变和突变体酶学性质分析, 明确Tre-1B在进化过程中关键氨基酸的变化对Tre-1B酶学特征的影响。采用胚胎期RNA干涉技术和CRISPR/Cas9技术探明和比较两类典型Tre-1B的生物学功能，明确鳞翅目昆虫海藻糖酶基因的进化和功能分化机制, 为系统揭示鳞翅目昆虫体内海藻糖的代谢机制提供理论基础, 为进一步阐明鳞翅目昆虫能量供给与碳循环提供理论依据，并为鳞翅目害虫防治提供新思路和新靶点。

昆虫海藻糖酶在昆虫生长发育，飞行能量供给，几丁质合成调节以及机体抗逆等方面发挥重要的作用。普遍认为昆虫中存在两种不同类型的海藻糖酶基因。最新研究发现, 鳞翅目昆虫除了含有两种典型的海藻糖酶基因外，还存在另外一种新的海藻糖酶基因Trehalase1b (Treh1b)。但关于它的表达方式、酶学性质和生理功能却鲜有文献报道。本项目系统解析了鳞翅目昆虫海藻糖酶基因的进化机制，明确了家蚕、三角斑褐蚕蛾和柑橘凤蝶不同藻糖酶基因的表达模式，酶学性质和亚细胞定位。通过功能分歧和选择压力分析，获得了导致鳞翅目昆虫海藻糖酶基因功能分化的氨基酸位点。从分子进化角度阐释了鳞翅目昆虫藻糖酶基因家族的功能分化机制，证明了正选择作用是适应性进化的重要驱动力。采用CRISPR/Cas9技术对家蚕3种海藻糖酶基因进行特异性敲除，以家蚕为代表研究了鳞翅目昆虫Treh1b的生理功能。研究结果表明家蚕Treh1b对维持体内海藻糖的代谢平衡发挥重要作用。本研究解析了鳞翅目昆虫海藻糖酶基因的进化和功能分化机制,为系统阐明鳞翅目昆虫体内海藻糖的代谢,能量供给与碳循环提供了重要理论基础。