家蚕蔗糖特异性水解酶的适应性进化机制及其功能研究

Insect membrane-associated sucrase, is not only a key enzyme in the metabolism of carbohydrates, but also plays an important role in physiologic processes such as the osmotic regulation, stress response and signal transduction. However, it is difficult to purify membrane-associated sucrase, because membrane-associated sucrase trapped in the cell glycocali or inserted into the membrane. So the enzymatic properties remain unknown. Recently, we identified a novel membrane-associated, sucrose-specific hydrolase gene from Bombyx mori, named as BmSUH. The evolutionary mechanism of the adaption function and physiological function of BmSUH is unclear. To examine effects of key amino acid changes on enzymology characteristics of BmSUH, site-directed mutagenesis will be performed. The mutant and wild-type BmSUH will be expressed in insect cell, purified and characterized. In order to explore the biological functions of BmSUH in the process of growth and development, we will use the CRISPR/Cas9 system to generate BmSUH gene knockout mutants, analyze economic traits, physiological and biochemical properties of wild-type and the mutant silkworm. These results will provide a solid theoretical basis for understanding digestive system and nurtrient absorption of lepidopteran insects. Furthermore, it will greatly facilitate the improvement rate of digestion and absorption of silkworm feed, silk production and the artificial feed development with high quality and low cost.

鳞翅目昆虫膜结合型蔗糖酶不仅是蔗糖水解的关键酶, 而且在渗透压调节、胁迫响应和信号传导等多个方面发挥重要作用。昆虫膜结合型蔗糖酶因与细胞膜脂双层结合, 纯化困难, 具体酶性质一直鲜有报道。申请者在前期研究中首次鉴定了家蚕膜结合型蔗糖特异性水解酶基因BmSUH, 但其适应性进化机制和生物学功能尚不清楚。本研究以新发现的BmSUH为切入点, 通过点突变和突变体酶学性质分析, 研究在进化过程中关键氨基酸的变化对酶学特征形成的影响。应用CRISPR/Cas9技术对BmSUH 基因进行特异性敲除, 分析和比较正常和BmSUH 基因敲除家蚕的经济性状和生理生化性状, 探明BmSUH在家蚕生长发育过程中的生理功能, 为揭示鳞翅目昆虫特异性消化吸收机理提供理论基础和重要参考, 同时为我们提高家蚕的饲料消化吸收率、开发优质低成本人工饲料和提高叶丝转化率提供理论支撑。

昆虫膜结合型蔗糖酶不仅是蔗糖水解的关键酶, 而且在渗透压调节、胁迫响应和信号传导等多个方面发挥重要作用。昆虫膜结合型蔗糖酶因与细胞膜脂双层结合,纯化困难,具体的酶性质一直鲜有报道。本研究在前期工作的基础上，首次从分子进化角度阐释了鳞翅目昆虫SUH家族的适应性进化机制，证明了正选择作用作为适应性进化的重要驱动力，导致α-葡萄糖苷酶 GH13家族在鳞翅目昆虫中发生扩张和功能分化，产生了蔗糖特异性水解酶SUH。此外，采用CRISPR/Cas9技术对BmSUH基因进行特异性敲除，研究了家蚕BmSUH的生理功能。结果表明BmSUH突变体的糖代谢水平整体下降，中肠膜结合型蔗糖酶的活性明显降低，幼虫生长期延长。进一步研究表明BmSUH在杆状病毒经口感染过程中发挥作用。缺失BmSUH的突变体对BmNPV的经口感染具有一定的抵抗性，其存活率显著上升。本研究阐明了鳞翅目昆虫SUH的进化机制，明确了BmSUH在家蚕生长发育过程中的作用,为研究以家蚕为代表的鳞翅目昆虫特异性消化吸收系统提供了理论基础和重要参考。此外，本研究有望为阐明BmNPV与宿主的相互作用及侵入机制提供一个突破口，为防治BmNPV提供理论基础。