根皮苷水解酶与植物类黄酮互作影响亚洲小车蝗取食策略

Lactase-phlorizin hydrolase (LPH) is secreted from insects midgut epithelial cell. It can relieve the inhibition of insulin (ILP) metabolic pathways by breaking down flavonoids in the food and inhibiting PTP1B. We found that O. asiaticus maintained a specific dietary structure．Transcriptome and qPCR had indicated that lph expression level was significantly related to the flavonoids in different compositions of Stipa krylovii, Leymus chinensis, and Cleistogenes squarrosa from which we speculated that it is the interaction of Lactase-phlorizin hydrolase (LPH) and flavonoids affect Oedaleus asiaticus feeding strategy that lead to the specific dietary structure. Thus, we cloned lph gene and prepared antibody of LPH、PTP1B. In order to make clear the process of ILP metabolic pathway was negatively regulated by flavonoids via PTP1B, we plan to interfere the expression of lph by RNAi, to inhibit LPH by enzyme inhibitors, to determine flavonoids accumulation process in the body of grasshoppers when feeding different food composition by GC-MS, to detect enzyme activity of phosphate kinase (Akt) which acts as an key target protein of tyrosine phosphatase (PTP1B) and an important enzyme in ILP pathway. We will reveal the mechanism of the formation of food structure with LPH and plant flavonoids interaction by exploring the relation between food structure, development progress, viability, and flavonoids accumulation, LPH gene expression, LPH activity, PTP1B activity. This project will make clear the feeding strategy of O. asiaticus, and provide reference for grasshoppers’ gene control and ecological management.

根皮苷水解酶LPH由昆虫中肠上皮细胞分泌，降解食物中类黄酮，抑制蛋白酪氨酸磷酸酶PTP1B解除其对类胰岛素代谢途径ILP的抑制。申请者发现亚洲小车蝗有固定取食模式，取食羊草、针茅、隐子草不同组合处理时lph基因表达量与类黄酮含量相关。推测LPH与植物类黄酮互作影响亚洲小车蝗取食策略，导致固定食物结构的形成。为此，我们克隆了lph基因，制备了LPH、PTP1B抗体。拟利用RNAi干扰亚洲小车蝗lph基因表达，抑制剂抑制LPH活力，GC-MS测定不同食物组合亚洲小车蝗类黄酮积累，检测类黄酮靶标PTP1B与ILP途径中磷酸激酶Akt活力，明确类黄酮物质负调控ILP途径代谢过程。测定不同食物组合亚洲小车蝗食物结构、生活力指标，分析与类黄酮物质积累，lph基因表达，AKt、PTP1B活性间的关系，揭示LPH与植物类黄酮互作影响亚洲小车蝗取食策略的机制，为草原蝗虫种群基因调控及生态治理提供理论指导。

经过4年的系统研究，本项目在如下方面取得了进展。1）通过生态学、生理学和分子生物学研究，分析了食物因子与蝗虫灾害的关系，阐明食物驱动草原蝗虫发生机制为：a) 亚洲小车蝗食性研究方面，，针茅作为指示种，利于车蝗生长发育，决定发生密度及范围；冷蒿作为食物胁迫不利于生长发育与发生。b) 亚洲小车蝗解毒代谢方面，亚洲小车蝗表型可塑性明显，寄主营养及次生代谢物胁迫导致其生活力降低，解毒酶活性升高，消化酶活性降低，食物适应性降低。c) 分子调控机理方面，亚洲小车蝗通过ILP介导调控基因差异表达适应寄主。针对寄主营养及次生代谢物胁迫，负调控ILP途径，表皮形成、物质代谢等基因下调，而抗逆性相关（如热激蛋白Hsp、细胞凋亡抑制剂）、解毒代谢等相关（如乳糖酶-根皮苷水解酶LPH、细胞色素P450、羧酸酯酶）基因和蛋白表达量显著上调。2）研究发现亚洲小车蝗取食模式由植物次生代谢物决定，发育进度受类黄酮影响显著。亚洲小车蝗能够选择性取食调节类黄酮摄入，反式调控ILP，调节自身发育，调控种群同时爆发。3）昆虫中肠根皮苷水解酶LPH水解类黄酮为苷元，作用于NADPH氧化酶，降低O2-浓度，激活蛋白酪氨酸磷酸酶PTP1B，抑制蛋白酪氨酸激酶PTK，负调控类胰岛素途径ILP。测定了不同纬度亚洲小车蝗食物结构及类黄酮摄入量，克隆了LPH、PTP1B、PTK基因并合成dsRNA。a）干扰了LPH，HPLC定量分析了肠壁细胞苷元含量及活性氧浓度，定量PCR检测了PTP1B、PTK及ILP下游IR、IRS1、Akt、FOXO转录，ELISA检测了蛋白磷酸化，并通过抑制剂处理分析，揭示了LPH与类黄酮互作通过PTP1B与PTK反式调控ILP调节生长发育，不同纬度种群同时暴发的新机制。b) 干扰了PTK的5龄亚洲小车蝗，除成活率外，其体干质量、生长速度和综合性能均显著降低。而干扰PTPN4对所有这些生长参数均无显著影响。通过基因表达、蛋白磷酸化水平及酶活性检测发现，在亚洲小车蝗的ILP中，PTK能促进亚洲小车蝗的生长、体重和整体性能等，最终以较低的能量消耗促进昆虫的解毒代谢。