抗性水稻植株次生代谢物对褐飞虱类酵母共生菌的抑制作用研究

The Brown planthopper (Nilaparvata lugens) is an important rice pest in China. Yeast-like symbiotes (YLS) is very important for the growth and development of brown planthopper. Previous studies showed that the number of YLS in BPH decreased sharply after feeding on resistant rice, and the extracts of secondary metabolites from resistant rice could directly inhibit the in vitro culture of YLS. However, how do secondary metabolites of resistant rice inhibit YLS in brown planthopper? How do the secondary metabolites get into the fat body? Is it directly suppressed YLS? These has not yet been clarified. In order to solve the above problems, HPLC,LC/MS, artificial feeding techniques will be used to isolate and identify the secondary metabolites of inhibiting YLS in resistant rice. The secondary metabolites of inhibiting YLS were injected into the fat bodies of BPH by microinjection, and the inhibition of YLS and target strains was determined by in situ hybridization and laser confocal scanning. Isotope labeling technique was used to determine the transport pathway and distribution of secondary metabolites in brown planthopper after feeding. Clarifying whether the secondary metabolites of resistant rice can directly inhibit the YLS, is helpful to further guide the screening of resistant rice and bulid a theoretical foundation for the future control of insects by YLS.

褐飞虱是我国重要水稻害虫，褐飞虱脂肪体内类酵母共生菌（YLS）对其生长发育及致害性变异至关重要。前期研究表明，取食抗性水稻后，褐飞虱体内YLS数量会急剧下降；同时抗性水稻次生代谢物提取液可以直接抑制离体培养YLS。但是抗性水稻次生代谢物在褐飞虱体内如何抑制YLS（即何种成分抑制？如何进入脂肪体？对YLS及其多样性的抑制效果？）尚未阐明。针对以上问题，本研究运用HPLC、LC/MS、人工饲喂等技术分离鉴定抗性水稻抑制YLS次生代谢物；利用显微注射将抑制YLS次生代谢物注射到褐飞虱脂肪体内，同时利用原位杂交、激光共聚焦等明确其对YLS及其多样性的抑制作用，利用同位素标记技术明确次生代谢物在褐飞虱取食后体内的转运途径和分布情况。明确抗性水稻次生代谢物是否直接抑制褐飞虱YLS从而影响褐飞虱生长繁殖，将为有效筛选抗性水稻和构建抑菌治虫技术奠定理论基础。

褐飞虱是我国重要水稻害虫，褐飞虱体内共生菌对其生长发育及致害性变异至关重要。前期研究表明，取食抗性水稻后，褐飞虱体内YLS数量会急剧下降；同时抗性水稻次生代谢物提取液可以直接抑制离体培养YLS。但是抗性水稻次生代谢物在褐飞虱体内如何抑制YLS（即何种成分抑制？对共生菌多样性的抑制效果？对不同部位共生菌的抑制作用是否存在差异性？）尚未阐明。针对以上问题，本研究运用PCR-DGGE和绝对实时荧光定量PCR技术进一步研究了抗性水稻对褐飞虱体内共生菌的作用，结果表明，在敏感水稻品种TN1上饲养褐飞虱时，YLS的总数从1龄到成年期逐渐增加，但在5龄若虫中逐渐减少。抗性水稻品种Mudgo、ASD7和RH对褐飞虱第一代和第二代的三个显性YLS的抑制作用更为显著。然而，三个显性YLS的数量均从第三代褐飞虱中恢复。Ascomycetes symbionts是3种YLS中最占优势的菌株。然后利用人工饲喂的方法筛选出抗性水稻中具有抑制褐飞虱及其共生菌活性的次生代谢物，结果表明茉莉酸甲酯对褐飞虱及其共生菌活性的抑制效果最为显著，其次为肉桂酸、苯甲酸苄酯和没食子酸等，这些次生代谢物均在不同方面对褐飞虱表现出抑制作用。最后，建立基于数字PCR快速检测褐飞虱共生菌的方法，并选择茉莉酸甲酯进行检测应用研究，结果表明，茉莉酸甲酯对褐飞虱雌成虫共生菌的胁迫作用具有显著性，会在一定程度上影响褐飞虱雌成虫的菌群变化，且随着时间变化而改变，从而威胁褐飞虱的生存。褐飞虱不同部位的优势菌群存在差异性，Acinetobacter共生菌在肠道中的数量最多，Arsenophonus共生菌在血淋巴的数量最多，Noda共生菌在脂肪体中的数量最多。因此，明确抗性水稻次生代谢物是否直接抑制褐飞虱共生菌从而影响褐飞虱生长繁殖，将为有效筛选抗性水稻和构建抑菌治虫技术奠定理论基础。