乳汁中次生代谢产物在生菜抗虫中的功能

Plant latex is a sticky emulsion exuded from laticifer once plant is damaged. Secondary metabolites with defensive function are rich in latex. Lettuce is an important vegetable worldwide and contains plenty of latex, which is one of few plants developed as vegetables in Asteraceae. Lettuce is rarely suffered from insect pest in the field. However, little is known about the anti-herbivore mechanisms of lettuce. Our preliminary study showed that latex is crucial for insect-resistant of lettuce. The activity against insects of excised lettuce significantly decline, compared to live lettuce, and methanol extract of latex exhibited significant insect-resistant activity. In this study, the chemical constituents of secondary metabolites of lettuce latex will be isolated and elucidated by modern isolation and spectroscopy techniques, directed by pharmacological activity detection. Effects of isolated compounds on the insect-resistant will be assayed. Analysis method will be established to detect the content of active substance in latex during different period of lettuce. The effects of different treatments on the latex secretion and the content of secondary metabolites in lettuce latex, especially the active substances will be study, such as mechanical damage, insect feeding (mechanical damage add insect saliva), jasmonic acid, and different levels of nitrogen. The study will clarify the anti-herbivore mechanisms of lettuce and reveal the ecology function of lettuce latex, and are likely to supply lead compound for the synthesis of new insecticides.

植物乳汁是植物受到损伤后从受伤部位释放出来的粘性汁液，其中富含具有防御功能的次生代谢产物。生菜为菊科植物中为数不多的蔬菜，富含乳汁，深受全世界人喜爱。生菜在种植过程中极少遭受虫害，但其抗虫机理尚缺乏研究。申请人前期研究发现健康具有乳汁的生菜高度抗虫，去除乳汁后生菜抗虫作用大幅度下降，还发现乳汁的甲醇提取物具有很好的抗虫活性，可见乳汁对生菜抗虫至关重要。据此，本项目拟在生物活性跟踪指导下用现代分离和波谱技术对乳汁中的化学成分进行分离和结构鉴定；对单体化合物进行抗虫活性测定；建立生菜乳汁化学成分分析方法，测定不同生长时期生菜乳汁中抗虫活性物质含量；并研究机械损伤、模拟昆虫取食(机械损伤+昆虫唾液)、茉莉酸处理以及不同施氮水平对生菜乳汁分泌量以及乳汁中次生代谢产物、特别是抗虫活性物质含量的影响。研究成果将阐明菊科蔬菜作物生菜抗虫的机理，揭示生菜乳汁的生态学功能，并为合成新的杀虫剂提供先导化合物。

前期研究发现健康具有乳汁的生菜高度抗虫，但乳汁中具有抗虫活性的物质基础不明。本项目通过柱色谱、薄层色谱、制备液相等方法从生菜乳汁甲醇提取物中分离得到7个单体化合物。用核磁共振波谱和质谱等现代波谱技术，结合其理化性质及相关参考文献确定了化合物的结构，分别为2,5-dihydroxybenzaldehyde (1), Naproxen (2), 3β-hydroxy-4,15-dehydrograndolide (3), annuolide D (4), lactucin (5), lactucopicrin (6), hanphyllin (7)。分别测定了这7个化合物对斜纹夜蛾生长的影响。结果表明，化合物1对斜纹夜蛾的生长具有显著的抑制作用，在100 mg/kg的浓度下对斜纹夜蛾体重增长抑制率达到52.36%。化合物2 和化合物7在100 mg/kg的浓度下对斜纹夜蛾体重增长抑制率分别为23.17%和10%；化合物4-6对斜纹夜蛾的生长无显著抑制作用；化合物3对斜纹夜蛾的生长具有显著促进作用。.对用化合物1喂养的斜纹夜蛾 (RT组) 和用于对照的斜纹夜蛾 (CK组) 进行转录组测序发现：RT组和CK组之间共表达的基因共有9405个，差异表达的基因有5622个，其中RT与CK 相比上调的基因有3006个，下调的基因有2616个。GO分析及KEGG富集通路分析的结果显示，RT组中下调的基因主要是参与肽酶活力、丝氨酸型内肽酶活性酶活、丝氨酸水解酶活力、细胞色素P450代谢、谷胱甘肽代谢、药物代谢、氨基酸的生物合成等，上调基因主要参与信号转导、细胞通讯、角质层的结构成分、几丁质代谢过程、结构分子活性、溶酶体、过氧化物酶体、内吞作用等。.通过荧光定量PCR验证了40个与解毒酶相关的差异表达基因，其中13个属于UGT家族，20个属于P450家族，7个属于CarE家族，RT组与CK组相比，绝大部分解毒酶基因显著下调。酶活测定发现RT组与CK组相比，P450酶、尿苷二磷酸葡萄糖醛酸基转移酶 (UGT) 和羧酸酯酶 (CarE) 的酶活显著下调，谷胱甘肽S-转移酶 (GST) 的酶活显著上调。化合物1主要通过抑制斜纹夜蛾自身解毒酶的酶活达到抑制其生长的作用。