Nepenthes is a genus comprising carnivorous tropical pitcher plants that have evolved trapping organs at the tip of their leaves for nutrient acquisition from insect trapping. Recent studies have applied proteomics approaches to identify proteins in the pitcher fluids for better understanding the carnivory mechanism, but protein identification is hindered by limited species-specific transcriptomes for Nepenthes. In this study, the proteomics informed by transcriptomics (PIT) approach was utilized to identify and compare proteins in the pitcher fluids of Nepenthes ampullaria, Nepenthes rafflesiana, and their hybrid Nepenthes × hookeriana through PacBio isoform sequencing (Iso-Seq) and liquid chromatography-mass spectrometry (LC-MS) proteomic profiling. We generated full-length transcriptomes from all three species of 80,791 consensus isoforms with an average length of 1,692 bp as a reference for protein identification. The comparative analysis found that transcripts and proteins identified in the hybrid N. × hookeriana were more resembling N. rafflesiana, both of which are insectivorous compared with omnivorous N. ampullaria that can derive nutrients from leaf litters. Previously reported hydrolytic proteins were detected, including proteases, glucanases, chitinases, phosphatases, nucleases, peroxidases, lipid transfer protein, thaumatin-like protein, pathogenesis-related protein, and disease resistance proteins. Many new proteins with diverse predicted functions were also identified, such as amylase, invertase, catalase, kinases, ligases, synthases, esterases, transferases, transporters, and transcription factors. Despite the discovery of a few unique enzymes in N. ampullaria, we found no strong evidence of adaptive evolution to produce endogenous enzymes for the breakdown of leaf litter. A more complete picture of digestive fluid protein composition in this study provides important insights on the molecular physiology of pitchers and carnivory mechanism of Nepenthes species with distinct dietary habits.

猪笼草是一种由肉食性热带捕虫草组成的属，该植物在其叶尖处进化出捕集器官，以从昆虫诱捕中获取营养。最近的研究已应用蛋白质组学方法来识别投手液中的蛋白质，以更好地了解食肉动物的机制，但蛋白质的识别受到有限的物种特异性转录组的阻碍。猪笼草。在这项研究中，由转录组学（PIT）方法提供的蛋白质组学被用于鉴定和比较猪笼草的水罐中的蛋白质。壶腹猪笼草， 莱佛士猪笼草，以及它们的混合体 猪笼草 × 胡克安娜通过PacBio亚型测序（Iso-Seq）和液相色谱-质谱（LC-MS）蛋白质组学分析。我们从所有80,791个共有同工型的三个物种中生成了全长转录组，平均长度为1,692 bp，作为蛋白质鉴定的参考。比较分析发现，在杂种中鉴定出转录本和蛋白质N. × 胡克安娜 更像 莱佛士猪笼草，与杂食性相比，两者都是食虫性的 壶腹猪笼草可以从枯枝落叶中获取营养。检测到先前报道的水解蛋白，包括蛋白酶，葡聚糖酶，几丁质酶，磷酸酶，核酸酶，过氧化物酶，脂质转移蛋白，类索马汀蛋白，发病相关蛋白和抗病蛋白。还鉴定了许多具有多种预测功能的新蛋白质，例如淀粉酶，转化酶，过氧化氢酶，激酶，连接酶，合酶，酯酶，转移酶，转运蛋白和转录因子。尽管发现了一些独特的酶壶腹猪笼草，我们没有发现有力的证据证明适应性进化会产生内源酶来分解凋落物。这项研究中消化液蛋白质组成的更完整图片提供了有关投手的分子生理学和食肉动物食肉机制的重要见解。猪笼草 有不同饮食习惯的物种。