Table_3_Transcriptomic and Proteomic Analyses of Nepenthes ampullaria and Nepenthes rafflesiana Reveal Parental Molecular Expression in the Pitchers of Their Hybrid, Nepenthes × hookeriana.XLSX

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.

猪笼草属（Nepenthes）是一类食肉性热带猪笼草，其叶尖演化出特化的捕虫结构，通过诱捕昆虫获取养分。近年来已有研究借助蛋白质组学方法鉴定捕虫笼液中的蛋白质，以深入解析其食虫机制，但受限于该属物种特异性转录组资源匮乏，蛋白质鉴定工作常受阻碍。本研究采用转录组辅助蛋白质组学（proteomics informed by transcriptomics, PIT）策略，结合PacBio异构体测序（PacBio isoform sequencing, Iso-Seq）与液相色谱-质谱联用（liquid chromatography-mass spectrometry, LC-MS）蛋白质组谱分析，对苹果猪笼草（Nepenthes ampullaria）、莱佛士猪笼草（Nepenthes rafflesiana）及其杂交种胡克猪笼草（Nepenthes × hookeriana）的捕虫笼液蛋白质进行鉴定与比较分析。我们为三个物种构建了全长转录组，共获得80791个一致性异构体，平均长度为1692 bp，以此作为蛋白质鉴定的参考序列。比较分析结果显示，杂交种胡克猪笼草的转录本与蛋白质谱更接近莱佛士猪笼草；二者均为专性食虫物种，而杂食性的苹果猪笼草可通过落叶腐殖质获取养分。本研究检测到此前已报道的水解蛋白，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、类甜蛋白、病程相关蛋白及抗病蛋白。同时还鉴定出多种功能多样的新型蛋白质，如淀粉酶、转化酶、过氧化氢酶、激酶、连接酶、合酶、酯酶、转移酶、转运蛋白及转录因子。尽管在苹果猪笼草中发现了少量独特酶类，但未找到强有力的证据表明其存在适应性演化以产生内源酶来分解落叶腐殖质。本研究更全面地解析了猪笼草消化液的蛋白质组成，为理解不同食性猪笼草物种的捕虫笼分子生理学及食虫机制提供了重要见解。