MCP：中国农科院研究小菜蛾蛋白质基因组取得突破
2016年2月22日，著名蛋白组学权威期刊《molecular & Cellular Proteomics》在线发表了中国农业科学院蔬菜花卉研究所蔬菜虫害防控创新团队在小菜蛾蛋白质基因组方面取得的研究成果，为阐明小菜蛾对Bt杀虫剂抗性调控网络提供了重要的遗传信息。论文第一作者为蔬菜所博士后朱勋，通讯作者为张友军研究员。
随着高通量测序技术的不断发展，上万种生物的全基因组DNA序列被测序完成。其基因组的注释主要基于预测软件和已知蛋白的同源搜索，不可避免地导致编码蛋白质基因的错误注释和丢失、假基因广泛存在。另外，由于缺少肽段等信息，大量基因被注释为未知功能或假设的蛋白质。蛋白质基因组(Proteogenomics)是一种利用蛋白质组学的相关数据对基因组进行注释校正的新方法，由于其结合了大量生物体蛋白质氨基酸信息，从而有力地弥补基因组上基因寻找和注释的不足。小菜蛾是一种世界性重要农业害虫，每年因小菜蛾造成的损失高达40亿–50亿美元。小菜蛾几乎对所有的杀虫剂均产生了不同程度的抗药性，包括微生物杀虫剂Bt。该团队利用鸟枪法高效液相色谱-电喷雾-质谱/质谱(shotgun HPLC-ESI-MS/MS)对小菜蛾幼虫中肠蛋白质进行测定，结合其基因组数据，完成了首个小菜蛾中肠蛋白质基因组分析。利用获得的小菜蛾BBMV蛋白质谱图，鉴定到了大量与小菜蛾杀虫剂抗性相关的蛋白质，同时，还纠正了小菜蛾的基因注释，鉴定了大量新基因以及新的基因剪切形式。
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原文摘要：
The diamondback moth, Plutella xylostella (L.), is the major cosmopolitan pest of brassica and other cruciferous crops. Its larval midgut is a dynamic tissue that interfaces with a wide variety of toxicological and physiological processes. The draft sequence of the P. xylostella genome was recently released, but its annotation remains challenging because of the low sequence coverage of this branch of life and the poor description of exon/intron splicing rules for these insects. Peptide sequencing by computational assignment of tandem mass spectra to genome sequence information provides an experimental independent approach for confirming or refuting protein predictions, a concept which has been termed proteogenomics. In this study, we carried out an in-depth proteogenomic analysis to complement genome annotation of P. xylostella larval midgut based on shotgun HPLC-ESI-MS/MS data by means of a multi-algorithm pipeline. A total of 876,341 tandem mass spectra were searched against the predicted P. xylostella protein sequences and a whole-genome six-frame translation database. Based on a dataset comprising 2,694 novel genome search specific peptides, we discovered 439 novel protein-coding genes and corrected 128 existing gene models. In order to get the most accurate data to seed further insect genome annotation, more than half of the novel protein-coding genes, i.e. 235 over 439, were further validated after RT-PCR amplification and sequencing of the corresponding transcripts. Furthermore, we validated 53 novel alternative splicings. Finally, a total of 6,764 proteins were identified, resulting in one of the most comprehensive proteogenomic study of a non-model animal. As the first 4 tissue-specific proteogenomics analysis of P. xylostella, this study provides the fundamental basis for high-throughput proteomics and functional genomics approaches aimed at deciphering the molecular mechanisms of resistance and controlling this pest.
作者：朱勋
