基于iTRAQ-PRM技术筛选不同类型土壤烤烟根系差异表达蛋白

  目的  从蛋白质水平揭示烤烟根系对不同类型土壤的响应差异。  方法  以烤烟品种K326为材料，选取石灰岩土（沙壤土）、水稻土和红壤土，测定3种类型土壤烤烟根系活力、硝酸还原酶（NR）、谷氨酰胺合成酶（GS）和天冬氨酸合成酶（AS）活性；利用iTRAQ技术鉴定3种类型土壤烤烟根系差异表达蛋白，对所获得的差异蛋白进行生物信息学分析，从中选择16个蛋白进行平行反应监测（PRM）验证。  结果  （1）红壤土的烤烟根系活力、NR、GS和AS活性均高于沙壤土和水稻土；（2）从水稻土vs沙壤土、沙壤土vs红壤土、水稻土vs红壤土3组对比中分别鉴定出699、650、569个烤烟根系差异表达蛋白，其中上调/下调蛋白分别为412/287、291/359和323/246个；（3）差异蛋白质主要有催化、结合、转运活性等功能；（4）差异蛋白功能富集于次生代谢产物的生物合成、代谢途径和苯丙烷类生物合成等代谢途径；（5）红壤土烤烟根系与抗逆性、碳水化合物和能量代谢、蛋白质合成等相关的差异蛋白表达均高于沙壤土和水稻土。  结论  利用iTRAQ标记技术，结合PRM验证技术筛选出不同类型土壤烤烟根系差异表达蛋白，为获取响应关键蛋白奠定基础。 

Screening of differentially expressed proteins in flue-cured tobacco roots in different soil types based on iTRAQ-PRM

  Background  This study aims to reveal the response of flue-cured tobacco roots to different types of soil at the protein level.  Methods  Using flue-cured tobacco variety K326 as material, the root activity, nitrate reductase (NR), glutamine synthetase (GS) and aspartate synthetase (AS) activities of flue-cured tobacco roots in three types of soil including calcareous soil (sandy soil), paddy soil and red soil were measured. iTRAQ technology was used to identify the differentially expressed proteins of flue-cured tobacco roots in three types of soil, and the obtained differential proteins were analyzed by bioinformatics. On this basis, 16 proteins were selected for parallel reaction monitoring (PRM) validation.  Results  (1) The root activity and activities of NR, GS, AS of flue-cured tobacco roots in red soil were higher than those in sandy soil and paddy soil; (2) 699, 650 and 569 differentially expressed proteins were identified from the comparison of paddy soil vs sandy soil, sandy soil vs red soil, paddy soil vs red soil, respectively. The up-regulated/down regulated proteins were 412/287, 291/359 and 323/246, respectively; (3) Differentially expressed proteins are mainly involved in metabolic process, cellular process, stress response and other biological processes. They exist in cells, cell components and organelles, and have catalytic activity, binding activity and transport activity; (4) Their functions are enriched in the biosynthesis and metabolic pathways of secondary metabolites and phenylpropanoid biosynthesis; (5) The stress resistance, carbohydrate and energy metabolism, and protein synthesis of flue-cured tobacco roots in red soil were higher than those in sandy soil and paddy soil; (6) PRM analysis showed that results of iTRAQ proteomics were correct and reliable.  Conclusion  iTRAQ labeling technology was used to screen the root differentially expressed proteins of flue-cured tobacco in different types of soil combined with PRM verification technology, which provided some theoretical basis and technical support for choosing the appropriate soil type, formulating reasonable cultivation technology and producing high-quality tobacco.