基于蛋白质组学的高温干旱下Bt棉杀虫蛋白表达下降机制

Mechanism of reduced insecticidal protein expression in Bt cotton under high temperature and drought based on proteomics

研究高温干旱对Bt棉杀虫蛋白含量的影响及其蛋白质表达差异,可为Bt棉抗虫性安全表达提供理论参考.本研究以转基因抗虫棉泗抗3号为材料,设计38℃、土壤含水量为最大持水量40%处理(HD)和32℃、最大持水量60%处理(CK).使用非标定量(label-free)蛋白质组学技术分析HD处理后Bt棉棉铃中蛋白表达差异.结果表明:与CK相比,HD处理后Bt棉棉铃中杀虫蛋白下降了38.2 ng·g^(-1) FM,且差异达到显著水平.HD处理有83种蛋白质比CK显著或极显著上调,104种蛋白质显著或极显著下调;新出现蛋白质122种,不表达167种.京都基因和基因组百科全书通路分析表明,两者的差异蛋白富集通路有14条受到显著影响,其中与Bt棉杀虫蛋白合成有关的通路可能有3条,分别为碳水化合物消化吸收、蛋白外运、内质网的蛋白质加工等.碳水化合物消化吸收途径中,HD处理后泗抗3号水解淀粉能力上升,但催化己糖、葡萄糖、果糖磷酸化能力下降;蛋白质外运通路中,HD处理后肽链合成未受明显影响,但合成的肽链向内质网内转移加工过程受到限制;内质网蛋白质加工通路中,HD处理棉铃中泛素介导的蛋白酶体降解能力加强.

To provide theoretical basis for the safety of insecticidal efficiency in Bt cotton, the effects of high temperature and drought stress on insecticidal protein expression and protein differently expression profile was studied. In this study, the Bt cotton cultivar Sikang 3 was used as experimental material, with two treatments（40% field capacity and 38 ℃, HD, and 60% field capacity and 32 ℃,CK）. Differences in proteomics of Bt cotton between HD and CK were compared using label-free quantitative proteomics technology. The results showed that high temperature and drought caused a significant reduction of insecticidal protein content in bolls, with a decrease of 38.2 ng · g-1The analysis of differential protein expression by label-free quantitative proteomic approach showed that 83 proteins were significantly up-regulated, but 104 proteins were significantly down regulated in HD stressed cotton plants compared with CK. 122 new proteins were detected and167 proteins expression was not observed under stressed conditions. Results from the enrichment analysis of differently expressed protein between two treatments showed that 14 KEGG（Kyoto Encyclopedia of Genes and Genomes） pathways were affected under stress. Three KEGG pathways were related to the Bt protein synthesis and degradation ： carbohydrate digestion and absorption pathway,protein export pathway, and protein processing in endoplasmic reticulum pathway. In the carbohydrate digestion and absorption pathway, the starch hydrolysis ability of HD treated cotton plants increased, while the ability to phosphorylate the hexoses, fructose and glucose decreased. In protein export pathway,the peptide synthesis in HD treatment was not significantly affected, while the process of transferring peptides into the endoplasmic reticulum was prohibited. In the protein processing in endoplasmic reticulum pathway,the ability of ubiquitin mediated proteolysis was increased in HD treatment.