基于iTRAQ技术对棉花叶片响应化学打顶的差异蛋白质组学分析

【目的】比较化学打顶和人工打顶方式下棉花植株生理变化及蛋白质的差异表达,为化学打顶的作用机理提供理论依据。【方法】以黄河流域大面积推广的冀棉863为试验品种,于2015—2016年设置人工打顶、化学打顶和未打顶3种处理,于7月20日进行统一打顶处理,化学打顶剂为人工喷施,用量为1.125 L·hm~(-2)。打顶处理后定期测定各处理间棉花株高与主茎功能叶内源激素含量。株高测量为子叶节到主茎生长点的高度,使用直尺测量。使用酶联免疫法测定棉花功能叶的生长素(IAA)、赤霉素(GA_3)、脱落酸(ABA)和玉米素核苷(ZR)含量。采用iTRAQ技术对人工打顶和化学打顶处理的打顶后20 d的主茎功能叶进行差异蛋白质组学分析。【结果】与人工打顶的棉花相比,化学打顶处理株高显著高于人工打顶处理,两年试验中分别高11.8%和14.5%,但显著低于未打顶处理,两年试验中分别低6.0%和6.5%,喷施化学打顶剂有效抑制了棉花株高的增长。不同打顶处理对棉花功能叶GA_3含量影响较大,打顶后GA3含量变化为单峰曲线,处理30 d各处理之间达到显著差异,GA3含量为未打顶化学打顶人工打顶,30 d后化学打顶与未打顶处理呈下降趋势,人工打顶处理则在20 d时出现下降趋势,在处理后50 d时各处理GA_3含量无显著差异。2016年IAA含量峰值出现在处理后40 d,化学打顶处理峰值显著低于其他两个处理,2015年3种打顶处理间无显著差异。ABA含量在处理后40 d时达到最大值,未打顶处理峰值显著低于其他两个处理。3种打顶处理的ZR含量无显著差异。化学打顶与人工打顶处理相比,iTRAQ标记方法检测到69个差异表达蛋白,29个上调表达,40个下调表达,其中碳水化合物和能量代谢相关的蛋白多下调表达,降低了植株的长势;与GA调节正相关蛋白多上调表达,增强GA效应。【结论】化学打顶能有效控制棉花株高,对棉花功能叶的GA含量影响较大,化学打顶处理含量显著高于人工打顶处理,与人工打顶处理相比,化学打顶与植物生长发育相关蛋白多下调表达,可能是植株通过降低碳水化合物合成,减少能量代谢,增加GA含量,激活GA效应来实现株高的控制。

Differential Proteomics Analysis of Cotton Leaf Response to Chemical Topping Based on iTRAQ Technique

【Objective】The objective of this study is to compare the effects of manual topping and chemical topping on physiological changes and proteins differentially expression of cotton. The results of this study would provide a theoretical basis for the mechanism of chemical topping. 【Method】Manual topping, chemical topping and no top pruning were practiced on cultivar GM863 which was planted in large area of the Yellow River basin in 2015-2016. Cotton was treated on July 20th simultaneously. Artificial spraying chemical topping agent and the dosage was 1.125 L·hm^-2. The plant height and hormone contents of functional leaves were determined regularly after topping. The plant height was measured with the height from the cotyledon node to the growth point of the main stem by using ruler. The hormone contents of functional leaves, including IAA, GA₃, ABA and ZR were measured by enzyme-linked immunosorbent assay. The differential proteomics analysis was carried out on the functional leaves of manual topping and chemical topping by using iTRAQ technique. 【Result】 Compared with the cotton of manual topping treatment, chemical topping treatment was significantly higher than manual topping treatment, which was 11.8% and 14.5% higher than others in the two-year trial, but was significantly lower than no top pruning, which was 6.0% and 6.5% lower than others, vertical growth was significantly inhibited after spraying. Different topping treatments greatly affected the GA₃ content of cotton functional leaves, the change of GA₃ content was a single peak curve, the contents of GA₃ were no top pruning＞chemical topping＞manual topping, and reached significant difference. The GA₃ contents of chemical topping and no top pruning were decreased at 30 d after topping and manual topping was decreased at 20 d. There was no significant difference among three treatments at 50 d after topping. The maximum IAA content in chemical topping treatment was significantly lower than that in the other two treatments at 40 d after topping in 2016, and there was no significant difference among the three treatments in 2015. The maximum ABA content in no top pruning treatment was significantly lower than that in the other two treatments at 40 d after topping. There was no significant difference among the three treatments of the contents of ZR. Compared with manual topping treatment, 69 differentially expressed proteins were detected by using iTRAQ in leaves of chemical topping treatment, 29 up-regulated and 40 down-regulated expressions, in which carbohydrate and energy metabolism related proteins were most down-regulated, and it was indicated that the growth of plants was decreased. The up-regulation of most GA regulated positive correlation proteins enhanced the GA effect. 【Conclusion】Chemical topping can effectively control the plant height of cotton, and which has a great effect on the GA content of functional leaves. The contents of GA in chemical topping treated leaves were significantly higher than that of manual topping treatment. Compared with manual topping treatment, the proteins related to plant growth and development in chemical topping treatment were most down-regulated expression, may be the plants reduced carbohydrate synthesis and energy metabolism, and activated GA effect by increasing GA content to control plant height.