基于Label-free技术的高州油茶铝胁迫蛋白质组学研究

Proteomic analysis on the aluminum stress response of Camellia drupifera based on Label-free technology

【目的】南方地区拥有大面积的酸性红壤,导致土壤中铝元素多以离子的形式溶出,严重抑制油茶的生长及高产。研究采用Label-free技术探究高州油茶在铝胁迫下的蛋白质组学响应,为揭示高州油茶应答铝胁迫的分子响应机制奠定了理论基础。【方法】以高州油茶两年生实生苗为试验材料,测定不同浓度铝处理条件下高州油茶叶片部分代谢指标及抗氧化生理相关指标,同时比较对照组(CK)和4 mmol/L铝处理组(GZ4)差异蛋白质的表达。【结果】生理试验结果表明,不同浓度铝胁迫下油茶幼苗都会发生氧化胁迫导致细胞活性氧(ROS)积累,2 mmol/L铝浓度下油茶叶片通过增加渗透调节物质含量和增强多种抗氧化酶活性以清除细胞活性氧,使细胞免受氧化胁迫的损害;而4 mmol/L铝浓度下叶片丙二醛(MDA)含量和脯氨酸(PRO)含量明显升高,积累的活性氧超出了自身所能清除的范围,叶片可溶性糖含量和可溶性蛋白含量显著减少,抗氧化酶活性均有不同程度减弱,油茶正常的生理功能受到抑制。蛋白质组学数据表明,CK组和GZ4组共鉴定到4282个蛋白质,与CK组相比,GZ4处理组有207个蛋白质的表达丰度显著增加,129个蛋白质的表达丰度显著降低。差异表达蛋白质的通路富集和蛋白质互作网络分析结果表明,4 mmol/L铝胁迫下高州油茶主要通过提高谷胱甘肽代谢,增强细胞清除活性氧和自由基负离子的能力缓解铝胁迫造成的氧化损伤;4 mmol/L铝浓度下高州油茶蛋白质合成和光合作用受到明显抑制,抗氧化酶活性减弱可能与细胞供能能力不足有关。【结论】铝胁迫下高州油茶主要通过能量及碳水化合物代谢、氨基酸合成及代谢、生物碱合成、蛋白质加工、卟啉和叶绿素代谢等代谢途径抵抗铝毒。

【Objective】The southern region has a large area of acidic red soil,which leads to the dissolution of aluminum in the soil in the form of ions,seriously inhibiting the growth and high yield of Camellia drupifera.This study used Label-free technology to explore the proteomic response of Camellia drupifera to aluminum stress,which laid a theoretical foundation for revealing the molecular response mechanism of Camellia drupifera to aluminum stress.【Method】The partial metabolic indexes and antioxidant physiological indexes of Camellia drupifera leaves under different concentrations of aluminum treatments were used as experimental materials,and the expression of differential proteins in the control group(CK)and 4 mmol/L aluminum treatment group(GZ4)was compared.【Result】The results of physiological tests showed that oxidative stress would occur under different concentrations of aluminum stress,leading to the accumulation of cellular reactive oxygen species(ROS).Under the 2 mmol/L aluminum treatment,Camellia drupifera leaves could remove cellular ROS by increasing the osmoregulatory substances and enhancing the activities of various antioxidant enzymes,so as to protect cells from oxidative stress damage.Under the 4 mmol/L aluminum treatment,the contents of malonaldehyde(MDA)and proline(PRO)in leaves were significantly increased,and the accumulated reactive oxygen species(ROS)exceeded the range of self-clearance,the contents of soluble sugar and soluble proteins in leaves were significantly reduced,and the activities of antioxidant enzymes were weakened to varying degrees,which inhibited the normal physiological function of Camellia drupifer.The proteomic data showed that a total of 4282 proteins were identified in the CK group and GZ4 group.Compared with the CK group,the expression abundance of 207 proteins in the GZ4 treatment group was significantly increased,and the expression abundance of 129 proteins was significantly reduced.The results of pathway enrichment and protein interaction network analysis of differentially expressed proteins showed that under the 4 mmol/L aluminum treatment,Camellia drupifera alleviated the oxidative damage caused by aluminum stress mainly by improving glutathione metabolism and enhancing the ability of cells to remove reactive oxygen species and free radical negative ions.Under the 4 mmol/L aluminum treatment,the protein synthesis and photosynthesis of Camellia drupifera were significantly inhibited,and the decreased activity of antioxidant enzymes may be related to the insufficient cell energy supply capacity.【Conclusion】Under aluminum stress,Camellia drupifera mainly resists aluminum toxicity through energy and carbohydrate metabolism,amino acid synthesis and metabolism,alkaloid synthesis,protein processing,porphyrin and chlorophyll metabolism.