摘要
苎麻是中国重要的天然纤维作物之一,苎麻具有高生物产量,一年可收获三季,强大根系等特点,需肥量较大。现今对苎麻肥料利用主要通过栽培途径进行研究,尚缺乏通过分子手段对NPK的吸收、转运和代谢机制的相关研究,苎麻蛋白质组学的研究将提升其竞争力。建立一种适用于苎麻蛋白提取及双向电泳技术体系,并能同时适用于根、茎、叶等器官,达到质谱鉴定蛋白的要求,是实现蛋白质组学研究的关键步骤。通过建立的双向电泳体系研究苎麻响应缺N、P、K的差异蛋白质组学,以此研究苎麻对NPK吸收、利用和代谢分子机制,为研究苎麻N、P、K高效利用机制提供重要的参考信息。本文以“华苎五号”为实验材料,优化建立了适于苎麻的双向电泳技术体系,并进行了响应缺N、P、K的蛋白质组学研究,其主要结果如下：
1.优化建立的双向电泳技术体系如下：蛋白提取制备方法为改良TCA/丙酮法,蛋白裂解液为：(7M Urea,2M Thiourea,4%CHAPS,1%DTT),蛋白裂解后使用4倍预冷丙酮(-20℃C)进行蛋白纯化,等电聚焦电压为50000VH。该方法较之传统的蛋白提取方法能有效去除苎麻非蛋白杂质,并能同时适用于苎麻不同器官(根、茎、叶)。茎中蛋白最少(280±6),根中蛋白点最多(1093±20),叶片居中(765±16)。叶片中含有一定量的高丰度蛋白,在一定程度上会影响低丰度蛋白的分离,茎韧皮部作为输导组织蛋白含量较少。苎麻蛋白主要分布在PH4.5-6.5,分子量40-80KDa范围内,30%-38%蛋白分布在20-40KDa区域,主要为低分子量蛋白。考马斯亮蓝染色对苎麻叶片进行验证,同样能得到清晰的蛋白图谱,其蛋白点为780±17,说明该方法能满足质谱蛋白鉴定要求。
2.苎麻在缺N、P、K下,相对叶绿素含量不断下降,生长速度也逐渐停止,通过蛋白质组学分析了苎麻在缺N及缺K处理6d、缺P处理3d的叶片差异蛋白,MALDI-TOF/TOF质谱鉴定分别得到32、27和51个差异蛋白。其差异蛋白可分成10类,包括光合作用、蛋白定向与储藏蛋白、能量代谢、初级代谢、疾病防御、信号传导、细胞结构、转录、次生代谢、蛋白合成等功能。
3.苎麻在N、P、K胁迫时,细胞对C源和NADPH减少,光合作用相关蛋白下调如酮糖-1,5-二磷酸羧化酶/加氧酶大亚基以适应营养胁迫。同样能量代谢也整体下降,但缺K通过增加二磷酸核苷激酶和甘油醛-3-磷酸脱氢酶活性而加强糖酵解与光合作用的联系增强抗逆性。缺P可能通过增强己糖激酶的活性促进激素的合成,激动蛋白上调促进根生长从土壤中吸收更多的P,缺P和缺K都通过积累和分泌柠檬酸盐溶解土壤中的营养而增加抗逆性。缺N则通过维持TCA循环提供能量来适应胁迫。
4.苎麻在NPK胁迫时通过提高次生代谢物质来抵抗胁迫,缺P时苎麻通过增强亮氨酸氨基肽酶活性以促进C、N在细胞中的流动和利用抵抗P胁迫,缺K则通过促进无机硫化物合成半胱氨酸来提高抗逆性。
5.苎麻大量HSP家族蛋白下调,苎麻在缺素期间可能产生一些过氧化物和ROS,但在缺N和缺P时分别通过提高Cu-Zn过氧化物歧化酶、光谷氨酸过氧化物还原酶B活性对此进行清除。
6.P胁迫下抑制蛋白下调可能对苎麻的纤维生长影响较大,同时苎麻的细胞分化和生长都在一定范围内受到抑制。苎麻在N胁迫时通过加强信号传导调节ATP和DNA的合成而调控各种生命活动,以提高对缺N的耐性,但缺P和缺K蛋白合成酶下降。
Ramie (Boehmeria nivea) is a natural fiber crops characterized by high biomass yield (three harvests per year) and a strong root system. Large amount of NPK is required for ramie growth. The current research main focus on high-efficiency NPK utilization thourgh cultural management, however, there has been little research on the molecular mechanisms of ramie related to the absorption, utilization and metabolism of nitrogen (N), phosphorus (P) and potassium (K).The study of ramie proteomics will lead to enhanced competitiveness. Establishes a two-dimensional electrophoresis system and protein extraction method that is suitable for the proteomic analysis of ramie's various parts (roots, stem and leave), also it is suitable for the requirement of protein spot identification by Mass Spectrum, which is key to the success of proteomics studies. Through the established two-dimensional electrophoresis system studies the molecular mechanisms of ramie related to the absorption, utilization and metabolism of NPK by comparative proteome analysis of the response of ramie under N, P and K deficiency, and it will provide important information for further study on the high-efficiency NPK utilization mechanism of ramie. In this study, the experimental matrial of Hua Zhu#5was used to improve and establish a two-dimensional electrophoresis system and study the comparative proteome analysis of the response of ramie under N, P and K deficiency. The main results were following:
1. This article improves and establishes a two-dimensional electrophoresis system that is suitable for the proteomic analysis of ramie's various parts by optimizing the traditional TCA/acetone protein extraction method: Protein lysis buffer (7M urea,2M thiourea,4%CHAPS,1%DTT), protein purification (4times volume of acetone), and IPG strips (pH4-7,17cm). This system is also applied to the roots, stems and leaves of ramie. Compared with traditional method, this system can effectively remove the non-protein impurities from ramie, the protein spots obtained from the improved (823±15) significantly higher than the traditional(320±14) method, and it is suitable for the different parts of ramie and the requirement of protein spot identification by Mass Spectrum, where clear protein two-dimensional electrophoretogram can be obtained; The number of protein in the stem, roots and leaves were280±6,1093±20and765±16respectively. Higher-abundance proteins were found in the leaves, which affected the resolution of lower-abundance proteins to some extent, the stem with less protein content is the major transport tissue and less involved in other physiological functions. Ramie protein was mainly distributed in the pH range4.5-6.5with molecular weight40-80KD;30%-38%of protein was distributed in the20-40KDa region, mostly low molecular weight protein. The Coomassie Brilliant Blue procedure was used to stain2-DE gels can suitable for the requirement of protein spot identification by Mass Spectrum, which can obtain780±17protein spots.
2. The SPAD values in N-, P-and K-deficient treatments consistently declined, and the growth of ramie finally stopped. The differentially expressed proteins in the leaves of ramie were analyzed by proteome analysis after6d of N-and K-deficient treatments and3d of P-deficient treatment by using MALDI-TOF/TOF mass spectrometry and32,27and51differential proteins were obtained, respectively. The differential proteins were functionally classified into ten categories: photosynthesis, protein destination and storage, energy metabolism, primary metabolism, disease/defense, signal transduction, cell structure, transcription, secondary metabolism and protein synthesis.
3. Under NPK deficiency, both carbon source and NADPH supply declined in ramie. The proteins related to photosynthesis such as ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit were downregulated to adapt to nutrient stress. At the same time, the energy metabolism was generally weakened. For the K-deficient treatment, the Nucleoside diphosphate kinase and GADPH activities increased and so enhanced the connection between glycolysis and photosynthesis, thus enhancing resistance. The P deficiency may induce hormone synthesis by enhancing hexokinase activity. Actin level was upregulated and promoted root growth and absorption of more P from soil. Both P and K deficiency cause the secretion and accumulation of citrate to dissolve the nutrients from the soil and increase the stress resistance. As a response to N deficiency, the TCA cycle was sustained to provide energy to adapt to the stress.
4. Ramie generally showed higher secondary metabolism under NPK deficiency. The P deficiency may facilitate the flow and utilization of carbon and N in the cells to enhance leucyl aminopeptidase activity and to sustain plant growth. Ramie responded to K deficiency by facilitating the synthesis of cysteine using inorganic sulfides, thus increasing resistance to K deficiency.
5. Large quantities of proteins of the HSP family were downregulated in ramie. Due to the nutrient deficiency, some peroxides and ROS were also produced. However, in N-and P-deficient treatments the peroxides and ROS were cleared in ramie by increased activities of copper-zinc superoxide dismutase and2-cysteine peroxiredoxin B.
6. The upregulated level of profilin due to P deficiency may significantly affect fiber growth of ramie. The cell differentiation and growth of ramie were inhibited within a certain range of P. A variety of life activities of ramie were regulated by enhancing the signal transduction responsible for synthesis of ATP and DNA under N deficiency and this may help counteract the decline of protein synthase caused by P and K deficiency and increase resistance to N deficiency.
引文
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