【作者】 程霞；

【导师】 刘飞虎；

【作者基本信息】 云南大学 ， 植物学， 2016， 博士

【摘要】 盐害是全球农业区域最重要的非生物胁迫之一,预计全球陆地面积的20%和超过50%的农业灌溉土地正受到盐害的影响,如何解决土壤盐碱化是当今研究的热点之一。工业大麻为重要的天然绿色纤维作物之一,具分布广、根系发达、生长快、生物量大、抗逆性强、易栽培等特点,并能适于盐碱地胁迫环境。现今对大麻耐盐研究主要通过栽培、生理途径,尚无对大麻耐盐机制的研究,大麻对盐胁迫的适应分子机制尚不清楚。本文以耐盐强品种巴马火麻(BM)、盐敏感品种云麻5号(YM)为实验材料,优化适于工业大麻叶片的蛋白样品制备方法,并进行基于iTRAQ蛋白质组学技术研究工业大麻对盐胁迫的蛋白应激机制,其主要研究结果如下：1.建立并优化了大麻叶片蛋白质制备体系：优化后的水化液为5M Urea,2 M Thiourea,1% Triton X-100,50mM Tris-Hcl(PH 8.8),1%DTT;蛋白水化后加入4倍体积100%预冷丙酮进行蛋白纯化,通过双向电泳检测大麻叶片总蛋白数为1027±12,且蛋白质量可满足下阶段iTRAQ差异蛋白组学要求。2.盐敏感型品种YM可溶性糖、可溶性蛋白、SOD活性皆在盐胁迫第2d迅速下降,叶绿素含量则随着胁迫不断下降,胁迫第2d后下降速度加快。两品种大麻Fv/Fm、ETR、qP在NaCl胁迫下整体呈下降趋势,qN值则呈上升趋势。生理学数据表明敏感型品种YM在盐胁迫第4d对胁迫反应差异最显著,可作为本次差异蛋白质组学取样时间点。3.通过对盐胁迫第4d的BM和YM的对照和盐处理叶片的差异蛋白质组学研究,共鉴定3799个蛋白,并在NCBI中成功匹配3599个蛋白,匹配上的蛋白注释物种有34.5%来自蔷薇科,其中BM、YM的差异蛋白分别为403和252个。差异蛋白分别可分成14类：初级代谢、能量、蛋白定向与储存、疾病／防御、蛋白合成、光合作用、转运、转录、信号传导、细胞结构、次生代谢、细胞内交通、细胞生长／分化、未知。4.大麻可能通过提高ATP代谢,根据光照强度调节光合代谢,加强叶绿素合成,促进细胞松弛、膨大,促进渗透调节物合成,增强无机硫在体内的流动,调控水通道蛋白,加强离子运输信号传递,提高蛋白间、蛋白与细胞膜间的信号传递和有机分子和无机分子选择吸收和运输速度,降解半纤维素细胞壁,控制细胞物质的进出,促进新陈代谢和细胞稳定性来适应胁迫。试验结果可为研究大麻适应盐胁迫分子机制研究提供重要的参考信息。更多还原

【Abstract】 Salt damage is one of the most important abiotic stresses in global agricultural regions. There are 20% of global land area and over 50% of agricultural irrigated land influenced by salt damages,how to cope with soil salinization has become one of research hotspots nowadays. Industrial hemp is one of the important natural green fiber crops, which has the characteristics of wide distribution, deep root system, fast growth, high biomass, strong resistance to stress and ease cultivation. It can adapt to the stress environment of saline alkali land. The research of salt-tolerant of hemp mainly get through cultivation and physiological way, but the molecular mechanisms of adaption to salt stress remain unclear. In this study,We used salt-tolerant (BM) and salt-sensitive(YM) industrial hemps as experimental materials, established a protein sample preparation method and studied the comparative proteome analysis of hemp under salt stress. The main results were as following:1. This article establishes a protein extected system that is suitable for the proteomic analysis of hemp leaf after optimizing the traditional method:Protein lysis buffer (5 M urea,2M thiourea,1% Triton X-100,50mM Tris-Hcl(PH 8.8),1%DTT),4 times volume of acetone added after protein purification; The number of protein in the leaves were 1027±12 by two-dimensional electrophoret,and the quality of protein can meet requirement of next iTRAQ proteomics.2. The soluble sugar, soluble protein content and SOD content in salt-sensitive YM rapidly decreased in 2d after salt stress;and chlorophyll content continuously decreased with salt stress, with an increased rate of descent after 2d.The contents of Fv/Fm, ETR, qP in two varieties have a downward trend on the whole under salt stress, but qN is on the rise. Physiological datas indicated salt-sensitive variety YM was most sensitive to stress at the 4th day of salt stress, therefore the differential proteomics analysis was conducted at the 4th day of each treatment in this experiment.3. Through study on differential proteomics in the leaves of control, salt-treated BM and YM at 4d of salt stress,3799 proteins were identified, and 3599 proteins were successfully matched by BLAST (NCBI). The result demonstrated that 34.5% of the matched protein annotation species were from the Rosaceae, with 403 and 252 differential proteins in BM and YM, respectively. These differential proteins could be classified into 14 categories:primary metabolism, energy, orientation and storage of protein, disease/defense, protein synthesis, photosynthesis, transport, transcription, signal transduction, cell structure, secondary metabolism, intracellular transport, cell growth/differentiation and unknown.4. Hemp may adapt to stress mainly through improve ATP metabolism, regulate the photosynthesis according to light intensity, strengthen chlorophyll synthesis, promote cell relaxation and enlargement, improve the synthesis of osmoregulation substances, enhanced the flow of inorganic sulfur in body, regulate aquaporins, strengthen the transmission of ion transport signals, improve the signal transmission between proteins, as well as between proteins and cytomembranes, improve the selective absorption and transportation speed of organic and inorganic molecular, degrade hemicellulose cell wall, control cellular material in and out, promote metabolism and cell stability. The test results can provide important reference information for the researches on molecular mechanism of salt-stress adaption in hemp.更多还原