基于微萃取技术的根系分泌物分析及其化感作用研究.doc

博士学位论文基于微萃取技术的根系分泌物分析及其化感作用研究RESEARCH ON ROOT EXUDATE ANALYSISBASED ON MICROEXTRACTION ANDALLELOPATHIC EFFECT孙海燕哈尔滨工业大学2014年 4月 国内图书分类号：O652.6国际图书分类号： 540学校代码：10213密级：公开工学博士学位论文基于微萃取技术的根系分泌物分析及其化感作用研究博士研究生：孙海燕导师：王炎教授申请学位：工学博士学科：化学工程与技术所在单位：理学院答辩日期： 2014年 4月授予学位单位：哈尔滨工业大学 Classified Index: O652.6U.D.C: 540Dissertation for the Doctoral Degree in EngineeringRESEARCH ON ROOT EXUDATE ANALYSISBASED ON MICROEXTRACTION ANDALLELOPATHIC EFFECTCandidate：Supervisor：Sun Haiyan Prof. Wang Yan Academic Degree Applied for： Doctor of EngineeringSpeciality：Chemical Engineering and Technology Affiliation：School of Science April, 2014 Date of Defence：Degree-Conferring-Institution： Harbin Institute of Technology 摘要摘要根系分泌是化感物质释放的重要途径之一，根系分泌物一般浓度较低、不易溶于水、化学性质复杂，受外界因素影响较多，因此，根系分泌物的收集、分离、鉴定以及化感作用的研究始终是该领域研究的前沿和难点。因此，开发简单、绿色、原位的微萃取方式，对于还原根系分泌化感物质的真实种类，有效地研究其化感作用是非常重要的。本文以辣椒作为模式植物，根据根系分泌物的性质，建立了不同形式的分离方法，比较开发的方法与固相萃取（ SPE）、液液萃取（ LLE）的分析特性，研究了开发的方法在不同种类根系分泌物中的应用，并通过生物测定方法研究了根系分泌物对不同受体植物的化感响应，确定出最合适的受体植物和潜在的化感物质，系统研究外源潜在化感物质单独以及协同作用受体植物的化感作用。采用顶空固相微萃取（ HS-SPME）方法分析辣椒根系培养液中的邻苯二甲酸二甲酯（DMP）、邻苯二甲酸二乙酯（DEP）、邻苯二甲酸二异丁酯（DIBP）、邻苯二甲酸二丁酯（ DBP）、邻苯二甲酸二辛酯（DOP）等 5种邻苯二甲酸酯类物质。10 mL的样品溶液中加入 18%的 NaCl，在 1000 rmin-1的搅拌速度下，使用 65 m PDMS/DVB的萃取纤维在 80的温度下萃取 50 min，5种分析物的萃取效率最大。将萃取纤维在 250的 GC进样口温度下解析 10 min，萃取物被完全脱附。采用 HS-SPME萃取根系分泌液中低浓度的邻苯二甲酸酯类物质，5种分析物的回收率在 83.4%104.7%之间，相对标准差 8.74%。因此，选择 HS-SPME分析邻苯二甲酸酯类物质取得了较满意的萃取效果。开发了衍生后 PDMS管微萃取方法分析辣椒根系分泌的 2,3-二甲基苯酚（2,3-DMBP），2,6-二叔丁基苯酚（2,6-DTBP）、4,4-（1-甲基亚乙基）双酚（4,4-(1-M)BP）等 3种酚类化合物，研究不同衍生试剂及衍生试剂体积对萃取效率的影响， 200 L五氟吡啶作为衍生试剂可使 3种分析物被完全衍生，采用PDMS管微萃取衍生产物， 1.5 mL异辛烷可将 PDMS微管吸附的分析物完全洗脱下来。在 0.220 gmL-1的线性浓度范围内，比较衍生后 PDMS管微萃取与SPE方法在回收率、相对标准差和检出限等方面的萃取特性，结果表明，衍生后 PDMS管微萃取根系分泌的 3种酚类物质，均得到了低的检出限、满意的回收率和良好的重现性。建立 3种不同衍生方式与中空纤维液相微萃取（ HF-LPME）联用分析根系分泌的 1-萘胺（1-NA），二苯胺（DPA）和 N-苯基-2-萘胺（N-P-2-NA）3种胺- I - 哈尔滨工业大学工学博士学位论文类化合物，确定了原位衍生 HF-LPME是最合适的萃取方法。研究了不同衍生试剂对原位衍生 HF-LPME萃取效率的影响，15L甲苯与乙酸酐（80:20，v/v%）作为中空纤维的萃取剂，在 pH=12，在 50的温度下萃取 30 min，3种分析物的萃取率达到最大。原位衍生 HF-LPME在回收率、相对标准差和检出限等方面的萃取特性明显优于 LLE，采用原位衍生 HF-LPME分析了根系分泌物中的 3种胺类物质，回收率在 90.2%96.1%之间，相对标准差均小于 9.1%。因此，原位衍生 HF-LPME是一种操作方便、环境友好、富集倍数高的萃取方法，可以较好的分析水溶液的中胺类化合物。以辣椒根系分泌液为研究对象所建立的 3种萃取方法为化感物质分析提供了新的分离技术。将不同时期的根系培养液分别作用于番茄、黄瓜、生菜、南瓜、萝卜和辣椒等 6种不同的受体植物，研究分泌液对受体植物发芽率、发芽势、胚根长度、胚芽长度等指标的影响，通过化感隶属函数值和响应指数相结合的方法确定生菜为最合适的受体植物。采用 GC-MS与 GC相结合的方法鉴定出根系分泌液含有 28种有机化合物，根据测得的浓度将 0、2、4、6、8、10 gmL-1等 6种不同浓度的外源疑似化感物质溶液分别作用于生菜，根据种子萌发与幼苗生长相结合的生物方法确定了 N-苯基-2-萘胺（N-P-2-NA）和邻苯二甲酸（PA）是辣椒根系分泌的两种潜在化感物质。将 0 (CK)、5、10、15、20、25 gmL-1等 6种不同浓度的两种外源物质（ N-P-2-NA和 PA）单独以及互作于生菜幼苗7 d，分析潜在化感物质对受体植物幼苗内光合与呼吸作用，内源激素和矿质营养元素含量的影响，研究幼苗碳氮代谢及抗氧化能力，结果表明，在 010gmL-1，潜在化感物质 N-P-2-NA和 PA抑制了生菜种子的萌发和幼苗生长，N-P-2-NA的化感作用大于 PA的作用；在 1525 gmL-1时，PA的作用大于N-P-2-NA；两者互作时表现化感叠加抑制效应。关键词：微萃取技术；根系分泌物；衍生方法；潜在化感物质；化感作用- II - AbstractAbstractRoot exudate is one of the important releasing ways of allelochemicals, most ofallelochemicalsareorganiccompoundswhichareinsolubleinwaterwith low concentration. The research of collection, isolation, identification of rootexudate and allelopathic effect has been the frontier and difficulty in the field due tothe complex chemical properties and many influencing factors. Therefore, it isimportant to develop a simple, friendly and in situ extraction method for restoringthe realcomponentsand studying the allelopathic effect. Root exudate was studiedbased on hot pepper as a model plant. Three analytical methods were used to extractdifferent components from root exudate, respectively. The analytical performancewas estimated comparing with solid phase extraction (SPE) and liquid-liquidextraction (LLE). The higher sensitivity and lower limit of detection were obtainedwhen the developed methods were applied for the real sample under the optimizedcondition. Seed germination and seedling growth were comprehensively used tostudy allelopathic potential of different components from root exudate. The mostappropriate donor plant and potential allelochemicals were determined withdifferent concentrations according to biometric indicator. The exogenous potentialallelochemicals were applied solely and synergistically in the donor plant to studyallelopathic effect.Headspace solid phase microextraction (HS-SPME) with gas chromatographywas developed to extract root exudative dimethyl phthalate (DMP), diethyl phthalate(DEP), diisobutyl phthalate (DIBP), dibutyl phthalate (DBP) and dioctyl phthalate(DOP) of Capsicum annuum in hydroponic solution. Parameters affecting extractionand desorption efficiency of 5 PAEs were optimized. The result showed that theoptimal condition is extraction fiber of 65 m PDMS/DVB, extraction temperatureof 80, extraction time of 50 min, stirring rate of 1000 rmin-1, sodium chlorideof18%, sample volume of 10 mL. The highest extraction efficiency of 5 PAEs wasobtained, and 5 PAEs were completely desorbed at the 250 for 10 min. Analyticalperformance of HS-SPME and LLE were compared from relative standard deviation(RSD), and recovery. Recovery ranging from 83.4% to 104.7%, and the RSDs below8.74% were obtained when HS-SPME can be applied for the determination of5PAES in real sample under the optimum condition.PDMS tube microextraction after derivatization was applied for thedetermination of 2,3-dimethyl phenol (2,3-DMP), 2,6-di-tert butyl phenol(2,6-DTBP) and 4,4-(1-methylethyliden) bisphenol (4,4-(1-M)BP) in aqueous- III - 哈尔滨工业大学工学博士学位论文solution using gas chromatography-mass spectrometry (GC-MS). Differentderivatization reagents and their volume were studied.The phenolic compoundswere fully derivatized using 200 L pentafluoropyridine as derivative reagent.PDMS microtube was applied to extract derivative product at the extra ction time of30 min and extraction temperature of 60. 1.5 mL isooctane can completely elutethree derivative products from PDMS microtube. The high sensitivity, goodreproducibility, low detection limit and satisfactory recovery were obtained fortarget analytes by PDMS tube microextraction after derivatization under optimalconditions comparing with SPE method when PDMS tube microextraction afterderivatization was used to determine 2,3-DMP, 2,6-DTBP and 4,4-(1-M)BP in rootculture solution of hot pepper.Hollowfiberliquidphasemicroextraction(HF-LPME)withthreederivatization methods was developed to extract 1-naphthylamine (1-NA),diphenylamine (DPA) and N-phenyl-2-naphthaleneamine (N-P-2-NA). HF-LPMEwith in situ derivatization was the appropriate extraction method. The effect ofdifferent derivatization reagents on extraction efficiency was studied. When 15Ltoluene and aceticanhydride (v/v=80:20,v/v%) were used in the hollow fiber asextraction and derivatization reagent, the optimum extraction efficiency of 1-NA,DPA and N-P-2-NA was obtained using the following parameters: extraction time of30 min, sample solution pH of 12; extraction temperature of 50; stirring rate of800 rmin-1. Analytical performances of HF-LPME with in situ derivatization for1-NA, DPA and N-P-2-NA from RSD, LOD and recovery was better that of LLEThe recovery rate ranging from 90.2% to 96.1% and the RSDs below 9.1% wereobtained when HF-LPME with in situ derivatization was applied to determine rootexudate 1-NA, DPA, and N-P-2-NA. This proposed method is simple, inexpensive,and environmentally friendly .Therefore, HF-LPME with in situ derivatization canbe applied for the determination of the for phenylamine compound in aqueoussamples.Three developed analytical methods provided new separation technique for theallelochemicals analysis based on root exudates of hot pepper. Six donor plantsincluding Lycopersicon esculentum , Cucumis sativus, Lactuca sativa, Cucurbitamoschata , Raphanus sativu and Capsicum annuum were used to study the allopathicpotential of root exudates in hot pepper by bioassay. Lactuca sativa was theappropriate acceptor plant using the combination of subordinate function values andallelopathic response index. 28 compounds (similarity80%) including alkanes,phthalateester, phenolsanilinesand carboxylic acid were isolated and identified fromaqueous root exudate by the combination of GC-FID and GC-MS. Six- IV - Abstractconcentrations of exogenoussuspected substances (0, 2, 4, 6, 8,10 gmL -1) wereseparately applied for lettuce seed for the bioassay according to themeasuredconcentration range in sample solution. N-P-2-NA and phthalicacid (PA) wereconsidered as root exudative potential allelochemicals by the research ofgermination rate, germinationpotential, root length and shoot length. Six differentconcentration of N-P-2-NA and PA including 0, 5, 10, 15, 20, 25 gmL -1 weresolely and synergistically applied for hydroponic lettuce seedling for 7 days,Ttheeffect of two potential allelochemicals on photosynthesis, respiration, plant hormonecontent, carbon and nitrogen metabolism, ion absorption and antioxidant ability oflettuce seedling was studied. The result showed that potential allelochemicals ofN-P-2-NA and PA inhibited seed germination and seedlings growth in theconcentration range from 0 to 10 gmL -1, and the allelopathic effect of N-P-2-NA isgreater than that of PA. However, the allelopathic effect of PA is stronger that ofN-P-2-NA between 15 and 25 gmL -1. The allelopathic interaction of N-P-2-NAand PA showed inhibitory synergistic effect.Keywords: Microextraction, root exudate, derivatization method, potentialallelochemicals, allelopathy- V - 哈尔滨工业大学工学博士学位论文目录摘要 . IAbstract . III第 1章绪论 . 11.1课题研究的目的和意义 . 11.2根系分泌物的萃取方法 . 11.2.1溶剂萃取法 . 21.2.2树脂法. 31.2.3固相根区萃取法 . 31.3固相微萃取技术的发展 . 41.3.1纤维式固相微萃取 . 51.3.2管内固相微萃取 . 71.3.3搅拌棒式吸附萃取 . 81.3.4固相微萃取条件的优化 . 81.3.5固相微萃取的应用 . 101.4液相微萃取技术 . 121.4.1液相微萃取形式 . 121.4.2液相微萃取理论 . 141.4.3液相微萃取参数的选择 . 161.4.4液相微萃取的应用 . 181.5化感潜力及作用机理的研究 . 201.5.1抑制细胞分裂与幼苗伸长 . 201.5.2影响酶系统的功能活性 . 211.5.3影响离子和水分的吸收 . 221.5.4影响植物内源激素的含量 . 221.5.5影响植物光合与呼吸作用 . 231.6主要研究内容 . 24第 2章实验材料与方法 . 252.1实验材料 . 252.1.1实验药品 . 252.1.2实验材料 . 26- VI - 目录2.1.3实验仪器 . 262.1.4溶液的配制 . 272.2实验方法 . 282.2.