【作者】 李锋；

【导师】 陈英旭；

【作者基本信息】 浙江大学 ， 环境工程， 2009， 博士

【摘要】 海州香薷（Elsholtzia splendens）为唇形科香薷属一年生草本植物,对铜有较高的耐性和累积能力,被鉴定为铜耐性/富集植物。近年来,利用海州香薷进行Cu污染土壤的修复研究及海州香薷对铜吸收累积与耐性解毒的分子机理研究受到广大生物和环境科学学者的关注。本论文以海州香薷为材料,采用水培试验,运用cDNA-AFLP、实时荧光定量PCR、SDS-PAGE、2D-PAGE、MALDI-TOF MS和LTQ-ESI-MS/MS等技术,研究高铜和缺铜胁迫对海州香薷转录组和蛋白质组的影响,首次从转录组学和蛋白质组学的水平阐释了海州香薷对铜吸收累积与耐性解毒的分子机理。此外,还应用SMART技术构建了海州香薷高铜和缺铜胁迫的全长cDNA文库,为开展海州香薷功能基因组学研究提供了宝贵资源。主要研究结果如下:1、海州香薷高Cu、缺Cu胁迫的转录组学研究在转录组学研究中,采用cDNA-AFLP技术筛选海州香薷根尖（约3～5cm）基因在缺铜（0μmol/L CuSO₄）和高铜（100βmol/L CuSO₄）处理期间,四个时间点（0h、3h、6h和24h）基因的差异表达,获得了66个差异表达的TDF片断,Blast分析发现62个单基因,其中有43编码已知功能蛋白,6个编码未知功能蛋白,其余13个TDFs未能找到显著同源的序列信息。随机选择5个TDFs进行实时荧光定量PCR检测,结果与cDNA-AFLP表达谱带所示表达趋势一致。在62个单基因中,12个基因受高Cu胁迫的特异诱导,1个基因受缺Cu胁迫的特异诱导,其余的49个基因的表达在缺Cu和高Cu处理时都受到影响。在43个已知基因中,2个参与胞内氧化还原调控,6个与细胞内的信号转导有关,2个转录因子,1个翻译起始因子,9个核糖体基因,3个蛋白代谢相关基因,1个能量代谢相关,9个逆境胁迫防御基因,3个金属耐性与解毒相关基因,4个细胞壁代谢相关基因,2个次生代谢相关基因,以及1个叶主要表达蛋白基因。对这些基因在海州香薷铜吸收与耐受过程中的机制作了探讨。2、海州香薷蛋白质组学技术研究本研究对TCA/丙酮法、匀浆法和酚提取法三种蛋白提取方法进行比较发现酚提取法提取的海州香薷根、叶蛋白适于双向电泳研究。不同pH范围胶条的2-DE图谱比较研究表明胶条pH值范围为5-8时,海州香薷根、叶蛋白的2-DE分离效果最好。3、海州香薷高Cu胁迫的蛋白质组学研究本研究发现100μM Cu胁迫处理6天,海州香薷根系Cu含量增加了92倍,达5170.5mg/kg·干重,而叶部Cu含量仅增加了3倍。对海州香薷香薷根、叶蛋白的SDS-PAGE分析发现,Cu处理后,海州香薷根系蛋白谱带发生变化,而叶部蛋白的谱带变化不大。进一步对根叶的蛋白质组研究发现,Cu胁迫处理后,根系有45个蛋白点的表达发生变化,其中16个上调,29个下调;而叶部仅有6个蛋白点的表达发生变化,上调3个下调3个。对根系蛋白进行GO分析表明,这些蛋白具有多种分子功能,参与多个生物学过程,说明海州香薷的Cu耐性与解毒过程具有复杂的分子机制。海州香薷根系蛋白可以分为8类:氧化还原动态平衡相关蛋白、与转录翻译调控相关蛋白、信号转导和调控相关蛋白、与能量代谢相关的蛋白、细胞壁结构相关蛋白、细胞骨架相关蛋白、转运相关蛋白和胁迫相关蛋白。根据这些差异表达的蛋白我们阐释了海州香薷Cu耐性与解毒机制分子机理。4、海州香薷缺Cu胁迫的蛋白质组学研究本研究发现,缺Cu胁迫处理6天,海州香薷根系Cu含量下降了15.55%,而叶部Cu含量下降了25.98%。SDS-PAGE分析发现海州香薷根、叶的部分蛋白条带的亮度稍有上调。进一步对根叶的蛋白质组研究发现,根系有79个蛋白点的表达发生变化,其中49个上调,30个下调;叶部有14个蛋白点的表达发生变化,上调9个下调5个。对根系蛋白进行GO分析表明,这些蛋白具有多种分子功能,参与多个生物学过程,说明海州香薷对Cu的吸收过程同样具有复杂的分子机制。根系差异表达蛋白主要参与淀粉代谢、信号转导、氧化还原动态平衡调控、转录与翻译调控、能量代谢调控、蛋白质的修饰、降解与转运、疾病抗性、细胞壁代谢与细胞骨架调控等过程;叶部差异表达蛋白主要参与液泡离子转运、能量代谢和碳同化,另外Rubisco蛋白降解比较明显。这些蛋白质组学数据展示了海州香薷通过改变体内生化反应和代谢途径来适应缺Cu胁迫的分子机理。对海州香薷高铜和缺铜胁迫时根、叶的蛋白质组学研究结果表明根系在Cu的吸收耐性与解毒机制中具有重要的意义,可能起到Cu库的作用。海州香薷适应缺铜和高铜胁迫时,能够诱导一些类似功能的蛋白,诸如氧化还原调控、信号转导、转录与翻译调控、能量代谢调控、细胞壁代谢与细胞骨架调控、离子转运等相关的蛋白,但这些蛋白在两种胁迫时处理Cu离子的机理不尽相同。5、海州香薷高Cu、缺Cu胁迫根cDNA文库的构建本研究分别以缺铜和高铜处理3h、6h和24h的海州香薷根尖为建库材料,采用SMART技术首次构建了海州香薷缺铜和高铜胁迫的cDNA文库。缺铜胁迫cDNA原始文库滴度为1.22E+08 pfu/mL,文库所含独立克隆数为6.41E+07个,扩增文库滴度为3.96E+12 pfu/mL,重组率为98%,插入片段82%大于500bp。高铜胁迫cDNA原始文库滴度为3.44E+07pfu/mL,文库所含独立克隆数为1.81E+07个,扩增文库滴度为4.86E+12 pfu/mL,重组率为99%,插入片段79%大于500bp。更多还原

【Abstract】 Elsholtzia splendens is an annual herbaceous plant,belonging to the family Labiatae,which can endure elevated copper concentration and accumulate high level copper content.In China,it has been identified and accepted as Chinese native Cu-tolerant and accumulating plant,in the last few years,more research works have been conducted on the phytoremediation of copper contamination soil and its mechanism in copper tolerance and accumulation.In this study, hydroponics was used to investigate the transcriptomic changes of E.splendens in response to Cu-excess(100μmol/L) and Cu-deficiency(0μmol/L) stresses by cDNA-AFLP and Real-time quantity PCR technique,to explore the proteomic changes of E.splendens in response to Cu-excess (100μmol/L) and Cu-deficiency(0μmol/L) stresses by SDS-PAGE,2D-PAGE,MALDI-TOF MS and LTQ-ESI-MS/MS technique.The mechanisms of Cu uptake,accumulation,tolerance and detoxification in E.splendens were elucidated at the transcriptome and proteome levels for the first time.In addition,the fully length cDNA libraries of E.splendens treated with 100μmol/L and 0μmol/L Cu were constructed,which provided costful resources for functional genome research such as clone sequencing and gene screening and so on.The main results are listed as follows:1.Study of transcriptomics in E.splendens upon Cu-excess and Cu-deficiency stressesIn the transcriptomic study,cDNA-AFLP analysis was used to investigate the gene expression profile in E.splendens root tips(about 3～5cm) at four time-points(0h,3h,6h and 24h) during the Cu-excess and Cu-deficiency stresses.Sixty-six transcript derived fragments(TDFs) shown differential expression pattern were obtained.62 unique genes were found by Blast analysis,which were composed of 43 function-known genes,6 function-unknown genes and 13 novel genes.Five TDFs were selected random and the expression levels were assessed using real-time quantitative reverse transcription PCR(qRT-PCR) and the results were consistent with that of cDNA-AFLP. Among the 62 unique genes,12 genes were induced by Cu-excess stress,one gene was induced by Cu-deficiency stress,and other 49 genes were induced by both Cu-excess and Cu-deficiency stress. Of the 42 function-known genes,two genes are involved in redox homeostasis,six for signal transduction,three for protein metabolism,one for energy metabolism,nine for stress resistance, three for metallic tolerance and detoxification,four for cell wall metabolism,two for secondary metabolism,and includes one eukaryotic translation initiation factor,two transcription factors,nine ribosomal genes and one predominantly leaf-expressed protein gene.These Cu-induced genes indicate that Cu toxicity affected different physiological and biochemical pathways of the plant.2.Study of proteomics technique of E.splendensThe results of comparison on three extraction methods(TCA/acetone method;homogenization buffer method;phenol extraction method) of E.spIendens total protein showed that the E. splendens root and leaf protein extracted using phenol extraction method were adapted for two dimensional electrophoresis.E.splendens root and leaf proteins were Separated by two dimensional electrophoresis using IPG strips with different pH,and the maps of 2-DE using IPG strip with pH5-8 have the best results with high resolutions and much more protein spots.3.Study of proteomics in E.splendens upon Cu-excess stressThe present study investigated the Cu accumulation and proteome changes in E.splendens tissues upon Cu stress.After 6 days of treatment with 100μM Cu,Copper contents were increased with the 100μM Cu treatment in both leaves and roots,the concentration in the roots being 92-fold increased compared to controls,reaching 5170.5 mg/kg dry weight,but only 3-fold increased in the leaves.SDS-PAGE analysis indicated that the proteins changed more intensively in roots than that in leaves proteins upon Cu treatment.2-DE and image analyses found that 45 protein spots, including 16 up- and 29 down-regulated protein spots were significantly changed in roots,but only 6 protein spots with equal up- and down-regulated protein spots in leaves.Gene ontology analyses showed that these root proteins have many molecular functions and are involved in many biological processes,which indicates that complicated physiological and biochemical changes were involved in the mechanism of Cu tolerance and detoxification in E.splendens.The identified E.splendens root proteins were classified into several functional categories including signal transduction,redox homeostasis,regulation of transcription and translation,energy metabolism,cell wall metabolism, cell cytoskeleton rearrangement,transport and stress resistance.The roles of these different expressed proteins gained insight into the molecular mechanisms of E.splendens Cu tolerance and detoxification.4.Study of proteomies in E.splendens upon Cu-deficiency stressThe present study investigated the Cu accumulation and proteome changes in E.splendens tissues upon Cu-deficiency stress.After 6 days of treatment with 0μM Cu,the decrease of copper contents was 25.98 percent in roots and 15.55 percent in leaves.SDS-PAGE analysis showed that the intensity of some root and leaf protein bands was slightly increased.To further investigate the changes of protein profiles during Cu-deficiency stress,2-DE analysis of the total proteins in E. splendens roots and leaves were carried out.Quantitative image analysis revealed a total of 79 and 14 protein spots that changed their intensities significantly by more than 2.0-fold in roots and leaves respectively.There were 49 up-regulated spots and 30 down-regulated spots in roots,9 up-regulated spots and 5 down-regulated spots in leaves.Gene ontology analyses showed that these root proteins have many molecular functions and are involved in many biological processes,which indicates that complicated physiologiCal and biochemical changes were involved in the uptake process and response to Cu-deficiency stress.The identified E.splendens root proteins under Cu-deficient condition are closely associated with starch metabolism,signal transduction,energy metabolism,redox homeostasis,regulation of transcription and translation,energy metabolism, protein modification、degradation and transport,disease resistance,cell wall metabolism and cell cytoskeleton rearrangement.The differently expressed proteins in E.splendens leaves were manly involved in ion transport across the tonoplast,energy metabolism and carbon assimilation.In addition,Rubisco might undergo enhanced degradation under Cu stress.The results of these proteomic data indicated that E.splendens changed the biochemical reactions and metabolic pathways to enhance copper uptake,reutilization and other adaptive changes.All together,the proteomic analysis of the differentially displayed proteins in E.splendens roots and leaves upon Cu-excess and Cu-deficiency stresses indicated that E.splendens root organs played an important role in Cu uptake and tolerance,which might act as a Cu-sink.E.splendens adaptation to Cu-excess and Cu-deficiency stresses can synthesize some proteins involving in the same processes such as redox homeostasis,signal transduction,transcription and translation, energy metabolism,cell wall metabolism,cell cytoskeleton rearrangement and ion transport.But, the roles of these proteins between Cu-excess and Cu-deficiency stresses might be different.5.Construction of cDNA library from E.splendens roots challenged by Cu-exeess and Cu-defieieney stressesIn this study,root tips of E.splendens treated with 0 and 100μmol/L Cu for 3h,6h and 24h were used for construction of cDNA library.Two nonnormalized full-length cDNA library were constructed based on SMART technique.The titer of unamplified Cu-deficient library is 1.22E+08pfu/mL.Individual clones of the library are 6.41E+07.The titer of amplified Cu-deficient library is 3.96E+12 pfu/mL.The percentage of recombinant clones is 98%.82%of the inserted cDNA is long over 500bp.The titer of unamplified Cu-excessive library is 3.44E+07pfu/mL. Individual clones of the library are 1.81E+07.The titer of amplified Cu-excessive library is 4.86E+12pfu/mL.The percentage of recombinant clones is 99%.79%of the inserted cDNA is long over 500bp.更多还原