【作者】 董蔚；

【导师】 夏光敏；

【作者基本信息】 山东大学 ， 细胞生物学， 2012， 博士

【摘要】 近年来,农业生态环境不断恶化,土壤的高盐逆境愈发严重,对农作物产量造成巨大威胁。因此,培育耐盐作物新品种是现代农业的一项重要而紧迫的任务,已成为全世界的研究热点之一。小麦是世界上分布最广、种植面积最大、加工制品最为丰富的重要粮食作物。但它属于甜土植物,耐盐能力较差。本实验室在前期研究中,建立了通过不对称体细胞杂交方法创制小麦渐渗系的技术体系(国家发明专利：ZL200410075774.7),并将小麦近缘优质牧草、单子叶植物耐盐性最强的长穗偃麦草的染色质渐渗到普通小麦,创建了一批高产、抗逆、抗病和优质的渐渗系新种质和新品系,从中选育出杂种渐渗系高产、耐盐新品种山融3号(SR3)(鲁审[2004]030),获得了研究小麦渐渗系机制和功能基因的“突变体”新材料。转录组和蛋白组分析表明SR3抗逆性与其较强的抗氧化能力密切相关。盐胁迫能诱导细胞产生大量的活性氧(Reactive Oxygen Species, ROS),形成次级氧化胁迫。植物在进化过程中形成了一套复杂有效的ROS清除系统,包括酶促和非酶促抗氧化剂及调控其表达的各种信号分子和转录因子。ROS能诱导脂质过氧化(lipid peroxidation),形成多种氧化产物,部分含有不饱和键,引起膜氧化损伤等毒性伤害,因而氧化的脂肪酸(义称为羟脂,oxylipids),是膜氧化损伤的优良指标及细胞感知氧化胁迫的信号。细胞中的羟脂种类较多,其中最受关注的是12-羰-植物二烯酸(12-oxo-phytodienoic acid, OPDA)及其代谢产物茉莉酸(jasmonic acid, JA),而二者都是十八碳烷酸合成途径的代谢物。据此,本实验分析了SR3和JN177中十八碳烷酸合成途径相关基因的胁迫应答表达潜,克隆了小麦SR3十八碳烷酸合成途径相关基因TaOPR1, TaOPR2以及TaAOC1,研究了其过表达对拟南芥发育和抗逆能力的影响,确定其在胁迫下的表达变化与SR3抗逆性的关系、探讨该途径的基因在植物发育和抗逆中的作用机制。主要研究内容和结果包括：1植物十八碳烷酸合成途径基因的表达谱分析为了进一步认识植物十八碳烷酸合成途径的羟脂物质在植物应对盐胁迫中所起的作用,我们对该途径基因的胁迫表达谱进行了分析。首先我们运用本实验室SR3和JN177200mMNaCl和18%PEG胁迫下的芯片信息,分析了该途径关键酶基因AOS,AOC, OPR的胁迫表达状况。结果显示,在小麦芯片中,这3个基因都以多拷贝存在,不同探针所对应的基因对于NaCl和PEG胁迫的响应趋势与变化量都不一样。根据其响应模式的不同,将每种基因的变化模式归类,发现有一类OPR基因,在NaCl胁迫下表达上调达20倍以上,有的高达几十倍甚至于上百倍,并且它们在SR3中的上调趋势比JN177更加明显。暗示了这类基因在SR3的NaCl胁迫响应中发挥了重要的作用。另外,我们通过TAIR网站在线分析了拟南芥相对应的AOS, AOC, OPR基因在NaCl和十早胁迫下的表达趋势变化。结果显示,AOS基因只有1个拷贝呈轻微上调的表达趋势；AOC基因有四个拷贝,其变化模式各不相同；OPR有3个拷贝都呈现出显著地上调表达,其中AtOPR1和AtOPR2属于OPR基因的第一亚家族,其上调趋势比属于OPR第二亚家族的AtOPR3上调更为明显。这些结果说明,小麦植物十八碳烷酸合成途径的基因在植物盐胁迫响应中发挥了一定的功能,其中OPR第一亚家族基因的功能可能更为重要。2TaOPR1和TaOPR2的克隆及其在非生物胁迫应答中的功能研究根据基因在SR3和JN177中表达量的差异,本实验室从小麦SR3中克隆了2个第一亚族的12-羰-植物二烯酸还原酶基因TaOPR1和TaOPR2。RT-PCR和Real-time PCR结果显示,对照条件下TaOPR1在SR3中的表达量高于JN177；200mMNaCl、18%PEG6000、100μM ABA和10mM H202胁迫条件下SR3和JN177中该基因的响应趋势基本相同,但是SR3的表达量更高；100μM的ABA合成抑制剂Norflurazon可抑制TaOPR1在200mMNaCl中的上调表达。TaOPR1小麦过表达(CE)株系以及拟南芥过表达(OE)株系在含有NaCl的培养基中的生长状态明显优于空载体对照(VC)株系,CE株系与OE株系相对于各自的对照株系而言,超氧化物岐化酶(SOD)、过氧化氢酶(CAT)等ROS清除酶活性显著提高,而丙二醛(MDA)含量则显著下降。这表明TaOPR1的过表达可以显著的提高植物的耐盐性。TaOPR1的拟南芥OE系在含有H202培养基中的生长状态也明显优于VC系,并且OE系中的ROS含量明显低于VC系；在含有ABA的培养基中,无论是种子萌发还是植株生长,OE株系都表现出了对ABA更为敏感的表型；Real-time PCR分析显示,OE株系中ABA合成途径关键基因AtABA1、AtABA2、AtNCED3和AtAAO3以及ABA依赖的胁迫应答响应关键基因AtRD22及其调控因子AtMYB2、AtMYC2等的表达均明显上调。这些结果表明TaOPR1可通过增强植物对ROS的清除能力,以及刺激ABA合成以及ABA依赖的胁迫应答途径来提高植物的耐盐能力。另外,TaOPR1的拟南芥OE系在含有JA的培养基中生长状态与VC系没有明显的差别,RT-PCR分析显示OE株系中JA合成以及信号转导途径MARKER基因的表达量均没有明显的变化。因此,我们认为TaOPRl与植物的JA途径没有关系,这与第一亚家族的OPR基因不参与JA合成的结论是一致的。Real-time PCR分析显示,NaC1、PEG和H202、胁迫下,以及胁迫响应激素ABA处理下,TaOPR2在SR3中均呈现持续上调的表达模式,说明TaOPR2也参与植物对非生物胁迫应答。作为同源基因,TaOPR2与TaOPR1在各种胁迫下的表达趋势相同；但是,TaOPR2在胁迫条件下的基因表达上调比TaOPR1明显,并且TaOPR2在渗透胁迫下的表达较盐胁迫下上调幅度更大。TaOPR2拟南芥OE系的表型分析显示,TaOPR2也可以通过刺激ROS清除途径以及ABA依赖的信号途径提高拟南芥对于盐胁迫、渗透胁迫以及过氧化胁迫的抗性。并且TaOPR2的作用与JA途径也没有关系。与TaOPR1比较,TaOPR1的OE株系在H202胁迫下不仅根长于VC株系,地上部分的生长状态也优于VC株系。Real-time PCR分析显示,在TaOPR2的OE株系中,ABA途径抑制因了AtABI1的表达被明显下调,表明TaOPR2主要通过ABA途径的负调控因子起作用。另外,TaOPR2还能通过DREB途径调控非ABA依赖途径胁迫响应基因(例如AtRD29A)的表达,这说明TaOPR1与TaOPR2两个基因虽然在功能上有着一定的冗余,但是也有着一定的分化,在植物胁迫应答中有独特的机制。3TaAOC1的克隆及其在非生物胁迫应答中的功能研究二烯氧化物环化酶基因基因(AOC)是OPDA合成途径的最后一个酶也是JA合成途径的关键酶,在植物的十八碳烷酸合成途径中具有重要的意义。本实验室从小麦SR3中首次克隆得到了一个二烯氧化物环化酶基因,命名为TaAOC1。该基因受到NaCl和PEG的诱导以后均呈现出短期上调的表达趋势。TaAOC1过表达(OE)转基因纯系的生长受到明显抑制,在正常的条件下呈现与外源施加JA的类似的根系表型,在盐胁迫培养基中,OE株系表现出对盐胁迫不敏感并且侧根增多的表型被抑制。这些结果进一步揭示了AOC基因以及植物十八碳烷酸合成途径在植物耐逆过程中发挥了重要的作用,但是其具体作用机制尚需进一步研究。更多还原

【Abstract】 In recent years, the agricultural ecological environment is deterisrating and the high salinity stress becomes more and more serious, which adversely affect the crop production. Therefore, to cultivate salt tolerant crops are an important and urgent task of modern agriculture and have becoming one of the world’s research focuses. Wheat is one of the most important crops worldwide, but its growth and productivity are obviously inhibited by abiotic stresses such as drought and salt. In our previous work, a new wheat introgression line Shanrong No.3(SR3) was generated using common wheat Jinan177(JN177) and Thinopyum ponticum, a salt and drought tolerant grass, via asymmetric somatic hybridization. Former results showed that SR3’s genome were integrated with some chromatin fragments of T. ponticum, and took place a high frequency of allelic variation, indicating that SR3is an excellent material for investigating genetic variation, isolating stress tolerance associated genes, and dissecting the mechanisms underlying stress response. Transcriptomic and proteomic analysis showed that the SR3’s resistance is closely related to its strong antioxidant capacity.Salt stress could induce cells to produce large amount of reactive oxygen species (ROS), which perform as intracellular secondary oxidative stress. Plants have evolved a complex and effective ROS scavenging system to cope with such stress, including enzymatic and non-enzymatic antioxidants as well as complicated signaling molecules and transcription factors to regulate their expression. ROS-induced lipid oxidation (lipid peroxidation) form a variety of oxidation products, some of which contain unsaturated bonds and cause membrane oxidative damage and other toxic injuries, so the oxidized fatty acids (also known as oxylipids) is the indicator of membrane oxidative damage degree, and serve as signals for cells to perceive oxidative stress. There have many types of oxylipids, of which the most characterized are the12-oxo-phytodienoic acid (OPDA) and its metabolite jasmonic acid, two intermediates of octadecanoid pathway, suggesting the possible role of this pathway in SR3’s high salt tolerance. OPDA reductases (OPRs) catalyze the reduction of OPRA, the first step of its conversion to JA, and they are classified into two subgroups, OPRI and OPRII; OPRII participates in JA synthesis, but OPRI’s function is still not known. Here, we performed a comprehensive comparison on the transcriptional patterns of genes involved in octadecanoid synthesis pathway, from which we cloned three octadecanoid pathway associated genes TaOPR1, TaOPR2, and TaAOCl from SR3, ascertained their roles in development and tolerance to abitoic stresses through overexpression in Arabidopsis and wheat, and uncovered the mechanisms of these genes in governing development and abiotic stress response in plants. The main research contents and results achieved in this work were summarized as follows.1. Screening expression patterns of octadecanoid pathway genesIn order to further know the relationship of oxylipids synthesized by octadecanoid pathway in the plant response to salt stress, we performed a screening of the expression patterns of genes involved in the pathway during abiotic stress. Firstly, we analyzed the expression profiles of genes encoding key enzymes in the pathway such as AOS, AOC, and OPR, according to the data from cDNA microarray of SR3and JN177. Bioinformatic analysis showed that AOS, AOC and OPR encoding genes have multiple copies in wheat genome, and the probes referring to these genes in cDNA microarray appeared differential expression patterns. We then divided these genes into several classes based on their stress responsive modes, and found there has one class of OPR genes that were dramatically induced by more than20or even100fold under salt stress, and the induction strength were much stronger in SR3than in JN177. This implies that these OPR genes may play important roles in salt stress response in wheat.In addition, we analyzed the expression patterns of Arabidopsis AOS, AOC and OPR genes under NaCl and PEG stresses with the online transcriptomic data at TAIR website. The results show that all of three OPR copies were significantly induced, with a more induction extent in OPRI subfamily AtOPR1and AtOPR1than in OPRII subfamily gene AtOPR3. Besides, all of four AOC copies were stress-responsive with different patternsonly, but one copy of AOS genes was slightly up-regulated.These results suggest that octadecanoid synthesis pathway associated genes of wheat and other plants certainly participate in plant salt stress response, of which the linkage between OPR genes, especially OPRI genes, and the response may be closer.2. Cloning and function analysis of TaOPRl and TaOPR1in abiotic stress toleranceComprehensive transcription comparison revealed the close relation between OPRI genes and salt stress response. Therefore, we cloned an OPRI subfamily gene TaOPR1and TaOPR2from SR3based on its most obvious induction by salt stress in cDNA microarray.RT-PCR and Real-time PCR showed that TaOPR1was induced by under NaCl, PEG6000, ABA and H2O2stresses in both SR3and JN177, with a more significant degreee in SR3. The induction of TaOPR1by NaCl can be inhibited by ABA biosynthesis inhibitor norflurazon, indicating the NaCl-induced TaOPR1expression is ABA-dependent.To know its role in salt stolerance, TaOPR1was transformed into wheat and Arabidopsis. TaOPR1overexpression did not affect the growth and reproduction ability of wheat and Arabidopsis. Under NaCl stress, wheat overexpression lines (CE) had a superior growth ability than the wildtype, and Arabidopsis overexpression lines (OE) also grew more vigorously than the empty vector control (VC) lines, indicating the positive contribution of TaOPR1in salt tolerance.In TaOPR1overexpressors, the activities of ROS scavengers superoxide dismutase (SOD) and catalase (CAT) were significantly increased, but ROS and malondialdehyde (MDA) contents were decreased evidently. Consistently, under H2O2stress, Arabidopsis OE seedlings were stronger than the VC line, and their seeds also had higher germination rate. This indicates that the contribution of TaOPR1in salt tolerance is partially achieved through its role in enhancement of ROS removal.Arabidopsis OE lines were hypersensitive to exogenous ABA, and their seedling root length and seed germination rate were both restricted more obviously than the VC line. Real-time PCR analysis showed that in ABA biosynthesis pathway associated genes AtABA1, AtABA2, AtNCED3and AtAAO3and ABA-dependent stress response pathway genes AtRD22, AtMYB2and AtMYC2all had higher transcript levels in OE lines. These results provide convinced evidence that besides through helping in ROS removal, TaOPR1enhanced stress tolerance by promoting the ability of ABA synthesis and ABA-dependent stress response pathway.In addition, Arabidopsis OE lines and the VC line showed similar response to exogenous JA. RT-PCR analysis demonstrated that JA biosynthesis and signal transduction pathway associated genes except for AtAOCl had no differential transcription patterns between the OE and VC lines. This indicates that TaOPR1has no crosstalk with JA pathway, which is in agreement with the knowledge that the OPRI subfamily genes are not involved in JA biosynthesis.Real-time PCR analysis showed that TaOPR2can be activated by NaCl, PEG, H2O2, and ABA in SR3, indicating that TaOPR2is also involved in abiotic stress responses. In comparison with TaOPR1, the response of TaOPR2to abiotic stresses was more distinguishable; TaOPR2accumulated more transcripts under PEG than NaCl stresses.Alike TaOPR1, TaOPR2overexpression also enhanced tolerance to salt, PEG and H2O2stresses, and sensitivity to exogenous ABA. But unlike TaOPR1, TaOPR2overexpressors not only had longer roots, but also larger leaves, indicating its excellent potential for crop breeding.Physiological and molecular analysis indicated that TaOPR2also promote the activities of ROS scavengers and ABA-dependent stress response pathway. But unlike TaOPRl, TaOPR1overexpression inhibited the expression of AtABI1, whose encoding protein is an inhibition factor for ABA signaling pathway, indicating that the acceleration of ABA-dependent stress response pathway by TaOPR2is accomplished through releasing its repression. Besides, TaOPR2but not TaOPR1induced the expression of DREBs, key components of ABA-independent stress response pathway. The similarities of TaOPRl and TaOPR2as well as their specificities in role of abiotic stress response indicating their functional conservation and differentiation during evolution 3. Cloning and function analysis of TaAOCl in abiotic stress toleranceAOC catalyzes the last step of OPDA biosynthesis, during which AOC can convert cis-OPDA to trans-OPDA, the in vitro substrate of OPRIs, suggesting its putative coordinated role in TaOPR1/TaOPR2-induced salt tolerance. Based on this speculation, we first cloned a wheat AOC gene TaAOC1from SR3, which shares the highest identity to AtAOCl, the AOC copy induced in Arabidopsis TaOPR1OE lines, among available wheat ESTs annotated as AOC. The gene was induced at early stage of salt and PEG600treatments. TaAOC1overexpression Arabidopsis (OE) lines showed a growth-inhibited and lateral root-increased phenotype, which is similar to the wild-type seedlings exposed to exogenous JA. TaAOC1OE lines had superior salt tolerance to salt stress and the lateral root-increased phenotype was controled. These results provide a primary glimpse to the role of AOC and octadecanoid pathway metabolites in plant tolerance to abiotic stresses, but how they perform this role needs to be further studed.更多还原