【作者】 胡伟；

【导师】 何光源；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 小麦（Triticum aestivum L.）是世界上最重要的粮食作物之一，也是我国的第二大粮食作物，为全球35%以上的人口提供每日所需的蛋白质和热量。非生物逆境胁迫是指由复杂的环境因素所导致的多种胁迫，主要包括强光照、紫外线、低温、冰冻、干旱、盐碱、重金属和低氧胁迫等。非生物逆境胁迫严重影响植物的生长发育，最终会影响作物产量和品质。其中干旱胁迫严重影响着我国多个麦区小麦的生产。所以，研究小麦对干旱胁迫的耐受机制对于小麦抗性遗传改良具有重要意义。近年来，脱落酸-胁迫-成熟响应蛋白（Abscisic acid ABA-, stress-, and ripening-inducedproteins, ASR）基因已经在多个物种中有报道，已经被证明能够增加植物对非生物逆境胁迫的耐受性。但是，ASR基因赋予植物对非生物胁迫的耐受性的相关机制还不清楚。在小麦中ASR基因的研究还未见报道。本研究旨在探索小麦ASR基因是否能够赋予植物对非生物逆境胁迫的耐受性，以及小麦ASR基因赋予植物对非生物胁迫的耐受性的生理和分子机制。所以，本研究首先分析了小麦模式品种中国春（Triticumaestivum L. cv. Chinese Spring）中ASR基因家族与渗透胁迫的关系，克隆了其中一个候选基因TaASR1；进一步利用遗传学、生理学及生物化学和分子生物学的方法研究了TaASR1在非生物胁迫中的功能和作用机制，取得的主要研究结果如下：1)小麦ASR基因家族EST序列与渗透胁迫的关系从DFCI小麦数据库中共获得了16个小麦ASR基因的EST序列，表达分析结果表明BE414974（group1），TC418737I（group2），CJ554788（group3）和CK154393（group6）被聚乙二醇（PEG）处理诱导。而且，BE414974（group1）在正常条件下表达量比其它ASR基因的EST序列表达量高，在PEG胁迫处理条件下能够被显著诱导。所以，我们以group1ASR基因作为候选基因进行进一步功能分析。2) TaASR1基因的克隆及生物信息学分析利用RACE技术获得了group1ASR基因TaASR1的全长cDNA序列，该基因的ORF为414bp，编码一个由137个氨基酸组成的蛋白质。TaASR1所编码蛋白具有ASR家族蛋白所具有的保守结构域及活性区域。进化分析表明，TaASR1与水稻和玉米ASR基因具有较近的进化关系。3) TaASR1基因的表达分析器官差异表达分析表明，TaASR1在营养器官根、茎、叶中表达量较高，在生殖器官雌蕊、雄蕊中表达量则较低。TaASR1基因的表达能够显著受到渗透胁迫、脱落酸（Abscisic acid，ABA）和双氧水诱导。茉莉酸甲酯（Methyl jasmonate，MeJA）、水杨酸（Salicylic acid，SA）和生长素能够轻微的诱导TaASR1上调表达。而且，ABA和双氧水信号可能参与PEG诱导TaASR1基因的上调表达。4) TaASR1转基因烟草对干旱胁迫的耐受性分析将TaASR1基因克隆到pCAMBIA1304植物表达载体，利用农杆菌转化方法成功获得了T2代转基因烟草株系。表型分析结果表明，过表达TaASR1能够增强转基因烟草对干旱胁迫的耐受能力。生理分析结果表明，在干旱胁迫处理条件下，与野生型相比较转TaASR1基因烟草植株具有较高的相对水含量（Relative water content，RWC）、超氧化物歧化酶（Superoxide dismutase，SOD）活力和过氧化氢酶（Catalase，CAT）活力；较低的离子渗漏（Ion leakage，IL）、丙二醛（Malondialdehyde，MDA）含量和双氧水含量。这些结果表明，过表达TaASR1能够显著增强植株抗氧化酶的活力从而使转基因植物能够更好的对抗干旱胁迫所导致的氧化损伤。5) TaASR1转基因烟草对渗透胁迫的耐受性分析在高渗培养基上进行种子发芽率分析和根长测定的结果表明，过表达TaASR1能够有效地降低渗透胁迫对烟草发芽和根长的抑制。二氨基联苯胺（Diaminobenzidine，DAB）和氯化硝基四氮唑蓝液(Nitrobluetetrazolium，Nitrotetrazolium blue chloride，NBT)染色、双氧水含量及抗氧化酶活测定结果表明，在渗透处理条件下过表达TaASR1能够降低烟草中活性氧（Reactive oxygen species，ROS）的积累，增加抗氧化酶活性。胁迫相关基因表达分析表明，在正常生长条件下，NtERD10C、NtERD10D和NtNCED1基因的表达量在转TaASR1基因烟草植株中显著高于野生型；在渗透胁迫处理条件下，NtERD10C、NtERD10D、NtNCED1、NtSOD、NtCAT、NtLEA5、NtLTP1、NtDREB3和TobLTP1基因的表达量在转TaASR1基因烟草植株中显著高于野生型。6) TaASR1转基因烟草对氧化胁迫的耐受性分析表型分析结果表明，在氧化胁迫处理条件下，转TaASR1烟草植株比野生型植株表现出较少的叶片黄化。而且，转TaASR1烟草植株比野生型烟草植株具有较高的叶绿素含量、较低的双氧水含量、较高的超氧化物歧化酶活力和过氧化物酶活力和表达。7) TaASR1转录激活分析我们将TaASR1、TaASR1-N和TaASR1-C克隆到酵母表达载体pGBKT7。结果表明，转pGBKT7-TaASR1和转pGBKT7-TaASR1-N的酵母菌能够在组氨酸缺陷型SD培养基上生长。而且，在组氨酸缺陷型SD培养基上加入5-溴-4-氯-3-吲哚基-β-D-吡喃半乳糖苷（5-bromo-4-chloro-3-indolyl-β-D-galacto-pyranoside，X-gal）后，转pGBKT7-TaASR1和转pGBKT7-TaASR1-N的酵母菌能够显示蓝色。8) TaASR1亚细胞定位分析将TaASR1转入洋葱表皮细胞中表明，TaASR1定位在细胞核中。转基因烟草根系切片观察也表明TaASR1定位在细胞核中。9) TaASR1转化小麦利用基因枪法将TaASR1基因导入小麦中，获得了T0代转基因阳性植株。综合以上结果，本研究的主要结论是从小麦中克隆了一个脱落酸-胁迫-成熟响应蛋白基因TaASR1，证明TaASR1作为一个转录因子通过调控抗氧化系统和胁迫防御相关基因的表达赋予植物对渗透、干旱及氧化胁迫的耐受性。更多还原

【Abstract】 Wheat (Triticum aestivum L.) is the foremost staple food crop in the world whichprovides both calories and proteins to over35％of the human population. Wheat is thesecond most important staple crops in China. The term abiotic stress‘includes numerousstresses caused by complex environmental conditions, e.g. strong light, UV, high and lowtemperatures, freezing, drought, salinity, heavy metals and hypoxia. Abiotic stresses leadto serious damage for plants, especially for crop yield and quality. Therefore,understanding the molecular mechanisms of abiotic stress responses is necessary forgenetic improvement of stress resistance in wheat. Recently, Abscisic acid ABA-, stress-,and ripening-induced proteins (ASR) have been reported to increase tolerance to abioticstresses in various species. However, the mechanisms for its action remain unclear and noresearch on ASR in wheat has been reported. The perpose of present study is to investigatewhether wheat ASR genes can confer drought stress tolerance and if so what ismechanisms underlying the tolerance? Firstly, we analysed the relationship between ASRfamily gene and osmotic stress in model wheat variety Chinese Spring and cloned acandidate gene TaASR1. Furthermore, we characterized the function of TaASR1in abioticstresses by using genetic, physiological and biochemical and molecular approavhes. Themain results are as follows.1. The relationship between the ESTs of ASR gene family and osmotic stressFrom the DFCI wheat gene index database,16ASR EST sequences were acquired.RT-PCR analysis showed that expression of BE414974(group1), TC418737I (group2),CJ554788(group3) and CK154393(group6) was induced by PEG treatment. Notably,group1exhibited higher expression in normal conditions and was strongly up-regulatedafter PEG treatment. Therefore group1was selected as a candidate for further functionalcharacterization.2. TaASR1cloning and bioinformatics analysisEmploying RACE-PCR the full-length cDNA of ASR gene, designated as TaASR1,was cloned using mRNA extracted from the leaves of wheat seedlings as template.TaASR1cDNA comprised of544bp with a414bp open reading frame (ORF). The deduced TaASR1protein contained137amino acid residues. Based on amino acidsequence alignment, two highly conserved regions were observed in TaASR1.Phylogenetic analysis indicated that16ASR proteins branched out into separate monocotand dicotyledon groups with TaASR1very close to OsASR and ZmASR2in monocotgroup.3. The expression analysis of TaASR1TaASR1was expressed in all tissues including root, stem, leaf, stamen, pistil andlemma with higher expression in root, stem, leaf and lemma. TaASR1was induced byosmotic stress, ABA and H2O2. TaASR1transcript induction by MeJA, SA and auxin wasmarginal and occurred much later. Moreover, the upregulation of TaASR1by PEGpossibly involves ABA and H2O2signaling.4. Analysis of drought stress tolerance in TaASR1-overexpressing tobacco andWT plantsTo further investigate the role of TaASR1in drought/osmotic stress tolerance,transgenic tobacco plants over-expressing TaASR1under the control of CaMV35Spromoter were generated. Over-expression of TaASR1in tobacco resulted in increaseddrought/osmotic tolerance, which was demonstrated that transgenic lines had lessermalondialdehyde (MDA), ion leakage (IL) and reactive oxygen species (ROS), but higherrelative water content (RWC) and superoxide dismutase (SOD) and catalase (CAT)activities than wild type (WT) under drought stress.5. Analysis of osmotic stress tolerance in TaASR1-overexpressing tobacco andWT plantsSeeds germination rate and root length were higher and longer in transgenic linesthan in WT and Vector Control (VC) treated with150or300mM mannitol.Over-expression of TaASR1reduced ROS accumulation by enhancing the SOD and CATactivities under osmotic stress. Stress related genes selected include NtSOD, NtCAT andNtPOX involved in ROS detoxifcation, NtNCED1involved in ABA biosynthesis,NtERD10C, NtERD10D and NtLEA5related to stress defense, the regulatory geneNtDREB3and lipid-transfer protein genes NtLTP1and TobLTP1. Under normal conditions,expression of NtERD10C, NtERD10D and NtNCED1were higher in OE12than in WT.Although expression levels of all tested genes were up-regulated by osmotic stress, they were higher in transgenic plants than in WT except for the NtPOX under osmotic stress.6. Analysis of oxidative stress tolerance in TaASR1-overexpressing tobacco andWT plantsTobacco plants were subjected to30μM methyl viologen treatment. This resulted insevere cotyledon bleaching or chlorosis in WT and VC plants than transgenic plants. Inaddition, transgenic lines displayed higher chlorophyll, lower H2O2contents and higheractivity and expression of SOD and CAT than WT and VC.7. Transcriptional activity of TaASR1Yeast strain AH109was transformed with the fusion plasmids pGBKT7-TaASR1,pGBKT7-TaASR1-N, pGBKT7-TaASR1-C and pGBKT7(Control)(Fig.12A) and thegrowth status of transformants was evaluated. Yeast cells containing pGBKT7-TaASR1and pGBKT7-TaASR1-N grew well in SD medium lacking histidine. Moreover, in thepresence of X-gal, yeast cells that grew well on the SD medium without histidine turnedblue.8. Subcellular location of TaASR1Fluorescence of the35S::TaASR1-GFP chimera was associated with cellular nucleusin onion epidermal cells, suggesting a nuclear localization. In the root cells of plantstransformed with TaASR1-GFP, fuorescence was also observed in the nuclei.9. Overexpression of TaASR1in wheatThe positive transgenic wheat plants were aquired by using gene gun method.In conclusion, the fndings of this study demonstrated that TaASR1function as atranscription factor in abiotic stress tolerance. TaASR1conferred drought, osmotic andoxdidative stress tolerance through regulating the expression of stress-, anddefense-associated genes and enhancing the antioxidant system, thus preventing plantsfrom oxidative damage.更多还原