【作者】 王顺才；

【导师】 束怀瑞；

【作者基本信息】 西北农林科技大学 ， 果树学， 2011， 博士

【摘要】 干旱胁迫是农业生产中存在的重要问题,对世界作物产量的影响,在环境胁迫中占首位。而国内外关于干旱对果树的影响及其适应机理研究落后于其它农作物。苹果是世界上重要的栽培果树之一,是我国北方落叶果树中栽培面积最大的树种之一。苹果是嫁接繁殖的果树,其抗逆性主要决定于砧木。在我国苹果砧木的抗性研究起步较晚,因此了解苹果砧木的耐旱能力,深入研究干旱胁迫对苹果砧木的伤害及其响应生理和分子机理,对抗旱遗传改良都具有重要的意义。本文以两年生楸子（Malus prunifolia（Willd）Borkh.）和平邑甜茶（M. hupehensis （Pamp） Rehd.）盆栽幼苗为试材,采用控水方式进行干旱处理。比较分析了干旱胁迫下这两种苹果砧木叶片的活性氧代谢变化、细胞形态解剖结构及亚细胞超微结构的变化及其两者的抗性差异。在此研究基础上利用抑制消减杂交（SSH）技术构建干旱处理的楸子叶片cDNA文库,筛选并鉴定出部分与抗旱相关的ESTs,并应用定量PCR（qRT-PCR）技术分析它们在干旱胁迫下的表达谱。采用菌液PCR筛选SSH cDNA文库与电子克隆方法,克隆抗性基因的全长cDNA序列,运用生物信息学方法分析抗性基因的核酸序列特征、基因编码的氨基酸序列特征及分子进化关系,使用同源建模技术预测和分析基因编码蛋白的空间结构与生物功能。另外,采用qRT-PCR和半定量PCR技术分析抗性基因在不同组织及不同逆境胁迫下的表达谱。主要结果如下:1.楸子和平邑甜茶对干旱胁迫的生理响应不同。耐旱性强的楸子受到氧化胁迫的伤害较小,其抗氧化防御能力强于耐旱性较差的平邑甜茶。正常水分条件下,楸子和平邑甜茶幼苗叶片内活性氧代谢系统均无明显变化。在干旱处理后,两种苹果砧木叶片中O₂产生速率和H₂O₂含量均增加,膜脂过氧化产物MDA含量上升,且随着干旱胁迫程度的加重呈上升趋势。抗氧化酶SOD、CAT和POD活性及AsA-GSH循环系统中的4种关键酶,即APX、GR、MDHAR和DHAR活性,在干旱胁迫前期和中期都有所提高,而在后期降低,部分抗氧化酶活性低于对照。干旱胁迫下,抗氧化剂AsA和GSH含量呈现先升后降的变化趋势,在干旱胁迫结束时（12 d）均低于对照（楸子GSH除外）。与平邑甜茶相比,楸子具有较低的ROS水平和较高的抗氧化系统的防御能力。这些结果表明,干旱胁迫下两种苹果砧木幼苗叶片中活性氧（reactive oxygen species,ROS）增加,诱导植株体内抗氧化防御系统启动以减轻（缓）或抵抗干旱胁迫对细胞所造成的氧化伤害;但随着干旱胁迫程度的持续加重,植株抗氧化防御能力下降,ROS大量积累,导致细胞膜脂过氧化加重,MDA大量产生。而楸子植株叶片的活性氧清除系统能力高于平邑甜茶。2.干旱胁迫下,两种苹果砧木叶肉细胞的形态解剖结构及亚细胞超微结构发生了适应性变化,但两者之间存在较大的差异。光学显微镜观察发现,干旱胁迫下苹果砧木的叶片厚度、栅栏组织厚度及叶肉组织结构紧密度（CTR）都减小,海绵组织厚度、角质层厚度及叶肉组织结构疏松度（SR）均增加。与平邑甜茶相比,干旱胁迫下楸子叶肉细胞形态解剖结构的变幅较小。电子显微镜观察发现,干旱胁迫下,两种苹果砧木叶片的亚细胞超微结构发生了明显的变化,细胞损伤程度随着干旱胁迫程度的加重而增加。其主要的变化有:细胞体积变小、细胞核浓缩、细胞质凝集、细胞器数目减少、细胞膜结构解体、叶绿体膨胀及空泡化、基质片层（stroma lamella）排列混乱、淀粉粒消失、线粒体肿胀及嵴变模糊、表皮细胞气孔密度增大、气孔开张度变小等。与平邑甜茶相比,干旱胁迫下楸子叶片亚细胞超微结构遭受的损伤程度较小,楸子能较好地维持其细胞结构的完整性。3.以干旱处理的楸子幼叶为tester,正常生长的楸子幼叶为driver,利用SSH技术构建了一个干旱胁迫下楸子叶片的消减cDNA文库。该文库的重组率高于95%,插入片段集中在250⁶00 bp之间。经蓝白斑抗性筛选和菌液PCR鉴定后,对所挑选的阳性克隆进行测序而获得大量的ESTs。BLASTN和BLASTP比对分析表明,大部分ESTs与己知逆境胁迫相关基因在核酸和蛋白水平上有较高的同源性,也有一些ESTs功能未知。通过GO（gene ontology）分类进行功能注释,ESTs编码产物涉及能量、光合、蛋白质水解、RNA转录、信号转导、逆境防御以及功能未知和没有匹配的序列等。对文库部分克隆进行测序发现,文库中含有半胱氨酸蛋白酶、金属硫蛋白、凋亡因子、富含甘氨酸RNA结合蛋白、叶绿素a/b结合蛋白、光系统2放氧复合物增强蛋白等大量的抗旱相关基因。采用qRT-PCR对部分抗旱相关基因在楸子叶片和根系的表达谱进行分析。结果表明,干旱胁迫下大多数的所检测基因都上调表达,也有个别基因下调表达,但它们的表达强度和时序有所不同。由此可见,干旱胁迫下楸子植株体内与干旱相关的基因被诱导表达,这些基因可能参与楸子对干旱胁迫的抗性反应。4.通过菌液PCR筛选SSH cDNA文库和电子克隆,从楸子和平邑甜茶叶片中分别克隆到1个富含甘氨酸RNA结合蛋白（glycine-rich RNA-binding protein,GR-RBP）基因,并分别命名为MpGR-RBP1（HM042682）和MhGR-RBP1（HQ380209）。楸子MpGR-RBP1和平邑甜茶MhGR-RBP1基因分别编码171和164个氨基酸多肽,它们都包含一个RNA识别结构域（RNA recognition motif,RRM）和一个富含甘氨酸区（glycine-rich region）。Genbank数据库检索结果表明,该同源基因在苹果属（Malus）中为首次分离。经SignalIP 3.0和PSORT预测,这2个同源基因所编码的产物可能为非跨膜蛋白,位于细胞核中,能与RNA结合,属于转录因子类蛋白。序列比对及分子进化关系分析表明,MpGR-RBP1与MhGR-RBP1、欧洲甜樱桃（93.0%,AAL13082）、大豆（86.0%,AAD48471）、天竺葵（84.9%,AAB63581）、烟草（84.3%,ACD03270）马铃薯（82.9%,ABB87126）、柑橘（82.6%,BAA92156）、拟南芥（80.2%,AAM62447）、苜蓿（80.7%,AAF06329）等蛋白的同源性较高。表达谱分析表明,在转录水平上,MpGR-RBP1与MhGR-RBP1基因具有组织特异性;均受干旱诱导上调表达。MhGR-RBP1基因受盐胁迫诱导上调表达;在外源激素ABA或JA处理下,其表达被抑制,推测MhGR-RBP1基因表达可能属于非依赖ABA或非依赖JA的调控途径。在干旱胁迫下,MpGR-RBP1与MhGR-RBP1转录产物迅速积累,参与干旱逆境胁迫的响应反应,而它们是否在转录后或翻译水平调控苹果砧木抗性反应还需要进一步研究。更多还原

【Abstract】 Drought is one of the most serious problems in sustainable agriculture, influencing plant growth and crop yield in agricultural production in many regions of the world. However, the mechanism research on drought influence of fruit trees is behind other crops. Apple is one of the world’s major fruit trees of cultivated species and one of the mostly cultivated deciduous fruit trees in northern China. Apple trees are propogated by crafting, therefore the resistance of apple trees to environmental stress depends on the resistance ability of rootstocks. In China, little has been done about the research of apple rootstocks resistance to environmental stress, thus understanding the resistance ability of apple rootstocks to drought stress and the physiological and molecular mechanism of their response to drought stress are becoming more and more important for genetic modification of apple trees under abiotic stress.In present thesis, two apple rootstocks with significant differences under drought stress were used as plant materials. Two-year old pot grown plants were treated to drought stress by withholding water. The accumulation of reactive oxygen species （ROS）, the oxidation of membrane lipid, the activities of antioxidant enzymes, the contents of antioxidants, the change of plant hormone and leaf anatomical structure as well as subcellular ultrastructure were studied, we were about to reveal the physiological mechanism of injury and the adaptive metabolism of apple rootstocks seedlings under drought stress, and we discussed the relationship between these changes and drought tolerance. To investigate how M. prunifolia, an excellent apple rootstock with strong drought tolerance, adapts to drought stress and to identify genes responsible to this important trait, we constructed a cDNA library with drought-treated leaves of M. prunifolia, using suppression subtractive hybridization （SSH） technique. A PCR-based screening method of SSH cDNA library then was used for identification of resistance-related expression sequence tags （ESTs）. To get an insight into their behavior and regulation by drought stress, we have characterized the expression profile of mRNA from these apple leaves and roots during drought treatment using real-time RT-PCR. To better understand their function at the molecular level in stress responses, we isolated and identified several novel full-length cDNA, using in silico cloning and reverse-transcription PCR. We characterized the nucleotide sequence and deduced amino acid sequence of these novel genes, and multiple alignment and phylogenetic analyses were made. To further characterize these genes, we also applied a model for predicting their homology of protein structure with other species, and their putative roles were discussed. Both organ-specific and stress-related expression profile of the genes were detected by quantitative real-time PCR and semi-quantitative RT-PCR. The main results are included as follows:1. The general responses of the two apple rootstocks to drought are different, and we identified significant differences in the adaptive metabolisms employed to achieve tolerance. Drought-tolerant M. prunifolia showed a better protection mechanism against drought damage by maintaining higher constitutive and induced activities of antioxidant enzymes as well as contents of antioxidants than drought-sensitive M. hupehensis.In this study, we compared responses of anti-oxidant defence system in the leaves of two contrasting apple rootstocks. Two-year old pot-grown plants were subjected to drought for 12 days by withholding water, until plants began to wilt. Under well-watered conditions, there are significant differences between the lipid peroxidation and relevant antioxidant parameters in both apple species during the whole control experiment. Water stress increased lipid peroxidation, hydrogen peroxide （H₂O₂） as well as superoxide radicals （O₂ ） production in leaves of stressed plants. Compared with M. prunifolia, M. hupehensis was more sensitive to drought stress, resulting in larger increases in the levels of H₂O₂, O₂ and MDA. The activities of antioxidant enzymes superoxide dismutase （SOD）, catalase （CAT）, ascorbate peroxidase （APX） and glutathione reductase （GR） displayed an initial increase with gradual development of water deficit, reaching the highest level after 6 days of withholding water, and then dehydroascorbate reductase （DHAR） and monodehydroascorbate reductase （MDHAR） activities peaking on Day 6 and 8, respectively. But peroxidase （POD） activity was substantially enhanced, and peaked on Day 10, and then decreased at the end of the experiment （Day 12）. In addition, the contents of antioxidant ascorbic acid （ASA） as well as glutathione （GSH） content peaked on Day 4 and 6, respectively. In responses to drought stress, the activities of SOD, POD, APX, GR, and DHAR as well as contents of ASA and GSH in M. prunifolia were more increased than that of M. hupehensis, but CAT and MDHAR activities changed. These results demonstrated that both antioxidant enzymes activities and antioxidant contents in leaves of M. prunifolia and M. hupehensis are increased in response to soil drought stress to minimize oxidative damage, and the former exhibited higher antioxidant ability and protective action during drought stress.2. We compared changes in lamina anatomical structure and subcellular ultrastructure in leaves of two apple rootstocks during drought treatment. Under drought stress, observation through method of light microscope showed that the palisade tissue thickness, leaf lamina thickness, and the ratio of palisade tissue to leaf lamina thickness （CTR） of two apple rootstocks decreased, but their sponge tissue thickness, leaf cuticle thickness and the ratio of sponge tissue to leaf lamina thickness （SR） increased. Changes of lamina anatomical structure in M. prunifolia was less than that of M. hupehensis.Ultrastructural observations on mesophyll cells showed, that under soil drought stress, drought cells exhibited obvious ultrastructural changes over control, and cellular damage rised with the increasing drought stress. Main ultrastructural changes included nucleus condensation, cytoplasm degeneration, decreased organelles, and damaged membrane systems, decreased the number of chloroplast, greater starch grain disappeared, deformation and vacuolization of chloroplasts, much less grana stacking, loosening and distortion of thylakoid, mitochondria swelling and cristae appear vague, and increased stomatal density, and so on. Our results demonstrated that M. prunifolia was able to maintain structural cell integrity longer than M. hupehensis under water stress.3. A cDNA library was constructed from drought-stressed and well-watered leaves of M. prunifolia, using suppression subtractive hybridization technique, in which the rate of recombination was 95% and the size of inserts ranged from 250 bp to 600 bp. Based on their blue/white coloring, recombinant colonies were then selected using PCR-based method of screening the SSH cDNA library. To identify which M. prunifolia genes are differentially expressed under drought treatment, positive clones obtained from the subtraction libraries were subjected to sequencing. BLASTN and BLASTP alignment analyses were used to search against database of NCBI, whose related-stress genes encoding proteins were involved in energy and metabolism, signal transduction and defense, protein metabolism, and no hit or unknown ESTs. Functional annotation and classification of these ESTs were made by GO method.Analysis of sequences from the positive clones picked randomly revealed that many drought stress associated genes, including cysteine protease, metallothionein, apoptosis facilitator, glycine-rich RNA-binding protein, chlorophyll a/b-binding protein, oxygen evolving enhancer protein, et al., were obtained. The successful construction of the subtracted cDNA libraries enabled us to identify new differentially expressed genes involved in the resistance mechanism of Malus plants. Some related-stress genes in leaves and roots of M. prunifolia were selected for further expression characterization under drought stress using quantitative real-time RT-PCR analyses. These results indicated that expression patterns of the genes revealed by real-time RT-PCR analyses were induced by drought stress, with different expression levels, inducing that these genes were involved in the adaptive mechanism to drought.4. Two apple drought-related protein genes, which named MpGR-RBP1 （HM042682） and MhGR-RBP1 （HQ380209）, were successfully identified from the drought-treated leaves of M. prunifolia and M. hupehensis, respectively, using screening library, in silico cloning, or RT-PCR methods. This is the first report of this class of proteins in Malus plants. The encoded proteins of two homologous genes, MpGR-RBP1 and MhGR-RBP1, consisted of 171 and 164 amino acids, respectively. Their deduced amino acids contain an amino-terminal RNA recognition motif （RRM） and a carboxyl-terminal glycine-rich domain, with structural similarity to a class of stress-induced GR-RBP proteins found in other plants. Phylogenetic analysis confirmed that both MpGR-RBP1 and MhGR-RBP1 proteins belong to the plant GR-RBP family, members of which play important roles in posttranscriptional regulation of gene expression under various stress conditions. The expression profiles of the two apple GR-RBP transcripts were detected by quantitative real-time RT-PCR and semi-quantitative RT-PCR. MpGR-RBP1 and MhGR-RBP1 were expressed in roots shoots, and leaves, and the expression level of MpGR-RBP1 in leaves is higher than that of MhGR-RBP1 under drought stress. MhGR-RBP1 in leaves was expressed both under drought and high salt stresses, with higher expression levels in the former than the latter. Additionally, MhGR-RBP1 was sensitive to ABA and JA treatment and down-regulated with different concentrations of ABA and JA, and indicated MhGR-RBP1 might play a role in the signal pathway of independent-ABA or independent-JA. And the putative roles of MpGR-RBP1 and MhGR-RBP1 proteins are discussed. These results indicated that MpGR-RBP1 and MhGR-RBP1 might be involved in apple rootstocks to stresses response.更多还原