【作者】 吴凯朝；

【导师】 杨丽涛；

【作者基本信息】 广西大学 ， 作物栽培学与耕作学， 2013， 博士

【摘要】 干旱是制约我国甘蔗生产的主要影响因子。选育抗旱能力强、地域适应性广的高产高糖新品种是解决季节性干旱给我国甘蔗生产带来不利影响的有效途径。挖掘抗旱力强、水分高效利用的基因并通过遗传转化进行分子育种是提高甘蔗抗旱能力研究的新方向。抗旱甘蔗品种既应具有在干旱胁迫状态下的耐受能力,还应具有干旱胁迫状态解除后恢复快速生长的能力,因此研究甘蔗对干旱和干旱后复水的生理和分子响应机理对如何提高甘蔗抗旱能力的遗传改良都具有同样重要的意义。甘蔗是高光效C4作物,具有耐旱、耐涝、耐瘠、抗病、高生物量等特性,是作物重要耐旱基因资源。为了研究甘蔗响应水分胁迫的生理和分子机理,挖掘甘蔗的耐早、水分高效利用及促进生长的基因,本研究选用优良抗旱育种杂交亲本GT11为试验材料,在渗透调节、保护酶活性、物质代谢和内源激素等几个方面探讨甘蔗伸长期在干旱及干旱后复水状态下生理响应；利用cDNA-AFLP和cDNA-SCoT两种基因差异分析方法分离和筛选甘蔗在干旱及干旱后复水诱导的差异表达基因,并对获得的部分特异基因片段进行克隆获得基因全长cDNA,为作物抗旱性的遗传改良提供相应的基因资源。主要研究内容和结果如下：1.在干旱胁迫下,甘蔗品种GT11伸长期在渗透调节、保护酶系统、物质代谢和内源激素等几个方面都做出积极的生理响应,表明了GT11在水分胁迫下可能有较强的适应能力。(1)干旱胁迫下,甘蔗的渗透调节物质可溶性糖和脯氨酸大量增加,P5CS活力增强,而可溶性蛋白质含量下降,ProDH活力受到限制。复水处理后,叶片中可溶性糖含量急剧下降后回调；脯氨酸含量很快下降；可溶性蛋白质含量快速回升；P5CS活力很快下降；ProDH活力急剧上升。(2)干旱胁迫下,甘蔗的丙二醛含量和超氧阴离子含量升高,保护系统酶POD、 CAT、SOD和GSH-R活力都在不同程度提高。复水处理后,丙二醛含量和超氧阴离子含量很快下降,保护系统酶SOD和GSH-R活力降低,CAT活性表现为先下降后升高。与对照相比,保护系统酶活力在复水前期保持较高活力水平。(3)干旱胁迫下,甘蔗的ABA含量随受干旱胁迫进程不断加大累积,PAO活力上升。复水处理后,ABA含量先上升后下降,PAO活力很快降低。2.使用cDNA-AFLP差异显示方法分离得到406个与水分胁迫相关TDFs,167个TDFs反向Northern印迹杂交验证为阳性。通过对27个TDFs进行克隆、测序、比对分析,涉及新陈代谢、能量代谢、转录调控、结合功能蛋白、环境互作等相关功能基因。3.使用cDNA-SCoT差异显示方法分离得到316个与水分胁迫相关TDFs,180个TDFs反向Northern印迹杂交验证为阳性。对177个TDFs进行克隆、测序、比对分析,其中107个TDFs在NCBI数据库有较高相似度已知功能基因,28个TDFs在NCBI数据库有相似性很高的未知功能基因和假想蛋白。135个TDFs可以分为14类：新陈代谢、能量代谢、运输途径、通信及信号转导、细胞循环及DNA加工、转录调控因子、蛋白质合成、蛋白质加工、结合功能蛋白、转座子、毒性及质粒蛋白、细胞分生物合成、防御和未分类蛋白。4.利用半定量RT-PCR对12个从cDNA-SCoT分离获得的TDFs进行表达验证分析,全部12个基因表达量都在一定程度上受到干旱诱导或干旱后复水的诱导。其中4个基因受到干旱胁迫诱导上调表达,为抗旱类基因；7个基因受复水诱导上调表达,为恢复生长调节类基因；1个基因在干旱和复水都被诱导上调表达,为双重功能基因。从12个基因的表达模式分类结果和该基因的生物学信息功能分析结果基本是一致的。5.以GT11的cDNA第一链为模板,采用同源克隆方法成功克隆到5个水分胁迫相关基因全长cDNA,分别为Sc-atpI基因、Sc-SUTl基因、Sc-SAMDC3基因、Sc-Tuba3基因和Sc-HSP70基因,并对这些基因的核苷酸序列及其编码氨基酸序列进行分析,结果表明5个基因在NCBI数据都有同源性很高的禾本科作物(玉米、高粱)基因。Sc-atpI基因、Sc-Tuba3基因、Sc-HSP70基因、Sc-SAMDC3基因为首次在甘蔗中克隆得到。更多还原

【Abstract】 Drought is the main factor that restricts the sugarcane productivity in china. The breeding and selection for new sugarcane varieties with new high yield, high sucrose, drought-resistance and wide regional adaptability are the effective way to improve the cane productivity under seasonal drought in China. Transgenic sugarcane with high drought resistance and water use efficiency gene is a new methodology to improve the sugarcane drought resistance. Drought resistance of sugarcane includes the drought tolerance and the ability to recover the growth after stress relief. Therefore, studies on the physiological and molecular mechanisms of sugarcane in response to drought stress and re-watering will aid in the understanding of the genetic improvement involved in drought resistance.Sugarcane is C4crop with high photosynthetic efficiency and many advantageous characteristics, such as drought resistance, waterlogging tolerance, barren tolerance, disease resistance, high biomass and so on, which is an important genetic resource for crop improvement, In order to understand the physiological and molecular mechanisms of sugarcane in response to water stress, and find drought-tolerant genes, the strongly drought resistant sugarcane variety GT11, was used as the plant material. The physiological characteristics including the osmotic adjustment, protective enzyme activities, physiological metabolisms, and endogenous hormones, etc. were analyzed to investigate the physiological response to drought stress and re-watering in sugarcane. The differential genes induced under drought stress and re-watering, were isolated and screened using two methods of cDNA-AFLP and cDNA-SCoT. The full-length cDNAs from a part of specific differential genes were obtained by cloning, which could provide gene resources for genetic improvement of the crop drought resistance. The main research contents and results are as follows:1. Sugarcane variety, GT11showed the physiological reposes to water stress including osmotic adjustment, protective enzyme activities, substance and endogenous hormones metabolism, etc. during elongating stage, which indicated the strong ability of GT11to adapt to water stress.(1) Under drought stress, osmotic adjustment substances, soluble sugar and proline were largely accumulated and P5CS activity was increased, while the soluble protein content and ProDH activity were decreased in sugarcane leaves. After re-watering, the soluble sugar content in leaves was decreased sharply and then increased to the level closed to control, proline content and P5CS activity were decreased rapidly, on the contrary, the soluble protein and the ProDH activity increased fast.(2) Under drought stress, the contents of MDA and the superoxide anion were increased and the activities of protection enzymes including peroxidase, catalase, superoxide dismutase and glutathione reductase were increased in different levels. After re-watering, the contents of MDA and the superoxide anion were decreased rapidly, and the activities of peroxidase, superoxide dismutase and glutathione reductase were also decreased, then increased, and the catalase activity was first decreased and then increased.(3) Under drought stress, abscisic acid content was highly accumulated as the drought intensifying, and polyamine oxidase activity was enhanced in a certain range. After re-watering, abscisic acid content was increased first and then decreased, and polyamine oxidase activity was decreased rapidly.2. Four hundred and six TDFs were isolated through cDNA-AFLP analysis. One hundred and sixty-seven among them were verified as positive by reverse Northern blotting. Twenty-seven TDFs were cloned, sequenced, BLAST analyzed and classified by functional analysis as seven categories, including energy metabolism, transcription regulation, protein binding, environmental interaction and other functional genes.3. There were316TDFs isolated from the cDNA-SCoT analysis for the gene differential expression induced by water stress. Among them180of positive TDFs were obtained using reverse Northern blotting excluding the false positives. One hundred seventy seven of positive TDFs were cloned, sequenced, and the BLAST analyzed. There were107TDFs showed high similarity to the known genes in NCBI database,28TDFs showed high similarity genes to the hypothetical protein with unknown functions in NCBI database. Total one hundred thirty five TDFS were divide into fourteen catagories by functional analysis, which involved in metabolism, energy, transport, communication and signal transduction, cell cycle and DNA processing, transcription, protein synthesis, protein fate, binding function protein, transposable elements, viral and plasmid proteins, cell fate, defence, and unclassified proteins.4. Twelve TDFs isolated with cDNA-SCoT were selected and verlidated by semi-quantitative RT-PCR analysis. The results showed that the expressions of the twelve genes were induced by drought stress and re-watering. The expression patterns among of those genes could be classified into three types by functional analysis. Four genes are induced by drought stress, as the drought resistance gene; seven genes were up-regulated by re-watering, as growth regulatory genes; one gene was up-regulated by both droutht and re-watering, as a dual functional gene.5. Five differentially expressed genes were cloned by homology cloning method with the first cDNA strain from sugarcane variety GT11. The genes included ATP synthase CFO subunit IV (Sc-atpl), sucrose transporter1(Sc-SUTl), heat shock protein70(Sc-HSP70), S-adenosylmethionine decarboylase3(Sc-SAMDC3), and tubulin alpha-3chain (Sc-Tuba3). The sequences of nucleotide acids and amino acids, domain, hydrophobicity, multiple alignment and phylogenetic tress of these genes were analysised. The results showed that five genes from sugarcane had a high homology with other gramineous crops in NCBI database, such as maize and sorghum. Sc-atpl, Sc-Tuba3, Sc-HSP70and Sc-SAMDC3were first cloned from sugarcane.更多还原