【作者】 李召春；

【导师】 程红梅；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 逆境胁迫,包括极端温度(低温、高温)、干旱、盐胁迫、化学毒害和光污染等,严重影响植物生长及产量,并触发植物在生理、形态、生化和分子结构等方面的一系列变化。逆境胁迫对植物生长和发育具有严重影响,传统的育种因为缺乏有效的资源而抵御逆境效果不明显,因此有必要对抗逆植物的基因资源进行深入发掘。作物在长期进化过程中获得一定的适应逆境的能力,当逆境出现时植物会有很多基因被激发表达来提高自身的抗逆能力,这些基因表达量的差异导致植物抗逆能力不同,只有成簇抗性相关基因的转录失活,才能有效提高作物的抗逆能力。沙冬青(Ammopiptanthus mongolicus( Maxim1)Cheng F1)是我国特有的荒漠植物,常年处于极端低温、高温和干旱等逆境环境,具有极强的抗逆性,是开展抗逆研究的理想材料。本文以沙冬青为研究对象,进行逆境转录表达谱的构建,并对部分差异表达片段进行了初步的验证和生物信息学分析,希望从中发现高效的抗逆调控基因,为转基因育种提供资源。在优化沙冬青种子萌发和幼苗逆境处理条件的基础上,对经逆境处理的沙冬青开展cDNA-AFLP研究。在对PCR反应条件、聚丙烯酰胺凝胶电泳(PAGE)、转录差异表达条带(TDFs)的回收及二次PCR条件等多方面的优化后,对沙冬青在低温、高温和干旱等逆境条件下的转录表达谱进行了分析。共回收了1487条沙冬青逆境胁迫下的TDFs,并对其中536条TDFs进行克隆并获得序列信息。在此536条TDFs中,有287条得到上调表达(up-regulated expression),249条得到下调表达(down-regulated expression)。利用Blastx进行比对后,选择其中201条与NCBI数据库中已报道基因具有明显同源性的片段进行了基因差异表达生物信息学分析。在这201条TDFs中,在低温逆境中出现85条,在高温逆境中出现175条,在干旱逆境中出现93条。根据同源性最高序列的预测功能,各逆境下的TDFs均可分为细胞代谢类(Metabolism)、信号传导(Signal transduction)、运输(Transporter)、转录(Transcription)、逆境胁迫/防御相关(Cell resecue / Stress-related / Defence)、细胞生长与结构(Cell growth and structure)、光合与能量(Photosyntesis and Energy)和功能未知(Function unknown)等8个部分。这些与抗逆性相关的功能蛋白为从分子水平研究和阐释沙冬青的抗逆机制,进而发掘其中的抗逆基因资源提供了依据。采取半定量RT-PCR方法对HSPs、CP43、EIN3a和CCR-4相关因子等功能蛋白编码基因同源的TDFs进行了初步验证。结果显示,在半定量RT-PCR实验中,各基因的表达量出现了与cDNA-AFLP实验一致的上调或下调。该结果进一步证实了相关基因在逆境处理下的表达变化。更多还原

【Abstract】 Abiotic stresses, including extreme temperatures (extremes of cold and heat), drought, salinity, oxidative stress and chemical toxicity, are serious threats to the plant growth and productivity and result in a series of physiological, morphological, biochemical and molecular changes. In view of the serious influences of abiotic stress on plant, it is important to carry out the research in molecular biology of stress-resistant plant and to gain the gene resourses from the plant with resistance. However, traditional breeding methods usually have little effection on the resistance to abiotic stresses because of the lack of effective gene resources. During the evolutionary, crops have gained certain resistance which can only be stimulated when stress occurs. The activation of cluster resistance-related genes transcription can effectively enhance the resistance of crops. Ammopiptanthus mongolicus, mainly distributing in the desert regions of Northwest China, which is capable of surviving unfavorable conditions such as cold/heat weather or lack of moisture, should be paid due attention as a highly adversity-resistant plant in the research of plant stress.In order to identify stress-regulated genes, A. mongolicus was chosen as the study object to estamblish its transcriptional profile under abiotic stresses, and some transcription derived fragments (TDFs) from the plant were identified and analysed. Based on the optimization of the conditions of seed germination and seeding stresses, a cDNA-based Amplified Fragment Length Polymorphism (cDNA-AFLP) approach was applied in this study. After optimizations of cDNA-AFLP such as the choice of primers combinations, the reaction conditions of polymerase chain reaction (PCR), ways of polyacrylamide gel electrophoresis (PAGE),recovery of the transcript derived fragments (TDFs) and the method of re-amplifications, the transcriptional profile on A.mongolicus with cold, heat and drought treating was then analyzed. A total of 1487 TDFs expressed in various stresses were gained in the study, of which 536 TDFs were cloned and sequenced. Among the sequenced TDFs, 287 had an up-regulated expression while 249 were down-regulated, and 201 which had significant homeology with the sequences in data bank were chosen to establish the transcriptional profile of A. mongolicus under abiotic stresses.Among the 201 TDFs, 85 could be found in low temperature stress, 175 could be found in high temperature stree, and 93 could be found in drought stress. According the Blast results of the sequenced TDFs, 201 showed significant similarity to the reported genes in the NCBI databases and were functionally annotated to be used in gene difference expression. The TDFs in each stress could be classified as 8 kinds: cellular metabolism, signal transduction, transporter, transcription, cell resecue/ stress-related /defence, cell growth and structure, photosyntesis and energy, and unknown function. The largest category of identified genes was in cellular metabolism.A semi-quantitative RT-PCR strategy was then carried out to verify the expression of some TDFs under stresses, which have significant homeology with the encoding genes of HSPs, photosystem II CP43 chlorophyll apoprotein, CCR4-associated factor. The results of semi-quantitative RT-PCR showed that the expression patterns of chosen TDFs were same with previous cDNA-AFLP, representing simultaneous up-regulation or down-regulation.更多还原