【作者】 刘志杰；

【导师】 林思祖；

【作者基本信息】 福建农林大学 ， 森林培育， 2014， 硕士

【摘要】 杉木是我国特有的速生用材树种,单产高,用途广,分布遍及我国南方16省(区)。受夏季太平洋副热带高压的影响,我国南方杉木人工林主产区季节性伏旱和秋旱频繁,严重制约着杉木林生长及南方林业产业的发展。根系是植物与根际环境“交流”的首要门户,在长期进化过程中,根系形成完善的生长机制来应答水分胁迫,但我们对其复杂的调控过程仍有诸多未知。本研究以杉木根系为材料,利用双向电泳-质谱分离的方法鉴定出杉木根系在PEG模拟水分胁迫下的差异响应蛋白,并从中发现植物中广泛参与信号转导的14-3-3蛋白,推测其可能作为重要的调控因子参与杉木根系应答水分胁迫。基于实验室前期克隆出的杉木14-3-3基因家族不同成员的cDNA全长序列,我们对其中两个14-3-3基因进行在大肠杆菌原核表达分析,并构建植物过表达载体,之后对野生型拟南芥材料进行遗传转化,探索杉木根系14-3-3蛋白在根系响应水分胁迫过程中扮演的角色。主要研究结果如下：1、利用双向电泳技术,研究了PEG模拟水分胁迫下,杉木无性系根系蛋白质差异表达情况,选取20个具有统计意义的蛋白点,对这些蛋白进行质谱分析,成功鉴定了11个蛋白点,主要分为6大类,分别为：参与植物体内氧化还原平衡、细胞信号转导、胁迫响应、能量代谢、光合作用相关蛋白及其他功能未知蛋白,其中包含在植物中广泛参与信号转导的14-3-3蛋白。2、通过设计带酶切位点的基因特异引物,分离克隆杉木14-3-3-0,14-3-3-4两个不同家族成员的cDNA全长序列,构建重组原核表达载体pET32a-14-3-3-0, pET32a-14-3-3-4,并转化大肠杆菌Rosetta,经IPTG诱导,14-3-3编码的蛋白可在大肠杆菌中高效表达,利用SDS-PAGE的方法也检测到分子量约为31kD的目的蛋白,与预测的蛋白分子量相符。3、分别克隆出不带终止密码子的14-3-3-0,14-3-3-4基因,将其构建到由花椰菜花叶病毒(CaMV)35S启动子诱导的植物表达载体pTEV7上,通过农杆菌介导浸花法转化拟南芥植株,卡那霉素抗性筛选与PCR检测显示获得转基因拟南芥阳性株,经过筛选、培养、鉴定,我们获得1个转14-3-3-0基因拟南芥纯和系,2个转14-3-3-4基因拟南芥纯和系。4、RT-PCR结果表明,杉木14-3-3基因整合到拟南芥基因组中并成功表达。利用目的基因与GFP融合蛋白共定位技术研究了杉木14-3-3蛋白的亚细胞定位。激光共聚焦显微镜观察显示,在根尖成熟区获得清晰的视野,杉木14-3-3蛋白主要定位于细胞核中。在甘露醇模拟水分胁迫下,和野生型拟南芥相比,转杉木14-3-3-0拟南芥植株弱小,主根伸长及地上部生长显著弱于野生型WT,一定程度上表现为水分胁迫敏感型；而转杉木14-3-3-4拟南芥植株幼苗的主根伸长较野生型有显著增加,地上部发育也好于野生型。因此,杉木14-3-3-4基因可能在响应水分胁迫中表现为耐受型,这种表型差异为进一步研究杉木14-3-3调控植物根系响应水分胁迫的分子机制提供了一定的参考,也为14-3-3在杉木分子育种上的应用奠定一定基础。更多还原

【Abstract】 Chinese fir, a characteristic of fast-growing, high yield and wide application timber species in our country, is distributed throughout16provinces(area) of southern China. In consequence of the Pacific subtropical high in summer, most of Chinese-fir plantation of southern China suffer from seasonal drought and autumn drought frequently, which has become a barrier to the Chinese-fir plantation growth, as well as the development of forestry industry. Root serves as the principal gateway for plant to perceive and respond to their environment. In the long-term evolution, plant root also forms a perfection mechanism in response to certain environmental signals and stresses, but the exactly signaling transduction underneath the complex mechanism control processes still remain unknown. This study choose Chinese fir root as the material, combining with the separation of two-dimensional electrophoresis and mass spectrometry, we successfully identified11differentially expressed protein spots in Chinese fir root conferring to water stress made by PEG-6000, and speculated the14-3-3proteins, widely involved in signal transduction in plant, maybe work as an important regulatory factor involved in Chinese fir root response to water stress. Based on laboratory early achievement on the isolation four14-3-3isoforms full length cDNA sequence, in this study, two of the14-3-3s,14-3-3-0and14-3-3-4were cloned into pET32a vector, and then expressed in E. coli. Besides, we respectively build the plant expression vector for Arabidopsis (Arabidopsis thaliana) genetic transformation, to further investigate what the roles of14-3-3proteins may play in root system in response to water stress. The main results were as follows:1、Proteomic analyses of Chinese fir root responding to water stress were conducted to identify proteins involved in such response. Uniform Chinese-fir seedlings were subjected to water stress made by PEG-6000, and proteins were extracted from roots and separated by two-dimensional polyacrylamide gel electrophoresis. The high-resolution proteome map demonstrated significant variations in about20protein spots detected on Comassie briliant blue-stained2-DE gels. Of these,11proteins were identified by mass spectrometry, which mainly divided into six categories:proteins related to participate in the plant body REDOX equilibrium, cell signal transduction, stress response, energy metabolism, photosynthesis related proteins and other functions of unknown protein.2、Two cDNAs encoding Chinese fir14-3-3-0,14-14-3-3were isolated by the gene specific primers contained two given restriction enzymes loci, respectively, and constructed recombinant prokaryotic expression vector pET32a-14-3-3-0, pET32-14-3-3-4. The combinative expression vector were successfully transformed into E. coli Rosetta. After IPTG induction, efficient expression of the14-3-3protein can be observed in E. coli. Through SDS-PAGE analysis, the molecular weight of about31kDa proteins were successfully detected, which consistent with the prediction of protein molecular weight.3\Chinese fir14-3-3-0,14-14-3-3genes without termination codon were cloned, respectively, and introduced into the over-expression vector pTEV7under the control of the plant cauliflower mosaic virus (CaMV)35S promoter element. The combinative over-expression vectors were then introduced into Arabidopsis by agrobacterium-mediated transformation. PCR and resistance screening were carried out to validate the integration of NPTlIand14-3-3-0,14-3-3--4gene in TO regeneration plants and the T1and T2progenies. Finally, one homozygous lines of14-3-3-0, two homozygous lines of14-3-3-4were obtained for the gene functional analysis.4、RT-PCR analysis showed that the expression level of Chinese fir14-3-3gene in over-expression transgentic plants was higher than that in wild type plants, which indicated that the Chinese fir14-3-3genes were integrated into Arabidopsis genome and expressed successfully. GFP fusion protein gene positioning technology was adopted to study the Chinese fir14-3-3proteins subcellular localization by laser confocal microscope, the results indicated that Chinese fir14-3-3-0and14-3-3-4proteins are both located in nucleus, and a bright fluorescent signal was easy to detected in the mature zone of root. The root phenotype of14-3-3over-expression transgentic plants was in sharp contrast with wild type Arabidopsis under water stress. The14-3-3-0over-expression transgentic plants showed a drawl phenotype, short root, and the leaves were much smaller than the wild types, indicating the Chinese fir14-3-3-0may be sensitive to water stress. Nevertheless, in comparison with wild type,14-3-3-4over-expression lines improved the primary root elongation significantly, phenotypic analysis show that over-expression of the Chinese fir14-3-3-4can improve the tolerance of water stress in Arabidopsis. The result of this research will help to shed light on the mechanism about14-3-3s integrate into other regulation network to regulate plant root in response to water stress, and make some advance in the application of Chinese fir molecular breeding.更多还原