【作者】 刘海浩；

【导师】 潘延云；

【作者基本信息】 河北农业大学 ， 细胞生物学， 2011， 硕士

【摘要】 微管蛋白是一种在真核细胞中普遍表达的蛋白,由α微管蛋白(TUA)和β微管蛋白(TUB)组成的微管蛋白异二聚体构成。在拟南芥中共有6种不同的TUA基因亚型和9种不同的TUB基因亚型。许多研究证实,不同的微管蛋白基因亚型在特定的发育阶段或组织细胞中高效表达,随着外界环境胁迫表达量也会发生变化。因此,微管蛋白基因不仅仅是持家基因,也参与众多的信号应答反应。本研究以TUA2基因亚型表达变化的突变体为材料。利用反向遗传学的方法,运用遗传转化,Tail PCR,半定量PCR等技术进行TUA2参与ABA胁迫下种子萌发过程的研究。主要结果如下:1.通过生理检测我们在若干个T-DNA插入突变体中发现了一个对ABA敏感的突变体。运用Tail PCR的方法找到了T-DNA的插入位点位于TUA2基因上游启动子区,并发现该位点的插入导致突变体中TUA2基因表达量提高。另外我们在SALK库也找到了相应位点的突变体。并通过遗传转化的方法得到了三种不同株系的TUA基因超表达体。2.构建了TUA2基因的亚细胞定位转基因表达载体,运用基因枪转化的方法将TUA2基因和GFP融合基因在洋葱表皮瞬时表达,结果显示TUA2主要在细胞膜上分布。3.通过对ABA胁迫下种子萌发率的检测发现TUA2基因突变体,超表达体均出现了种子萌发的延迟。推测TUA2可能参与了ABA对种子萌发的调控过程。4.通过RT-PCR的方法对ABA途径相关基因表达量进行分析的结果显示,与野生型相比突变体的HAB和ABI基因在ABA处理下的表达量明显降低,暗示了TUA2可能参与了ABA信号系统的负调控过程。更多还原

【Abstract】 Tubulin is a kind of protein which generally expresses in eukaryocyte , and it is made up of tubulin heterodimers composed byαtubulin andβtubulin.There are 6 different TUA subgroups and 9 different TUB subgroups in Arabidopsis . Many experimental results showed that different tubulin subgroups not only expressed effectively in specified developmental stage and tissue , but also change their expression level under environmental stresses. Thus , tubulin is a kind of housekeeping gene , and it participated in masses of singal respond reaction .Our research used the tua2 mutant of TUA2 subgroups as material , and we undertaked the research of germination under the TUA2-participated ABA stress by reverse genetics , genetic transformation , Tail PCR , semiquantitative-PCR and other technology.The major results showed as follow :1. We found an ABA-sensitive mutant from several T-DNA insertion mutants by phaenotype observation . Then we found the T-DNA insertion site ( located in the upstream promoter region of TUA2 ) with Tail PCR ,and the level of TUA2 genetic expression raised in this T-DNA insertion mutant. In addition we obtained the mutant inserted in the corresponding site form the SALK library ,and got three overexpression mutants in different strains by genetic transformation.2. We constructed TUA2 transgenic expression vector for subcellular localization , transiented expressed the fusion gene of TUA2 and GFP by particle gun transformation . The results showed that the TUA2 mainly distributed at cellular membrane.3. By observation of germinationrate under ABA stress , the TUA2 mutant is found , and the overexpressionmutant all emerged a phaenotype of germination delayed. We supposed that TUA2 may participate in the germination regulating process of ABA.4. We analyzed the expression level of ABA pathway related gene by RT-PCR, found that the expression level of mutant had been depressed compared to HAB and ABI in wild type under ABA stress. It implied that TUA2 may participated in ABA negative regulation process.更多还原