摘要
氧化胁迫是植物生长发育所面临的主要威胁之一,氧化胁迫是由植物体本身去除活性氧产物能力失衡导致的,所有的生物在其细胞层面上都必须维持还原性环境,过氧化物以及自由基的产生会打乱这种氧化还原状态,从而损伤生物分子,如蛋白质,核酸,脂质,进而影响新陈代谢等生命活动,严重的氧化胁迫会导致细胞的死亡。泛素蛋白酶体途径被认为能够有效去除过氧化蛋白。除此之外,泛素蛋白酶体途径对植物的生长发育调控也起着重要的作用,如调控植物激素的合成,参与光形态建成,自交不亲和以及细胞程序性死亡等过程。20S蛋白酶体是泛素蛋白酶体途径的核心元件,所有的20S蛋白酶体都由四层每层七瓣环状亚基组成,外面的两环各由七个α亚基组成,内侧两环各由七个β亚基组成,其中的β1,β2,和β5亚基具有水解蛋白质活性,并且针对不同类型的蛋白质底物,外层α亚基作用是作为与19S调节亚基的结合区域并且其N末端可阻止未经泛素标记蛋白质底物进入具有蛋白质水解活性的内空腔。在人类WI38/T和HL60细胞中过表β5亚基基因,能够提高其它β亚基的表达且提高蛋白酶体蛋白质水平的表达以及组装效率。序列分析显示,催化性的β亚基在进化过程中分化得比结构性的α亚基要早,并且β亚基在真核生物中有高度的序列相似性。
为研究拟南芥中β5亚基基因PBE的功能,本实验共设计了3个载体即PBE过量表达载体, PBE-GFP融合表达载体,并克隆了PBE上游1233bp的调控区,该序列含有TATA box及CAAT box等启动子基本元件,还含有1个HSE元件,一个涉及防御耐逆反应的TC-rich repeats元件以及生长素应答元件TGA-element,构建PBE基因启动子启动GUS表达载体。进而进行遗传转化,并获得子一代转化植株。在对转化植株的检测当中得到如下结果:PBE-GFP融合蛋白主要在细胞核内表达,这主要是因为蛋白酶体主要是通过对各种转录因子的水解以完成对相关生命活动的调节。PBE基因启动子与GUS表达转基因植株没有检测到GUS的信号,可能是与该启动子表达量低有关。在对过量表达转基因植株子二代的半定量RT-PCR检测发现,转基因植株PBE基因的转录水平较野生型有较大的提高。
Oxidative stress is one of the major threats to the plant living. The oxidative stress is caused by an imbalance between the production of reactive oxygen and a biological system's ability to clean it. All lives must maintain a reduction environment within cells which can be damaged by reactive oxygen and free radicals. Disturbances in this normal redox state can finally affect metabolism by the damage of the proteins lipids and DNA, severe oxidative stress can even cause cell death. The ubiquitin/26S proteasome pathway is considered to be an effective way of degrading unneeded or damaged proteins by proteolysis. Other than that, the ubiquitin/26S proteasome pathway also plays an important role in hormone signaling,photomorphogenesis, flower development and PCD by degrading regulatory proteins. 20S proteasome is the core particle (CP) of the 26S proteasome. All 20S particles consist of four stacked heptameric ring structures that are themselves composed of two different types of subunits: the outer two rings in the stack consist of sevenαsubunits each and the inner two rings each consist of sevenβsubunits. The outer two rings serve as docking domains for the regulatory particles and theαsubunits N-termini form a gate that blocks unregulated access of substrates to the interior cavity. Theβ1,β2, andβ5 subunits have protease activities which have three different substrate specificities. Sequence analysis suggests that the catalyticβsubunits diverged earlier in evolution than the predominantly structuralαsubunits and theβsubunits have high sequence identity in eukaryotic.
In order to study the function of gene PBE (the gene ofβ5 subunit in Arabidopsis thaliana ), in this experiment, three vectors had been constructed: the overexpression vector of gene PBE, the GFP transient expression vector of gene PBE , and the fusion construct of PBE: : GUS . For the generation of PBE: : GUS construct,, the PBE promoter sequence (1233bp) has been cloned from Arabidopsis thaliana genomic DNA. The sequence contains TATA box and CAAT box and it has a cis-acting element involved in heat stress responsiveness HSE, a cis-acting element involved in defense and stress responsiveness TC-rich repeats and a responsive element TGA-element. The three vectors were transformed into Arabidopsis thaliana by flora dip method. After the harvest and detection of T1 mutant, we found that PBE and the GFP transient expression were mostly located in cell nucleus probably because proteasome regulated the activities of life mainly through the hydrolysis of various transcription factors which are involved in plant growth and development. We didn’t detect any GUS expression in any tissue which was probably because of the low expression of the promoter. The semi-quantitative PT-PCR test indicated that the T2 mutant of overexpression line had a higher transcription level of PBE gene than the wild type does.
引文
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