【作者】 陈方见；

【导师】 张立新；

【作者基本信息】 兰州大学 ， 植物学， 2008， 博士

【摘要】 核糖体是合成蛋白质的细胞器,其功能是按照mRNA的指令高效且精确地合成多肽链。到目前为止,单个核糖体蛋白的功能大多数情况下是未知的。完整的叶绿体核糖体包含59个蛋白,6个是叶绿体特有的,它们是由核基因编码,然后转运到叶绿体。研究拟南芥中这些由核基因编码的特有蛋白以及它们的作用机制有助于我们进一步认识高等植物核糖体的功能以及其分子调控机理。在本研究中,我们采用Sense和Antisense技术手段构建了PSRP-3的超表达和反义抑制载体并转化Co10型拟南芥,进而对超表达的转基因植株从生理生化以及分子水平进行了详细的研究。主要的结果如下:PSRP-3(plastid--specific ribosomal protein 3)蛋白是叶绿体核糖体上的特有蛋白之一,该基因的超表达明显提高了拟南芥生长速度,据此我们选取四株具有生长梯度的Psrp-3超表达转基因苗,分别命名为L1、L3、L4和L5。其叶面积分别是野生型的1.37、1.5、1.46、1.16倍;鲜重是1.1、1.27、1.29、1.18倍,干重是1.12、1.34、1.32、1.21倍。RT-PCR实验结果表明在四个Psrp-3基因超表达植株中,该基因的转录水平是和生长状况成一定程度的正相关。为了从蛋白水平观察PSRP-3在转基因苗和野生型的表达情况,我们制取了PSRP-3多克隆抗体并进行免疫印迹,结果发现PSRP-3的蛋白表达和转录本情况相似,也是和转基因苗的生长状况成一定程度的正相关。表明转基因植物生物量的增加确实是由于PSRP-3超表达造成。为了进一步研究造成生物量增加的原因,我们首先测定了转基因植株的叶绿素荧光动力学,结果表明PSⅡ最大光化学量子产量Fv/Fm基本没有变化,说明光系统没有受到影响,和我们测定的色素含量变化结果是一致的;转基因植株的光化学淬灭系数qP高于野生型,说明转基因材料的电子传递活性比野生型强;而转基因植株的NPQ与PSRP-3表达成负相关,也就是PSRP-3表达量越高,非光化学淬灭系数NPQ越低,L1、L3、L4和L5分别是野生型的34.5、19.1、13.4、18.8%,表明吸收的能量用于热耗散的部分越小,相对整体而言,用于光合作用合成的部分就增加了,这就必然会提高生物量,这也与前述结果一致。然后我们测定了光合速率、蒸腾速率和气孔导度,结果同样显示与基因表达成正相关,L1、L3、L4和L5的光合速率分别是野生型的2.45、3.31、3.22、1.54倍,呼吸速率分别是1.44、1.65、2.0、1.30倍,气孔导度分别是1.65、2.38、2.35、1.57倍。转基因植株比野生型生物量明显增加,光合测定结果是和PSRP-3成正相关,推测可能与叶绿体的发育有关,带着这个问题我们观察了叶绿体的超微结构,结果显示转基因植株和野生型植株的叶绿体发育都很完好,类囊体垛叠在一起形成的基粒类囊体和没有垛叠的基质类囊体相互连接,均匀分布;但是转基因植株中基粒类囊体的片层数明显高于野生型对照,并且排列整齐、致密,野生型中的基粒类囊体的片层数为9±2.5,而转基因植株中为17±2.0。为了更深入的研究PSRP-3超表达对类囊体膜蛋白稳态水平影响,我们用蛋白免疫印迹的方法研究了转基因植株中类囊体膜蛋白的相对含量,结果显示PSⅡ的核心蛋白CP43和CP47,PSI的反应中心蛋白PsaA/B,核基因编码的放氧复合物亚基PsbO,细胞色素Cyt f,ATP合成酶复合物亚基CF1β和LMCII与野生型相比基本没变化。蓝绿温和胶电泳结果显示对类囊体膜蛋白复合物的影响也没有明显变化,缺失CP43的PSⅡ条带含量比野生型略为少些,则暗示了光系统Ⅱ复合物更加稳定。我们还发现转基因植株表现出对葡萄糖比较敏感。在野生型拟南芥中,只有当糖浓度高于7%才会表现出糖响应的特征:色素合成和莲座发育受到抑制;但是转基因植株不到4%就表现出了类似的特征。外源ABA实验暗示转基因植株对糖的敏感是由于对ABA超敏感造成的。因此,PSRP-3超表达可以增加基粒类囊体的片层数,使用于接受光的相对面积增大,提高光合速率,降低非光化学淬灭系数NPQ,单位时间内用于合成有机物的能力大大改善,最终表现叶面积变大,干鲜重生物量的增加。更多还原

【Abstract】 Ribosome is an organelle that syntheses proteins effectively and precisely according to the instructions of mRNA.Chloroplast ribosome is specific to plant,in which the function of individual proteins is not clear so far.Chloroplast ribosome comprises of 59 proteins,most of which are encoded by nuclear genes with the rest 6 encoded by chloroplast genes.Functional studying of these nuclear encoded chloroplast proteins will expand our knowledge of how ribosome is functioning and regulated at molecular levels. For this purpose,we have constructed the overexpression and suppression vectors of Psrp-3 for the use of sense and antisense study,transformed into Co10 Arabidopsis,and had a detailed study of transgenic plants.The main results are summarized as follows:Psrp-3 is one of specific ribosomal proteins encoded by nuclear gene and then translocated into the chloroplast.Overexpression of Psrp-3 in Arabidopsis significantly enhanced the growth rate.Therefore,we chose four transgenic plants,named L1,L3,L4, L5,which have gradient growth rates in our study,with leaf areas 1.37,1.5,1.46,1.16 timese;fresh weight 1.1,1.27,1.29,1.18 times;dry weight 1.12,1.34,1.32,1.21 times of wild type,respectively.RT-PCR results indicated that the growth rate positively correlated with the expression level of Psrp-3.The same result was obtained at protein level,indicating the increase in biomass is due to the expression of Psrp-3.To further investigate what causes the increase in biomass,first we measured the chlorophyll fluorescence induction kinetics.The result showed that Fv/Fm was unchanged in the transgenic plants,that is to say,photosystem has not been damaged, consistent with the changes of pigment contents.The photochemical quenching in transgenic plants was higher than wild-type,indicating the electron transport activity in transgenic plants was stronger than the wild-type;while the non-photochemical quenching was correlated with PSRP-3 level negatively,i.e.NPQ of L1,L3,L4 and L5, was 34.5,19.1,13.4,18.8%of wild-type.This result indicated that the less the heat dissipation,the more the photosynthesis efficiency,therefore biomass increases.We next examined net photosynthetic rate,transpiration rate and stomatal conductance,and the results showed all of them were correlated with PSRP-3 level positively.Then we wondered if the effect of Psrp-3 is related to the development of the chloroplast.With this in question,we observed the chloroplast ultrastructure.Both transgenic plant and wild-type chloroplasts displayed well-developed membrane systems composed of grana connected by the stroma lamellae,but the number of discs per grana stack in transgenic plant was evidently higher than wild-type,9±2.5 in wild-type, however,17±2.0 in transgenic plant.They are also orderly and compactly arranged in transgenic plant.To gain insight into the steady state levels of the thylakoid proteins in transgenic plants,immunoblot analyses were performed with the antibodies raised against specific subunits of the photosynthetic protein complexes.The results showed that levels of the tested thylakoid proteins per unit of chlorophyll,including CP43,CP47(PSⅡproteins), LHCⅡ,PsbO,PsaA/B of the PSI reaction center proteins,cytochrome f of the cytochrome b6f complex,and CF1βof ATP synthase,were not altered in the transgenic plants.BN-PAGE studies showed that the abundance of PSⅠ,PSⅡ,and LHCⅡprotein complexes were not changed in the transgenic plant.CP43-free PSⅡwas slightly less than wild-type,suggesting the PSⅡcomplex was more stable.We also found that the transgenic plant was sensitive to glucose.In the wild-type, dramatic glucose-responses such as inhibition of chlorophyll synthesis and developmental arrest of leaves were observed when plants were grown on medium containing 7% glucose.However,the transgenic plant showed similar glucose-responses when the glucose concentration in the medium was raised to more than 4%,indicating that the sensitivity to glucose in the transgenic plant may be due to hypersensitivity to ABA.Therefore,we propose that Psrp-3 functions to positively up-regulate photosynthesis at both physiological and developmental levels.更多还原