【作者】 侯蕾；

【导师】 赵彦修； 张慧；

【作者基本信息】 山东师范大学 ， 植物学， 2012， 博士

【摘要】 第一章拟南芥盐敏感突变体的筛选鉴定土壤盐渍化是严重影响农作物产量和质量的非生物胁迫之一。土壤中高浓度的Na⁺严重影响着植物的生长和发育，对植物造成很大的伤害，导致世界作物的大量减产。因此，分析植物对于盐胁迫的反应，寻找耐盐相关基因，研究其反应机制，不仅对于揭示植物耐逆的机理具有重要的理论意义，而且对于耐盐作物的培育具有重要的实践意义。然而，植物对盐胁迫的适应机制又是非常复杂的问题，提高农作物的耐盐性仍然面临着极大的挑战。近年来，一些拟南芥（Arabidopsis thaliana）盐敏感突变体的获得对盐胁迫信号途径相关基因的克隆做出很大贡献，并在一定程度上揭示了盐胁迫应答机制和信号转导通路。比如SOS途径突变体。在拟南芥中，SOS1-SOS5所组成的SOS信号通路的调控机制业已得到阐明，研究表明SOS信号通路在调控离子均衡和植物耐盐性中发挥重要作用。本文从拟南芥（生态型为Wassilewskija,Ws）T-DNA插入的功能获得型突变体库中，用75 mM NaCl的筛选压力分离筛选到了一个突变体dds1 （developmental defects and saltsensitive 1 ）。遗传分析表明，dds1是一个细胞核遗传的、单基因突变的隐性位点。用质粒拯救的方法获得了2个T-DNA插入位点，但是PCR检测及卡纳霉素抗性检测证明两个T-DNA插入位点与表型均不相关。利用图位克隆结合重测序的方法，DDS1被定位在第2号染色体末端的分子标记M183（62号染色体18361670 bp）和M188（72号染色体18878730bp）附近。本文对DDS1影响盐敏感的生理机制进行了初步的分析研究。DDS1突变造成了植株形态和发育特性方面的许多变异。在正常的生长条件下，突变体的主根明显比野生型短，侧根数目减少，叶片形状较小。进一步鉴定发现此突变体在种子萌发阶段和幼苗生长阶段均对150 mM NaCl的处理比野生型更为敏感，种子萌发率低，根生长停止。另外ABA、甘露醇、H₂O₂、MV等胁迫处理均加剧这种生长缺陷的表型。相比于sos1突变体对于较低浓度的NaCl （50 mM）和LiCl （20 mM）的处理更为敏感，dds1突变体在20 mM LiCl处理下没有发生明显改变而且能耐受稍高的NaCl（150 mM）处理。对sos1 dds1双突变体的研究发现其对盐胁迫的敏感程度和sos1一样，均表现强烈的盐敏感。认为在盐胁迫应答途径中，SOS1和DDS1二者不在同一途径，仅仅是sos1的盐敏感表型掩盖了dds1的盐敏感表型。通过对突变体和野生型第3片真叶叶片大小和下表皮细胞大小的对比，发现突变体叶片面积比野生型小，但是细胞大小却比野生型大，因此，dds1细胞的增大可能与细胞分裂缺陷有关。分别用生长素IAA和细胞分裂素KT处理盐胁迫下和非盐胁迫下的幼苗。发现较低浓度IAA处理下野生型根伸长受抑制，而对dds1的影响则不明显。但是更高浓度的IAA处理则突变体和野生型根的生长都受到抑制。较低浓度IAA处理同时给予150mM NaCl盐处理的情况下，dds1仍然表现盐胁迫敏感。而KT的处理对根的伸长影响未发现明显区别。突变体侧根数目的减少可能与生长素信号途径缺陷或者中柱鞘细胞分化及分裂活性有关。IAA对dds1的影响机制仍待探讨。依据上述结果，可以推测DDS1处于植物生长发育调控网络。更直接的证据还有赖于DDS1基因的克隆和进一步的生理分析。第二章蛋白酶体5亚基与植物抗氧化性的相关性研究在植物生长发育的过程中，氧化胁迫是主要的非生物胁迫之一。ROS（Reactive OxygenSpecies）的产生和积累以及清除能力的失衡会打乱胞内氧化还原的平衡状态，从而损伤蛋白质、DNA、脂质等生物大分子，进而影响细胞的新陈代谢生命活动，对植物产生伤害。泛素—26S蛋白酶体（proteasome）通过选择性的降解损伤和错折叠的蛋白、没有功能的蛋白，来维持细胞内大分子的动态平衡。泛素—26S蛋白酶体途径广泛地调节着生物的各种生长发育过程，如激素应答、光形态建成、花发育和细胞程序性死亡等等。20S蛋白酶体是26S蛋白酶体的核心部分。20S蛋白酶体能不依赖ATP供能和泛素标记来降解氧化胁迫受损蛋白，从而行使抗氧化胁迫的功能。所有的20S蛋白酶体都是由四层环状亚基组成，外面的两环各由七个α亚基组成，内侧两环各由七个亚基组成，其中的1、2、和5亚基具有水解蛋白酶活性，并且针对不同类型的蛋白质底物。在人类WI38/T和HL60细胞中稳定的过量表达蛋白酶体5亚基，能够提高其它亚基蛋白质水平的表达且促进蛋白酶体的组装效率，并提高了蛋白酶体的酶活性，从而增强了细胞的耐氧化性。但是蛋白酶体5亚基在植物抗氧化方面的研究还未见报道，因此将AtPBE基因在拟南芥中过量表达，对研究植物细胞抗氧化胁迫能力及运用基因工程手段有效地提高植物的抗逆性可能会具有重要意义。拟南芥蛋白酶体5亚基由两个基因编码（AtPBE1和AtPBE2），本实验共设计了4个载体即AtPBE1和AtPBE2的过量表达载体和GFP融合表达载体。通过RT-PCR的方法克隆到AtPBE1和AtPBE2两个同源基因，并将其构建到植物表达载体pCAMBIA3301中，导入农杆菌后，进行植物遗传转化，实现其在拟南芥中过量表达，用除草剂筛选转基因株系，并获得T₃代纯合转基因植株。另外从ABRC库订购了两个基因的T-DNA插入突变体，并筛选到基因敲除的纯合株系pbe1和pbe2。对拟南芥蛋白酶体5亚基的过量表达株系和基因敲除株系进行了分子生物学的验证及生理指标的检验，结果如下：1)通过对过量表达植株进行PCR扩增，得到了大约1 kb的特异条带，表明AtPBE已整合至拟南芥基因组中。RT-PCR和Realtime-PCR分析表明转基因植株AtPBE的表达量均升高。进一步说明AtPBE基因整合到拟南芥的基因组后已正常转录表达。半定量RT-PCR检测证明基因敲除株系pbe1、pbe2中检测不到AtPBE基因的表达。2)用Realtime-PCR的方法对蛋白酶体5亚基的过量表达株系和基因敲除株系pbe1、pbe2中蛋白酶体其他亚基的表达水平做了相对定量检测。发现过表株系中，PBA、RPN10和RPN12的表达均有所升高；而PAG的表达量有所降低。基因敲除株系中PBA的表达也有所升高，而PAG的表达量有所降低。PBF1和PAC1的表达量在各种株系中则没有显著区别。3)用NaCl、甲基紫精和H₂O₂对各株系的胁迫处理结果表明，无论在种子萌发、幼苗生长还是成苗生长阶段，过量表达株系和突变体的抗氧化胁迫表型都与野生型没有显著区别。4)AtPBE::GFP融合蛋白表达分析显示，融合蛋白主要在细胞核内表达，细胞质中也有少量表达。本文主要创新点：1)筛选到一株不同于sos1表型的盐敏感突变体，并对引起该突变体表型变异的生理机制进行了初步探索。2)通过对遗传背景基因型为Ws的dds1进行重测序，开发并鉴定了部分适用于Col-0和Ws生态型的分子标记。3)证明了拟南芥蛋白酶体5亚基的过量表达没有显著增强植物的抗氧化能力。更多还原

【Abstract】 Chapter 1: Isolation and Genetic Analysis of Arabidopsis Salt-Sensitive-MutantsSoil salinization is one of the major stress factors that limiting the productivity and qualityof crops. High sodium （Na⁺） concentration in soil is toxic to most higher plants, severely affectthe plants growth and development, accounting for large decrease in the yields of a wide varietyof crops all over the world. Thus, analyzing the mechanism of plants response to salt stress,exploring genes related to salt tolerance not only have important theoretical significance butalso have important practical significance for cultivation of salt-tolerant crops. The mechanismof plants adapt to salt stress is very complex, so improving the salt tolerance of crops still facedgreat challenges.For the past few years, many salt-sensitive mutants had been gained and had laid a goodresearch basis to further identify salt-sensitive mutants, example for the contribution of theseries of sos mutants. The modulating mechanism of the SOS signal pathway consisted ofSOS1-SOS5 in Arabidopsis has been illuminated, studies showed that the SOS signal pathwayplays an important role in ion homeostasis and salt tolerance of plants.In this dissertation work, T-DNA-mutagenized seeds of Arabidopsis thanlina inWassilewskaja genetic background was used to screen salt-sensitive mutants. By isolatingseedlings after treated with 75 mM NaCl conditions, we obtained the dds1 which hasdevelopmental defects and salt sensitive phenotype. Genetic analysis showed that the dds1carries a single, recessive nuclear mutation. We have obtained two sequence flanking T-DNA byplasmid rescue method, but PCR and kanamycin resistance assay showed that the mutationwere not co-segregated with the T-DNA insertion. Then the mutated DDS1 was mapped to thebottom of chromosome 2 and located in a region near M1836 and M1836 which are located in 18361670 bp and 18878730 bp of chromosome II.To determine the roles of DDS1 in plant growth and development, a few characteristic traitsrelated to growth and development were investigated physiologically in the dds1 mutant. TheDDS1 mutation causes a pleiotropic phenotype. Under normal conditions, the phenotype ofmutant exhibited reduced roots and rosette leaves elongation and reduced the number of lateralroots. Further analysis indicated that dds1 was more sensitive to 150 mM NaCl stress thanwild-type plant at seed germination and seedling stages. The dds1 mutant was also moresensitive to high ABA, Mannitol and MV stress. sos1 are more sensitive to low NaCl （50 mM）and LiCl （20 mM） stress, while dds1 showed no difference to 20 mM LiCl treatment andsensitive to high NaCl （150 mM） treatment. The results indicate that these two mutated genesmay be involved in different signaling pathways in plant response to salt stress.The leaf blades area and abaxial epidermal cells area was measured and compared betweendds1 and wild type. The area of dds1 leaves was smaller than wild type but abaxial epidermalcells area was wider than wild type. The increased of dds1 leaf expansion may associated withcell division defection. Finally the mutant responses to exogenous IAA and KT were alsoobserved. Incubation on low IAA containing MS medium has no effect on dds1 but reducedroot elongation in wild type, and low IAA has no effect on salt sensitive of dds1. But incubationon KT containing MS medium was no effect on dds1. Indicating that the IAA hormonesignaling pathway may changed in dds1 mutant.Taken together, these results suggest that DDS1 is a key component required for thenormal regulation of signal pathway during plant growth development.Chapter 2: The Proteasome 5 Subunit Relate to Plant Oxidative Stress ToleranceOxidative stress is one of the major threats to the plants. The oxidative stress is caused byan imbalance between the production of reactive oxygen and a biological system’s ability toclean it. Disturbances in this normal redox state can finally affect metabolism by the damage ofthe 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. The ubiquitin-26S proteasome pathway alsoplays an important role in hormone signaling, photomorphogenesis, flower development andPCD by degrading regulatory proteins.20S proteasome is the core particle （CP） of the 26S proteasome. All 20S particles consist offour stacked heptameric ring structures which composed of two different types of subunits: theouter two rings in the stack consist of sevenαsubunits and the inner two rings each consist ofseven subunits. The 1, 2, and 5 subunits have protease activities which have threedifferent substrate specificities.Stable overexpression of proteasome 5 subunit in WI38/T and HL60 cells increases theamount of assembled proteasome and confers ameliorated response to oxidative stress andhigher survival rates. So far, the overexpression of AtPBE in high plants has not been reported,so study the effect of overexpression AtPBE gene in Arabidopsis may give us muchinformations of improving the anti-oxidative ability of plant cells and the plant’s resistance toabiotic stress by genetic engineering means.In order to study the function of AtPBE （the gene of proteasome 5 subunit in Arabidopsisthaliana）, in this experiment, four vectors had been constructed: the overexpression vectors ofgene AtPBE, the GFP transient expression vectors of gene AtPBE. The AtPBE1 and AtPBE2genes were isolated from Arabidopsis thaliana by RT-PCR method and confirmed bysequencing. Than the AtPBE1 and AtPBE2 PCR products were inserted into binary plant vectorpCAMBIA3301. The resulting plasmids named pCAMBIA3301-AtPBE1 and pCAMBIA3301-AtPBE2 vectors, were introduced into Arabidopsis thaliana by Agrobaterium tumefaciens-mediated transformation with floral-dipping method. Transformants were selected for bastaresistance and gained T3homozygous lines. We also obtained mutant lines from ABRC andgained homozygous mutants pbe1 and pbe2. The homozygous lines were selected and used forfurther analysis molecularly and physiologically.The main results as following:1)The transgenic lines were detected by PCR, about 1 kb band was obtained while wild type has no band indicating that the AtPBE gene has been introduced into Arabidopsis genome.RT-PCR and Realtime-PCR analysis revealed the induced expression of AtPBE in T3transgeniclines than wild type Arabidopsis. RT-PCR analysis revealed no expression of AtPBE in pbe1and pbe2.2) Realtime-PCR analysis revealed that the transcript levels of several genes involved inPBA1, RPN10 and RPN12 subunits were constitutively up-regulated in the transgenic plants,but the transcript levels of PAG was reduced in transgenic plants and mutants.3) The analysis of the stress tolerance showed that the tolerance to high NaCl, MV andH₂O₂stress were the same between the transgenic plants and wild type in seeds germinationand seedling stages. The results implied that the overexpression of proteasome 5 subunit inArabidopsis were not results in increased oxidative stress resistance.4) The AtPBE::GFP transient expression were mostly located mainly in the nucleoplasm. Adispersed punctate pattern was also evident in the cytoplasm.The innovation of this thesis were shown as follows:1) We obtained the dds1, which has developmental defects and salt sensitive phenotype, afew characteristic traits related to growth and development were investigated physiologically inthe dds1 mutant.2) Abtained some markers suited for Arabidopsis thanlina mapping between Columbia-0and Wassilewskaja genetic background according to resequenceing.3) This work showed that overexpression of proteasome 5 subunit in Arabidopsis notresult in increased oxidative stress resistance.更多还原