【作者】 齐盛东；

【导师】 郑成超；

【作者基本信息】 山东农业大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 植物因为其固定生长的特点而必须适应周围不同的环境胁迫条件，如干旱、低温、高盐等。当胁迫危害来临时，植物会通过复杂的信号转导来激活或抑制体内的生理生化过程，以此来达到抵御胁迫的目的，在这个过程中植物激素扮演着重要的角色。RNA解旋酶是一类利用ATP水解产生的能量催化双链RNA解旋的酶，在生物体内广泛存在。虽然RNA在体内能够形成稳定的高级结构，但是这些具有高级结构的RNA一般都不具有生物学功能。RNA解旋酶能够催化其转变为有活性的RNA，被认为是最有效的RNA分子伴侣。RNA解旋酶功能发挥涉及到RNA的转录、翻译起始、mRNA的剪接、mRNA的降解、蛋白质的翻译、rRNA加工、核糖体的组装和核mRNA输出等过程，在基因表达调控方面发挥重要作用。在RNA解旋酶家族中研究最清楚的是DEAD-box家族成员。自2001年拟南芥首个冷诱导型DEAD-box解旋酶FL25A发现以来，LOS4、STSR1/2、AtRH9/25等一系列的DEAD-Box成员在非生物胁迫中的功能相继被鉴定。但是对于SKI2家族在植物中的研究却相对较少，与非生物胁迫之间的关系更是鲜有报道。本研究利用生物信息学和芯片分析技术，分离鉴定了拟南芥中的SKI2家族成员，并根据进化关系确定AtHelps为酵母Ski2p和人类Ski2w的同源基因。利用反向遗传学的方法对AtHelps生物学功能进行了分析，主要结果如下：（1）拟南芥AtHelps的鉴定与表达定位特性拟南芥AtHelps RNA解旋酶是拟南芥的SKI2家族成员，与ScSki2p和HsSki2w高度同源，核心区氨基酸同源性分别达53%和56%，并含有具有保守的DEVH-box型解旋酶基序与结构域。AtHelps基因在根组织和成熟的种子中表达量最高，在萌发的第二天表达量有明显的降低，之后慢慢恢复至正常水平。AtHelps基因的转录能够受到高盐和高渗透胁迫的抑制。AtHelps的启动子活性分析显示在各个时期的组织中都有表达，但是表达的强度不同：AtHelps在维管束组织中转录活性强，且随着组织的成熟逐渐消失；根中的转录活性持续很高。显微切片分析发现根中高表达主要在中柱鞘内，尤其是在韧皮部的薄壁细胞中表达强烈，可能在木质部的物质装载和卸载中发挥重要作用。在幼苗期，高盐和干旱胁迫抑制AtHelps基因表达。利用洋葱表皮、根尖观察和稳定株系叶组织的原生质体观察的方法对AtHelps的亚细胞定位进行研究，三种方法同时显示AtHelps的蛋白定位于植物的细胞质中，原生质体观察结果还表明在叶绿体中没有表达。（2）AtHelps参与萌发期和萌发后期对高盐的不同响应取决于不同的ABI基因通过RNA干扰技术获得特异的AtHelps的突变体，结合35S驱动的超表达植株进行表型分析。发现不同基因型在200mM的NaCl中有差异萌发的表型，其中突变体萌发快，而超表达株系则变慢。同样在萌发后的子叶变绿和最终植株的成活率方面突变体也表现出较好抗性。利用相同渗透势的KCl和一定浓度的LiCl处理作对照，发现AtHelps主要是对钠离子毒害有反应。不同种类的ABA合成抑制剂能够消除三种基因型在盐胁迫下的萌发差异，证明AtHelps对于盐胁迫的响应依赖于植物激素ABA的。利用定量RT-PCR技术检测了ABA合成基因ABA2、NCED3、AAO3和降解的关键基因CYP707A3，发现突变体中合成基因下调而代谢基因上调，但总体的效果是使ABA含量下降；而ABA含量的测定结果得出相同结果，证实ABA含量高低是引发不同基因型差异萌发的原因之一。三种基因型表现出对于ABA的不同敏感性。Western Blot结果显示在突变体中的ABI3和ABI5的蛋白水平降低，而这种蛋白水平降低所造成的表型能够被蛋白酶体抑制剂所减弱。对ABA敏感性的改变可能是引发不同基因型差异萌发的原因。突变体萌发后生长阶段的耐盐性主要是通过降低体内ABI4的水平，进而影响AtHKT1;1的转录，最终优化钠离子的分配，减少钠离子产生的活性氧伤害来实现的。（3）AtHelps参与对胞质mRNA稳定性的调节AtHelps能够影响特定mRNA的稳定性。突变体通过对CYP707A3和AtHKT1;1的mRNA稳定性的调节增强了其活性形式功能的发挥，进一步强化了盐胁迫抗性。更多还原

【Abstract】 Plants are sessile organisms capable of adapting to various environmental conditions,such as drought, cold, and high salt content in soil. When they encounter these stressfulconditions, the plant cells reprogram their cellular biochemical processes to defense it bytriggering a network of signaling events, during which phytohormone plays necessary roles.The RNA helicase is a kind of enzyme existed widely in all living organisms, whichcould catalyze double-stranded RNA to unwind using energy from ATP hydrolysis. And RNAhelicases convert RNA with advanced structure that makes it inactive to active functionalRNA, and is treated as the most effective RNA molecular chaperone. The function of RNAhelicase involves in RNA transcription, translation initiation, pre-mRNA splicing, mRNAdecay, protein translation, rRNA processing, assembly of ribosome and nuclear mRNA export,and so on. RNA helicase plays important roles in gene expression and regulation.The best known members in the RNA helicase family is the DEAD-box RNA helicases.Since the first cold induced DEAD-box helicase FL25A was studied in Arabidopsis thaliana,series of the DEAD-box helicase members were identified to function in abiotic stress, suchas LOS4, STSR1/2and AtRH9/25. But SKI2family is rarely studied in plants, and therelationship between SKI2family and abiotic stress is rarely addressed.Using biological information and gene-chip analysis, we identified the members of SKI2family in Arabidopsis. And we confirmed AtHelps as the homologous gene of Ski2p in yeastand Ski2w in human according to the evolutionary relationships. Then the biological functionof AtHelps was analyzed by the method of reverse genetics, and main results are as follow:(1) Identification and characterization of AtHelpsThe RNA helicase AtHelps was the member of Arabidopsis SKI2family, with closestphylogenetic relationship with ScSki2p and HsSki2w (53%and56%in core region aminoacid sequence), and contains a conserved DEVH-box motif.AtHelps expressed highest in root tissue and the mature seed; and the transcripts reducedobviously at the second day of germination process, and slowly returned to normal level after then. The transcription of AtHelps was inhibited by high salt and osmotic stress.Promoter activity of AtHelps was detected in all stages of Arabidopsis, but the intensitywas different: transcriptional activity was mainly found in the vascular tissue, and graduallydisappeared as the tissue turned older; Transcriptional activity in root was high all the time. Inthe root, transcriptional activity was mainly detected in pericycle, especially in theparenchyma cells of phloem. AtHelps might play an important role in loading and unloadingnecessary material to the xylem. High salt and drought stress repressed the transcriptionalactivity of AtHelps at seedling stage.The protein of AtHelps localized in cytoplasm by transient transformation assay withonion epidermal cells and protoplast of homozygous lines observation. However, it does notlocalized in chloroplast.(2) The responses to salt stress of AtHelps in germination and postgermination phasewere depended on different ABI genesWe got mutants by RNA interference technology and overexpression lines for furtherphenotypic analysis. The mutant germinated faster but the overexpression lines were slowerin200mM NaCl. The mutant also showed higher resistance to salt in cotyledon-greeningstage and final survival rate. The results of KCl and LiCl showed AtHelps was mainly inresponse to sodium toxicity.Different kinds of ABA synthesis inhibitor reduced the difference-germinationphenotype of three genotypes in salt stress. It proved AtHelps response to salt stress wasdependent on the phytohormone ABA.The expression of ABA synthesis key genes, ABA2, NCED3, AAO3and degradation keygene CYP707A3were detected by qRT-PCR. And synthesis key genes were reduced anddegradation key gene was induced in mutant. While the overall effect was to make the ABAcontent decrease in mutant; then the ABA content was messured and the results was in accordwith the qRT-PCR results. The difference of ABA content among genotypes was one ofreasons that caused the difference-germination.The sensitivity to ABA was diverse among three genotypes. Western Blot showed lowerABI3and ABI5protein levels in the mutant, and the phenotype caused by decreased proteinlevel can be reduced by proteasome inhibitors. In post-germination growth phase, the reduced transcripts of ABI4was the main reasonof phenotype, which directly enhanced the transcription of AtHKT1;1. AtHKT1;1can achievethe optimal allocation of sodium, which reduced active oxygen damage caused by sodium.(3) AtHelps participated in regulating the stabilization of mRNA in cytosol.AtHelps regulated the stability for some specific mRNAs, such as CYP707A3andAtHKT1;1. The active form was stabilized by protecting the mRNA from turnover, whichfurther played roles in resisting salt stress in mutant.更多还原