【作者】 赵吉强；

【导师】 赵彦修； 张慧；

【作者基本信息】 山东师范大学 ， 植物学， 2008， 博士

【摘要】 土壤盐渍化是目前影响农作物产量和质量的最主要原因之一。植物对盐胁迫的适应非常复杂,提高作物的耐盐性仍然面临着极大的挑战。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。到目前为止,拟南芥一直是研究植物分子生物学途径的最好模式材料,但拟南芥是真正的甜土植物,因此,使用拟南芥将只能揭示关于植物耐盐性较少的信息。作为拟南芥近缘的盐芥,是很有前景的植物耐盐模式系统。盐芥具有拟南芥很多同样的优点以作为模式系统:如类似的形态、小的基因组、短的生活史、丰富的种子和易于被转化等。而且,盐芥是真盐生植物,短时间内能耐受高达500mMNaCl的冲击,在盐适应前后既不产生盐腺也没有复杂的形态上的变化,表明其耐盐性很大程度上源于基本的生理和生化机制。此外,盐芥和拟南芥在cDNA和氨基酸水平上分别有90%和95%的同源性,可以方便的将拟南芥的很多信息(基因、蛋白质数据库以及突变体系等)移至盐芥耐盐性的分子生物学研究。目前,对盐芥生理生化的研究、基因表达的研究,以及各种cDNA文库、突变体库的建立,已经使盐芥成为一种非常有价值的耐盐研究模式系统。随着盐芥基因组测序的即将完成,盐芥功能基因组学研究将会成为研究工作的重点,而功能基因组学的研究是建立在获得大量突变体的基础上的,饱和诱变和饱和插入是植物基因组学研究中的两种策略,而通过基因标签法建立大规模的突变体库是目前植物功能基因组学研究的最直接有效的方法。其中,激活标记法不仅能产生一般意义的功能丧失突变体,更重要的是能得到功能获得突变体,对于功能冗余基因以及生命周期多阶段必需基因的研究具有独特的优势。酿酒酵母转录因子yap1(Saccharomy cescerevisiae)在酵母中调节多达70个与氧化胁迫相关的基因的表达,在抗氧化胁迫反应中起着重要的作用。但是YAP1基因在高等植物中异源表达及对其影响还未见报道,因此YAP1基因在拟南芥中过量表达,对研究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具有重要意义。本论文的主要目的是建立耐盐模式植物盐芥的激活标签突变体库,为发掘利用盐芥的耐盐基因,揭示盐芥耐盐的分子机理奠定基础,同时研究了酵母转录因子YAP1基因在拟南芥中过量表达的功能。主要结果如下:1.盐芥激活标记突变体库的建立以耐盐模式植物盐芥为实验材料,通过农杆菌介导的花浸染法将激活标记载体pSKI015导入盐芥基因组,试图建立一定规模的盐芥激活标记突变体库。主要结果如下:(1)通过农杆菌介导的花侵染法,利用激活标记载体pSKI015对盐芥进行了遗传转化,共获得转化T0代种子1000g左右,通过除草剂筛选,共获得抗性苗约2000株。(2)通过对所获得的盐芥除草剂抗性苗随机取约800株进行bar基因的PCR检测,阳性率在85%以上,证明采用除草剂对转化突变体进行筛选是可行的。(3)通过对获得的2000株激活标记抗性苗,采用TAIL-PCR的方法进行农杆菌T-DNA插入盐芥基因组侧翼序列的扩增,并进行测序,得到侧翼序列150余条,并进行了相关的序列分析。本实验的目的是建立饱和的盐芥激活标记突变体库,但由于各种因素的限制,所获得的体变体的数量还远远不够,大规模的突变体库还在构建之中。利用TAIL-PCR进行侧翼序列的扩增已获得初步成功,只是对于PCR产物的测序方法还有待于进一步完善。2、YAP1基因在拟南芥中的过量表达通过RT-PCR的方法克隆到YAP1基因,并将其构建到植物表达载体pROKII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,在含30mg/L的卡那霉素的培养基上筛选获得纯合转基因株系,自交一代获得足够的纯合转基因种子后,对其进行了分子生物学的验证及生理指标的检验,结果如下:(1)通过对转基因植株进行PCR扩增,得到了1.95Kb的特异条带,表明YAP1已整合至拟南芥基因组中;(2)Northern杂交分析表明转基因植株均有杂交信号,野生型植株无明显信号,进一步说明YAP1基因整合到拟南芥的基因组后已正常转录表达。(3)在含不同浓度的NaCl(0-150mmol/L)MS培养基上,YAP1基因的过量表达提高了转基因拟南芥的种子萌发率及幼苗的耐盐性。(4)在盐胁迫条件下,转YAP1基因拟南芥表现出比野生型更高的耐盐性,主要表现在:在盐胁迫下,转基因植株保持了更高的光反应效率, MDA、H2O2含量及细胞膜透性明显低于对照,表明YAP1基因减轻了植株的氧化损伤。(5)在盐胁迫条件下,转YAP1基因植株抗氧化保护酶SOD、CAT、POD、APX、GST及GR的酶活性都显著高于对照,表明酵母YAP1基因在高等植物异源表达能够起到类似转录因子的作用,在胁迫条件下,调控了一系列抗氧化保护酶的表达,增强了拟南芥的抗盐性。本论文的主要创新点:1.以耐盐研究模式植物盐芥为材料,通过农杆菌介导的花侵染法将激活标记载体pSKI015大规模的对盐芥进行转化,建立盐芥激活标签突变体库。目前已获得激活标记突变体2000株,并对所获得的突变体T-DNA插入盐芥基因组侧翼序列进行了扩增,Blast序列比对分析。在本实验室首次较系统的对盐芥激活标记突变体库的建立中的各个环节进行详细研究,为后续研究盐芥的抗盐机理,耐盐基因的发掘利用奠定了基础。2.从酿酒酵母克隆得到了转录因子YAP1基因,并首次在高等植物拟南芥中进行异源表达,对转化植株进行了相关的分子检测,并对转基因植株的耐盐水平进行了较全面的分析,为进一步培育耐盐作物奠定了基础。更多还原

【Abstract】 Soil salinization is one of the major stress factors that limiting the productivity and quality of crops. The mechanism of plants adapt to salt stress is very complex, so improving the salt tolerance of crops still faced great challenges. Thus, analyzing the mechanism of plants responses to salt stress, exploring genes related to salt tolerance not only have important theoretical significance but also have important practical significance for cultivation of salt-tolerant crops. Up to now, Arabidopsis is the popular model material for plant molecular biology research, but it is a glycophyte, and Arabidopsis can only reveal a little information of the plant salt tolerance. As a close relative to Arabidopsis, Thellungiella halophila (salt cress) is becoming a promising model of plant salt-tolerant research system. Similar to Arabidopsis Thellungiella halophila is also belong to Cruciferae and has good genetic features such as similar morphology, small genome size, short life cycle, high-yield seeds and an efficient transformation method. However, Thellungiella halophila is able to withstand dramatic salinity shock up to 500 mM NaCl. This plant does not have salt glands or other complex morphological alterations either before or after salt adaptation, indicating its salt tolerance, to a large extent, stems from the basic physiological and biochemical mechanisms. Besides, salt cress and Arabidopsis share 90% and 95% identities on cDNA and amino acid sequences respectively, so it is convenient to transfer informations fron Arabidopsis (gene database, protein database and mutant lines) to molecular analysis on salt tolerance of salt cress. Up to now, analysis on salt cress physiology, biochemistry and gene expression, and the construction of different cDNA libraries and mutant libraries have made salt cress a valuable model for the study of salt tolerance.With the genome sequencing of salt cress is nearing completion, functional Genomics study of salt cress will be the focus of research. The research of functional genomics is based on abundant mutants achieved, saturation mutagenesis and saturation gene insertion are the two main strategies of plant functional genomics, and constructing large scale mutant library using the method of activation tagging is the most direct and effective method of plant functional genomics research. The method of activation tagging can not only create mutants of lost of function but also can create gain of function mutants. Those gaining of function mutants have much predominance in study of functional redundant genes and those that are indispensable in multiple stages of the plant life cycle.The transcription factor YAP1 gene of Saccharomy cescerevisiae can regulate the expression of much for 70 genes related to oxidative responses and plays an important role in anti-oxidative stress. So far, the ectopic expression of YAP1 in high plants has not been reported, so study the effect of over-expression YAP1 gene in Arabidopsis can give us much information of improving the anti-oxidative ability of plant cells and the plant’s resistance to abiotic stress by genetic engineering means.The main objectives of the dissertation were to construct a activation tagging mutants library of salt cress, lay the foundation for exploitation and application of salt tolerance genes and revealing the mechanism of salt tolerance of salt cress; at the same time studied on the effect of over-expression YAP1 gene in Arabidopsis. The main results as following;1. The construction of activation tagging mutants library of salt cress(1) In this experiment, activation tagging vector pSKI015 was introduced into the salt tolerance model plants Thellungiella halophila by Agrobacterium tumefaciens-mediated flora dipping transformation with an intention to construct an activation-tagged mutant library, we have obtained transgenic T0 seeds about 1000g, selected by Basta, about 2000 Basta resistant salt cress seedlings were obtained.(2) The result of PCR on Bar gene of randomly selected about 800 seedlings of the Basta resistant seedlings showed the positive rate was above 85%, suggesting that use Basta to select transgenic seed is effective.(3) TAIL-PCR method were used to obtain the flanking genome sequences of T-DNA insertion site for the 2000 Bar-resistant salt cress seedlings, about 150 flanking sequence have been obtained, blast analysis of those sequences have been done.The aim of this study is to establish the saturation activation tagging library of salt cress, but due to variety factors, the number of mutants still far not enough, large-scale construction work are in progress. Though flanking sequences can be successfully amplified by TAIL-PCR, it is necessary to develop a more appropriate method of directly sequencing PCR products.2. The over-expression of YAP1 gene in ArabidopsisThe YAP1 gene was isolated from Saccharomy cescerevisia by RT-PCR method and confirmed by sequencing. The YAP1 PCR product was inserted into binary plant vector pROKII. The resulting plasmid, named pROK-YAP1, was mobilized to Agrobacterium tumefaciens strain GV3101 used for plant transformation. The yeast YAP1 gene was introduced into Arabidopsis thaliana by Agrobaterium tumefaciens-mediated transformation with floral-dipping method under the control of CaMV 35S promoter. Transformants were selected for their ability to grow on medium containing kanamycin (30mg/L), several homozygous lines that were all tolerant to kanamycin were selected and used for further molecular and physiological determination. The main result as following:(1) The transgenic lines were detected by PCR, a 1.95Kb band was obtained while wild type has no band indicating that the yeast YAP1 gene has been introduced into Arabidopsis genome.(2) Northern blot analysis revealed the expression of YAP1 mRNA in T3 plants several non-segregation transgenic lines while no signal was shown in wild type Arabidopsis.(3) On MS medium containing different levels of NaCl (0-150mM/L) over-expression of YAP1 in Arabidopsis improved seeds germination and seedling salt tolerance.(4) The MDA as well as the H2O2 content was obviously lower whereas the photosynthetic rate was higher in transgenic plants in comparison with that of controls under salt stress conditions. The results implied that the less oxidative stress might result from the transformants.(5) Under salt stress, in the transgenic plants the activity of ROS scavenging systems including SOD,CAT, APX,GST, and GR were obviously higher than that in wt plants, implying that YAP1 gene expressed in Arabidopsis act as an upstream regulating element and up-regulated much downstream genes expression.The innovations of this thesis can be summarized as follows:1. Activation tagging vector pSKI015 was introduced into the salt tolerance model plants Thellungiella halophila by Agrobacterium tumefaciens-mediated flora dipping transformation in large scale, with an intention to construct an activation-tagged mutant library, we have obtained about 2000 activation tagged lines, the T-DNA insertion flanking sequence was amplified by TAIL-PCR, and analysised by BlastN with Arabidopsis gene bank. This is the first time in our lab to study thoroughly the construction of salt cress activation tagging library, making the foundation for following study of exploitation and application of salt cress genes.2.The transcription factor YAP1 gene was cloned from Saccharomy cescerevisia, and for the first time heterogeneously expressed in Arabidopsis, the transgenic plants was identified by molecular analysis and the salt tolerance of the transgenic Arabidopsis was analyzed which lay a foundation for breeding new varieties in salt tolerance of crops.更多还原