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Construction Of Salt Cress Activation-tagged Mutant Library And Overexpression YAP1 Improved Salt Tolerance In Arabidopsis Thaliana

Posted on:2009-05-21Degree:DoctorType:Dissertation
Country:ChinaCandidate:J Q ZhaoFull Text:PDF
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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.
Keywords/Search Tags:Thellungiella halophila, activation tagging, mutant, transcription factor, YAP1
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