Construction Of Thellungiella Halophila Activation-tagged Mutant Library And Transposon-tagged Mutant Selection

The mechanism of plants adaptation to salt stress is very complex, so improving the salt tolerance of crops is still a great challenge. Therefore, 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.Thellungiella halophila (Salt cress) is a close relative of Arabidopsis thaliana, and has good genetic features such as a short life cycle, a small size of genome, an efficient transformation and high seed numbers, etc. Thellungiella halophila and Arabidopsis thaliana share 95% and 90% identities on cDNA and amino acid sequences respectively, so it is convenient to transfer informations of Arabidopsis (gene database, protein database and mutant lines) to molecular analysis on salt tolerance of salt cress. But, in sharp contrast with Arabidopsis, Thellungiella is able to withstand dramatic salinity shock up to 500 mM NaCl. This plant does not produce salt glands or other complex morphological alterations either before or after salt adaption. Up to now, Thellungiella has become a valuable model for the study of salt tolerance. And analysis on salt cress physiology, biochemistry and gene expression, and the construction of different cDNA libraries and mutant libraries have been made.Since Thellungiella sequencing is to be completed, the main task of this work will be functional genomic study, and the most direct and efficient way of functional genome study is to construct large-scale mutant library by various kinds of methods. From the current view, the mutant library construction methods used are mainly the following: spontaneous mutation, physical and chemical mutagenesis and insertional mutagenesis, in which insertion mutations can be divided into T-DNA insertional mutagenesis and transposon tagging methods.In this experiment, activation tagging vector pSKI015 and Ds launch pad T-DNA vector were respectively introduced into Thellungiella by Agrobacterium tumefaciens-mediated flora dip transformation with an intention to construct an insertional mutant library. Molecular analysis and cloning of the flanking genomic sequences have been conducted to the obtained resistant plants. Summarily, in this experiment, only primary research was made on construction of Thellungiella insertional mutant library and the library in large scale is being constructed now.The main objectives of the dissertation were to construct an activation tagging mutant library of Thellungiella and the transposon tagging method to obtain Thellungiella mutants had also been explored. The main results are as following:1.The construction of activation tagging mutant library of ThellungiellaIn this experiment, activation tagging vector pSKI015 was introduced into Thellungiella by Agrobacterium tumefaciens-mediated flora dip transformation with an intention to construct an activation-tagged mutant library. The main results are as following:(1) Activation tagging vector pSKI015 was introduced into the salt tolerance model plant Thellungiella halophila by Agrobacterium tumefaciens-mediated flora dipping method, we have obtained nearly 800g of T0 transgenic seeds, and 853 lines of Basta-resistant Thellungiella seedlings by Basta screening.(2) The basta-resistant Thellungiella plants were further identified by PCR detection of Bar gene, and the results showed that 685 of 853 resistant seedlings were positive, the positive rate reached 80%.(3) TAIL-PCR method were used to obtain the flanking genomic sequences of T-DNA insertion site from the 685 Bar-resistant Thellungiella seedlings, about 36 flanking sequences have been obtained, and BLAST analysis of these sequences have been done.2. The selection of Thellungiella transposon-tagged mutants Transposon tagged vector Ds was introduced into Thellungiella halophila by Agrobacterium tumefaciens-mediated flora dipping transformation.Then we selected transgenic T0 plants by Basta. And we obtained resistant Thellungiella seedlings. The main results were as below:(1) Transposon tagged vector Ds was introduced into Thellungiella halophila, we obtained transgenic T0 seeds, selected by Basta, about 87 Basta-resistant Thellungiella seedlings were obtained. (2) The results of PCR detection of Bar gene in resistant plants found that,76 lines of 87 resistant seedlings were positive.