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Screening And Gene Mapping Of Arabidopsis Oxidative Stress Sensitive Mutants

Posted on:2013-09-20Degree:MasterType:Thesis
Country:ChinaCandidate:P LiangFull Text:PDF
GTID:2230330371999750Subject:Cell biology
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Various environmental stresses such as drought, low temperature, salinity, heavy metals make plants develop a systemic defense mechanism. So it is very significant to screen some stress resistance gene to study the environmental defense mechanism of plants. This thesis consists of four chapters. The first chapter summarizes the biological characteristics and genetic characteristics of Arabidopsis, as well as experimental techniques involved in this experiment. Chapter II discusses in detail the screening of the oxidative stress-sensitive mutants of Arabidopsis and the gene mapping. We got twelve strains of H2O2sensitive mutants in this experiment. Then with the use of TAIL-PCR, the T-DNA insertion site flanking sequences of the mutants were amplified successfully. Finally, taking advantage of the three primer method, the insertion sites of T-DNA were verified. In addition, homozygotes were identified in the F3generation. The third chapter discusses the identification of the ICE1mutant of Arabidopsis, the construction of expression vector, and the genetic transformation of the revised vector. Here we identified the ICE1mutant and cloned the ICE1gene as well as its promotor. Based on the construction of two plant expression vectors, genetic tansformation was carried out.Chapter I Literature ReviewArabidopsis thaliana is conformed as the model plant in laboratory. With the completion of the genome sequencing, a large amount of sequence information was obtained. However, seldom was known about the function of these sequences.Utilizing mutants to study the gene function of the plant is an effective method. Mutants are good materials for functional genemics research.Chapter II Screening and Gene Mapping of Arabidopsis Oxidative Stress Sensitive MutantsActive Oxygen Species(AOS) with an active chemical activity refers to certain metabolic intermediates of oxygen and its derivatives, which is becoming the thesis of stress biology research, such as H2O2, an important signal molecular. However, scare research is related to the transduction detail of H2O2as a signal molecular and its relationship with other signal moleculars. In this experiment,H3, H4, H6, H7, H8, H9, H13, H14, H17,H18, H61,H71were obtained by screening the T-DNAinsertion mutants.500μM H2O2was employed to rescreen the seeds of F2generation to determine its phenotype. The T-DNA insertion site flanking sequence of the mutant were amplified successfully by TAIL-PCR. When compared the sequencing results with the NCBI database, the T-DNA insertion sites were identified as lipid-associate family, auxin, response factor of SIR, sulfite reductase lipid-associated family, auxin response factor, SIR sulfite reductase, leucine-rich repeat protein kinase, ML08calmodulin binding, disease resistance protein. In addition, homozygotes were identified in the F3generation.Chapter III Identification of Arabidopsis ICE1mutations and Construction and Genetic Transformation of Plant Expression VectorAs the upstream regulatory factors of cold response gene, ICE1may adjust the low-temprature-induced trans-acting factors CBF at low temprature, making the combination of the CBF with the core element CRT/DRE of COR promotor, with the elevesion of COR expression level. So that the cold tolerance ability of the plant will be improved greatly. Homozygote was obtained by three pair method. We also successfully constructed two plant expression vectors pEGAD-ICE1and pEGAD-PICEl::ICE1followed by the genetic tansformation.Chapter IV Modification of pFGC5941construct and Transfer of Double Antisense Genes of NAC1and SIP1into Arabidopsis thalianaAn unknown protein SIP1which has interaction with SOS2was obtained by yeast two-hybrid. Meanwhile we cloned both SIP1gene and NACI gene that can significantly promote the development of the lateral root of Arabidopsis thaliana And then we integrated SIP1gene with sense or antisense NACI gene into modified RNA interference carrier pFGC5941that includes two35S promoters, finally resulting in two dual gene expression vectors. These two vectors were successfully transformed into wild-type A. thaliana, giving birth to fifteen transgenic plants in total. which lays a good foundation for further study on the functions of SIP1and NAC1genes and the regulation of Arabidopsis lateral root development in salt stress.
Keywords/Search Tags:Arabidopsis thaliana, activation tagging, H2O2, mutant, TAIL-PCR, vector construction
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