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The Constructing Of Arabidopsis Thaliana Activation Tagging Pool And Its Screen For Salt Sensitive Mutants

Posted on:2011-08-29Degree:MasterType:Thesis
Country:ChinaCandidate:Y L GuanFull Text:PDF
GTID:2120360305955502Subject:Biochemistry and Molecular Biology
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Aboitic stress, such as high-salinity, drought, flood, low and high temperature, is very important factor to affect the plant normal growth and development, it is also the main limited factor to restrain the crop yield in the world. Therefore, understanding the molecular mechanism of plant adapting to abiotic stress, is very helpful to us to genetic modification crop resource for improving crop production.Activation tagging is a technique to big-scale of constructing the T-DNA insertion pool, causing activating or silencing individual gene related to certain phenotype or traits, it is a quick and fast way to isolate the functional gene from one species in short of enough genetic information. Constructing the T-DNA insertion activation tag pool basing on model plant arabidopsis thaliana , is an efficiently method to isolate functional gene.The main work of this experiment is to construct on activation-tag pool in Arabidopsis, screen mutants which are sensitive to salt , isolate the flanking sequence of T-DNA by Tail-PCR, and investigate their primary function. The main results are listed as following:(1) we have constructed an activation tagging mutantion pool, using Arabidopsis Columbia wide type as materials, transforming by Agrobacterium tumefaciens harboring an activation tagging plasmid (pCB260) , until now, over ten thousands transformed plants were generated.(2) During the screening basta-resistance T1 lines, we isolated some mutants with special phenotype including early or late flowering time, unmoral leaf shape, sterility, special flowers and thin seed capsule color et al. We selected ten mutants and isolated their T-DNA flanking sequnces by Tail-PCR.(3) Using the 75mM NaCl to screen the salt sensitive mutant from our mutants pool. So far, three salt sensitive mutants, namely 1985,1742 and 2012, have been isolated.Their T-DNA flanking sequence have been isolated by Tail-PCR. We test the correctness of T-DNA location and identify the homozygotes by three primers method. Then, detecting the change of target gene expression using semi-quantitative RT-PCR, we find that 1985 is an overexpressed mutant, however,1742 and2012 are knockout mutants. Using the Laser scanning confocal microscope,we detect the change of H2O2 and NO in mutants were significant by 6-h100mM NaCl salt treatment. Preliminary data indicate these mutants are member of salt pathway, which is mediated by H2O2 and NO. We have detect the change of ABA synthase during salt treatment, based on the result we proposed that At1985 is one member upstream of ABA pathway.
Keywords/Search Tags:Activation tagging, T-DNA, Salt sensitive, Tail-PCR, Mutant, Arobidopsis thaliana
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