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Construction Of Arabidopsis Activation Tagging T-DNA Insertion Mutant Library And The Screening Of D-Ala Resistant Mutants

Posted on:2012-02-09Degree:MasterType:Thesis
Country:ChinaCandidate:T GuoFull Text:PDF
GTID:2210330338470840Subject:Cell biology
Abstract/Summary:PDF Full Text Request
With the rapid development of biological sciences, the post-genomics becomes the focus of genomics research which identifying biological function of genes is regarded as targets. The mutants play an important role in the study of biological function of genes, and it is an important material of the research. To construct saturated mutant library, is one of the most direct and effective methods to go to the research. Arabidopsis thaliana is a plant annual herb of Cruciferae, used as the most popular Model plant in the study of plant molecular genetics, with its distinct genetic and molecular biological characteristics (such as small plant, short life period, high propagating, easy-to-artificial mutation and easy to transform, etc). Arabidopsis has become the focus of biological function of genes, with its genome has been fully sequenced, but 90% of the gene function has not yet been identified.Alanine (Ala) is classified as the protein amino acids, involved in protein composition, and there are two configurations:D-type and L-type because of its molecular spatial arrangement. The L-amino acid was found to be involved in natural protein composition, but current information suggests that D- amino acid metabolism is severely restricted in plants, and some D-amino acids such as D-Ala and D-Ser are toxic to organisms. The Proposed mechanism for D-amino acid toxicity is competitive inhibition or feedback inhibition, but its mechanism is still not well understood, and has only occasionally been studied in plants.The main work of this experiment is to construct an Arabidopsis gain of function mutants pool, screen mutants with D-Ala resistance, isolate the mutant genes by Tail-PCR, clone the genes, and study their primary function. Columbia wild type Arabidopsis thaliana was used as experimental material, T-DNA insertion mutant library was constructed by the activation tagging method (Agrobacterium tumefaciens with an activation tagging vector pCB260 was transformed into the plant by floral dip method). Six D-Ala resistant mutants were got, the mutant genes were isolated by Tail-PCR.The main results are as following: (1) T-DNA insertion mutant library was constructed by the activation tagging method. In this study, the Columbia (Col) wild-type Arabidopsis thaliana was used as experimental material, Agrobacterium tumefaciens of GV3101 carrying the activation tagging vector pCB260 with herbicide-resistant Bar gene as a selective marker was transformed into wild-type Arabidopsis plants by flower dip method. The mutants totally about 20,000 plants with T-DNA insert were screened by spraying herbicide Basta. And the mutant phenotypes are mainly as following:variation of the plant type and leaf shape; the changes of flowering time and fertility; the variation of color of seed, etc. And sever mutants with significant variations were got which the mutant genes were isolated by Tail-PCR.(2) First, the standard concentration (3mM) of screening D-ala resistant mutant was determined through the concentration gradient experiment. It was found that the effect of Arabidopsis plants from D-ala was related with the content of NO3-, the effect of plant has been weakened by stress with the increase of the content of NO3-. Then, six D-ala resistant mutants (A22, A36, A15, A20, A24, A46) were selected from the T-DNA insertion mutants pool, and the Phenotype of T2 generation was assured by re-screened on the MS medium containing 3mM D-ala.(3) The T-DNA flanking sequence was successfully amplified by Tail-PCR, and the results of NCBI data comparison indicated that the mutant genes were located at different chromosomes(2,5,2,1,3)。The concentration gradient experiment of the mutants (A22, A36) indicated that the mutants have a certain resistance to D-ala, and the mutant genes probability involved in Arabidopsis resistance to D-ala. The T-DNA location was verified, and the homozygotes were identified from T3 generation by three primers method.
Keywords/Search Tags:Arabidopsis thaliana, activation tagging, mutant, Tail-PCR, D-ala
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