الفهرس 
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Abstract
In this research thesis, we characterized a novel Medicago truncatula mutant, assigned it as black nodule mutant (blkn). This mutant is a Tnt1-retrotransposon mutant, Tnt1 is Nicotiana tabacum retrotransposon which is replicated via an RNA copy and integrated in Medicago truncatula genome. blkn is obtained from Noble Foundation Tnt1 -mutant collection. This line was produced in vitro through A. tumefactions transformation. blkn mutant is achieving a null function root nodules phenotype and exhibited about double contents of phenolic compound than the wild type, the mutated nodules displayed abnormality in infection and nitrogen fixation zones. The first characterization step for our mutant was using ABI 310 Genetic Analyzer to compare the polymorphism percentage between blkn and wild type. The Genetic Analyzer was used with a novel modification as we used the Tnt1 borders-specific oligonucleotide primers instead of using the universal oligonucleotide primers along with florescence labeled
Summary
oligonucleotide primers. Our next step was using reverse genetic tools to isolate all Tnt1-Flanking Sequence tags (Tnt1-FSTs) in our mutant line. AFLP type PCR was carried out to identify the mutated gene(s) in this mutant line which is supposed to be involved in nitrogen fixation process. More than 20 Tnt1-FSTs ranging from ~500 to ~100 bp were isolated, sequenced, analyzed, before submittion to Genebank accession numbers from MN529997 to MN530012. In The third step mutated gene was determined of in blkn mutant and its role in Medicago plant depending on the observed phenotype due to mutation in this gene. The mutation was precisely mapped in Medicago genome by determining the location of Tnt1 insertion next to Tnt1 insertion was precisely located next to the base number 303 post ATG start codon of M. truncatula L-type lectin-domain receptor kinase VII.2. The mutation on this locus was confirmed on both DNA and RNA levels. Plants homozygous for the blkn allele were crossed to R108 wild type plants. F2 plants were subjected to analyze the segregation pattern of blkn allele in order to
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confirm the relation between the phenotype and the mutation at this locus. The mutated Medicago seedlings resulting from the back cross was found 1/4 of F2 population individuals. The mutation of Medicago truncatula Lec_legB-RK caused alteration in plant cells unable to distinguish between the symbionts and invaders. A suggestion was made that although the Rhizobia- derived N-acetylglucosamine compounds were normally perceived by Medicago via LysM proteins otherwise the plant cells treated them as an attacker and consequently, produced a very high content of phenolic compound in blkn root and nodules comparing to wild type.
For more mutant characterization at the physiological level, Total carotenoid and phenolic compounds were found in higher concentrations in the mutant lines. Meanwhile the chlorophylls levels appear to be the same in both lines.
Some differences in the phenotype were observed between the control and the mutant as root length, nodule number and pod number were greater in the mutant line.