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Risultati sfruttabili
Lipooligosaccharides (LCOs) produced by strain A1 Rhizobium leguminosarum bv. viciae which is able to modulate Afghan peas were analyzed by means of chromatography and mass spectrometry. Strain Al produces LCOs in high amounts comparable to those of R.leguminosarum bv. viciae strain l089 used as a standard. Structural analysis revealed that strain A1 produces a large family of Nod factors, including all species described for R.leguminosarum bv. viciae strains, the nodX - mediated LCOs known to be necessary for nodulation of Afghanistan peas, and LCO species with specific fatty acids which are not very common for other R. leguminosarum bv. viciae strains. The host - specific nod genes of strain Al involved in LCO synthesis were cloned and sequenced. Comparison of the cloned genes with corresponding sequences from symbiotic plasmids pRL1JI and pRL5JI did not reveal significant differences between the structure of these nod genes. It was shown that fucosylation of the reducing terminus of Nod factor by the nodZ gene of Bradyrhizobium japonicum as well as acetylation of this site of Nod factor by the nodX genes from strains TOM or Al confers on the bacteria and ability to nodulate peas carrying sym2 -allele. A flavonoid - independent nodD gene is not required for the nodulation ability of nodZ - carrying strains but can improve this ability on more restrictive Afghan pea cultivars. It was also shown that the nodO gene which has been suggested to play a role in recognition of host range by the sym2 allele of Afghan pea is not involved in the complementation of by nodZ. Thus, it has been shown that the restriction of host range by the sym2 allele of Afghan pea is non - specific for the type of modification at the reducing terminus of nodulation signals.
The symbiosis is a result of a coordinated interaction between genomes of the legume plants and soil bacteria. The nodulin genes are known as early (ENOD) and late (NOD) nodulin genes. The homology between the genes of different biospecies for comparing two legume genotypes, namely pea (Pisum sativum) and broad bean (Vicia faba) was used.
ENOD12 is present in two copies, namely PsENOD12A and PsENODI2B. The research was aimed at revealing whether ENOD12 is a common legume gene or not. With primers homologous to 5' and 3' ends of cDNA copy of PsENOD12A it was shown that ENOD12 fragments of various lengths are present in several studied legume genomes, in most cases (broad bean, lupin, kidney bean) ENODl2 is represented on two copies like in pea. The data obtained show that the ENOD12A generally is more polymorphic than ENODl2B at least in pea. VfENOD12B fragment is 414 bp long and it contains 267 bp open reading frame. When comparing these two sequences, a small 12 bp insertion in VfENODl2B gene after the 224th nucleotide was found. As supposed, VfENODl2B homology with PsENODI2B is high - 82%. Identity between their proteins is 75.6%. VfENODl2B consists of 89 amino acids and its molecular mass is 9995 Da. This protein is hydroxyproline-rich (content of the proline is 16.8%) and it possesses 3 additional prolines as compared with the PsENOD12B. The VfENOD12B PCR fragment is essentially identical to the transcript sequence identified by differentially screening a nodule cDNA library of broad bean and to the corresponding gene. The 600 bp transcript was found exclusively in roots as early as 3 days after inoculation. The transcripts were located in the invasion zone II of the mature nodule.
Thus, it has been shown that different legumes do contain the sequences with homology to PsENOD12A and PsENOD12B genes. It cannot be claimed that all these sequences are normal transcribed genes. However, the facts that (i) sizes of PCR products were very similar to those of PsENOD12A/PsENOD12B and (ii) randomly chosen PCR fragment of broad bean does contain a full open reading frame which allows us to consider ENODl2 as a common gene. Further experiments using VfENODl2 promoter-GUS fusions in transgenic M. truncatula lines will give further insights in the gene expression of this nodulin gene.
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