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Mesorhizobium mediterraneum and Mesorhizobium
tianshanense – symbionts of sainfoin
S.A. Khapchaeva
(1),
N.V. Punina
(1,2),
V.S. Zotov
(1),
S.V. Didovych
(3),
A.F. Topunov
(1)
(1) A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia; е-mail: [email protected]
(2) Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow, Russia; [email protected]
(3) Institute of Agriculture of Crimea, National Academy of Agrarian Sciences of Ukraine, Simferopol, Crimea, Ukraine; е-mail: [email protected]
ABSTRACT
The Onobrychis genus comprises a few agronomically important forage legume species, with sainfoin being the most widespread. The Onobrychis
genus belongs to the Fabaceae family and Hedysareae tribe. It is widespread in temperate zones of North America, Europe, Mediterranean and
Western Asia. There are a lot of specific and ecological varieties of the sainfoin in natural flora. The main cultivated species are O. viciafolia, O.
antasiatica, O. arenaria.
Recent research works have highlighted several additional beneficial properties of Onobrychis for livestock, and this is mainly due to the nature of
its particular secondary metabolites. Furthermore, it is known to enhance diversity and stability of agroecosystems, representing a valuable pollen
and nectar source for honey production.
In comparison to other legumes, the nitrogen fixation rate of Onobrychis genus has been measured in terms of both the amount of nitrogen fixed and
expressed in terms of resultant increase in yield. Sainfoin is readily nodulated by many bacteria species such as Phyllobacterium, Rhizobium, and
Mesorhizobium, which can form the nodules on roots and can thus fix atmospheric nitrogen. However, they haven’t been examined in details. That
is why the previous investigations of taxonomy of sainfoin's nodule bacteria did not give the whole notion about the simbiont’s biodiversity. So the
investigation of phylogeny of sainfoin’s symbionts by complex approach targeted to investigation of housekeeping and symbiotic parts of a genome
is actual and perspective task.
RESULTS
INTRODUCTION
• The nucleotide sequences of the 16S rRNA for every
investigated strain had been determined. Strains were
referred into 2 species: M. tianshanense and M.
mediterraneum, that firstly was found for sainfoin
symbionts.
• Phylogeny by ITS-region revealed the presence of
frequent recombination events between species.
• The specific strain’s membership was confirmed by
analysis of gyrB gene sequences.
• By using of saAFLP analysis the significant interspecies
diversity was showed. Investigated strains were divided
into 6 genetically heterogeneous groups of strains.
• The investigated strains were historically developed
independently. Phylogeny of symbiotic genes (nodC
and nifH ) correlate with host legume systematics.
In prairie and foothill regions of Crimea for the first time 6
isolates of symbiotic bacteria from sainfoin’s nodules were
collected. These nodule bacteria were analyzed using
fingerprinting saAFLP with rare-cutting restriction enzymes
XmaJI, XbaI, and also by analysis of nucleotide sequences of
internally transcribed spacer region between the 16S and 23S
rRNA genes (ITS), 16S rRNA, gyrB, and symbiotic nodC
(synthesis of oligomers of Nod factors) and nifH (synthesis of
nitrogenase iron protein) genes.
Fig 1. Phylogenetic tree constructed based on sequences 16 S rRNA
gene of 18 strains Mesorhizobium sp. using algorithm NJ, bootstrap
value was 1000 replica
MATERIALS AND METHODS
Bacterial strains. 6 rhizobial isolates were obtained
from root nodules of legume plant – Onobrychis sp.
growing in Crimea, Ukraine.
DNA isolation. Total genomic DNAs of all rhizobial
isolates were obtained after lysozyme-sodium dodecyl
sulfate lysis followed by phenol-chloroform extraction and
ethanol precipitation.
PCR amplification 16S rRNA gene and 16-23S
rRNA (ITS). The primers fD1 and rD1 were used to
Fig 3. Phylogenetic tree constructed based on sequences nifH and nodC
genes of 54 strains Mesorhizobium sp. using algorithm NJ, bootstrap value
was 1000 replica
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amplify 16S rRNA genes (Weisburg et al., 1991). For ITS
amplification, the primers FGPS1490-72 (Normand et al.,
1992) and FGPL 132 (Navarro et al., 1992) were used. PCR
products were amplified, purified and directly sequenced.
PCR amplification gyrB gene. GyrB was amplified
with primers systems described earlier Yamamoto and
Harayama (1995). The gene fragments were amplified,
PCR-products were purified, were sequensed (Sanger et al.,
1977).
saAFLP. The AFLP procedure was performed as
described by Valsangiacomo (1995), with some
modifications. 80 ng of DNA was digested with XmaJI,
XbaI restriction enzymes and ligated with single adapter
specific for restriction half-site. The restriction-ligation
reaction was performed at 37°C for 2 h in a total volume of
20 μl (Zotov et al., 2012)
PCR amplification nodC and nifH genes. NodC
and nifH genes was amplified by PCR with primers systems
nodCF`, nodCI` and nifHF, nifHI (Laguerre et al., 2001),
respectively.
Fig 2. Phylogenetic tree constructed based on nucleotide sequences intergenic
region 16-23S rRNA (ITS) of 78 strains Mesorhizobium sp. using algorithm
NJ, bootstrap value was 1000 replica
ACKNOWLEDGEMENT
This research was made within the framework of contract about
scientific cooperation between A.N. Bach Institute of Biochemistry
RAS and Department of Microbiology, Institute of Agriculture of
Crimea NAAS of Ukraine, by financial support of «Greenwide»
company, and by target grant of Presidium RAS «Support of young
scientists».