Selection Rhizobial Strains
Development of Molecular Genetic Technology for Construction and Subsequent Selection of Highly Efficient Rhizobial Strains to Increase the Yields of Legumes
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
3 Approved without Funding
Research Center of Toxicology and Hygienic Regulation of Biopreparations, Russia, Moscow reg., Serpukhov
- Universita degli Studi di Milano / Departamento di Fisiologia e Biochimica Generali, Italy, Milan\nUniversity of Tennessee, USA, TN, Knoxville\nUniversity of Minnesota / Department of Soil, Water, and Climate, USA, MN, St Paul
Краткое описание проектаBacterial-plant interactions are of fundamental importance to our understanding of biological processes. The nitrogen fixing symbiosis of members of the family Rhizobiaceae (Rhizobium, Bradyrhizobium, Azorhizobium) species and legumes presents both theoretical and practical interest.
Industrial strains of (Brady-) Rhizobium are widely used in different countries as an economically profitable and ecologically harmless alternative to expensive nitrogen fertilizers.
Selection of efficient rhizobial strains is especially important in intensive agricultural production and is based on the knowledge of molecular genetic mechanisms responsible for nitrogen fixing (Brady-) Rhizobium - legume symbiosis.
The aim of this project is to develop a biotechnology for the directed construction and subsequent selection of efficient Bradyrhizobium. japonicum and probably other rhizobial strains.
The development of biotechnology will be includes the investigations of B. japonicum glt (glutamate syntheses, GOGAT) as well as a novel asm1, asm2 genes and GS (glutamine synthetase), Ntr (nitrogen regulation), Amt (ammonium transport), Asm (ammonium assimilation) genetic systems involved in nitrogen metabolism and simbiotic nitrogen fixation (Fix).
According to our previous investigation, the pBT53glt cosmid carrying these genes is able to complement still unknown gltY mutation in B. japonicum Asm Noes. 108, 109 strains ("Super Fix" strains), increasing the soybean yield by 15-20% as compared to the parental bacteria. The Asm 108, 109 mutants belong to two families of B. japonicum Asm Fix+ and Asm Fix- strains isolated earlier.
We plan to identify the gltX and gltY genes suggested to be mutated in B. japonicum Asm Noes. 103, 104 and Asm Noes. 108, 109 strains, respectively. The structural organization and functional role of the gltBD (GOGAT), gltX, gltY, asm1 and asm 2 genes in formation of the highly efficient phenotype by B. japonicum Asm Noes. 108, 109 mutant will be studied. The character of participation of the genes ("super fix" genes, symbolically) belonging to the GOGAT,GS, Ntr, Amt, Asm genetic systems in high efficiency of the "Super Fix" Asm 108, 109 strains will be elucidated.
The development of the biotechnology will be based on the results of the above investigations and subsequent construction of the effective (Brady-) Rhizobiuт will be achieved by directed and coordinated modification of the activity of glt Y, and/or gltBD, asm1, asm2 "super fix" genes in commercial mizobial strains.
Technical Approach and Methodology
The biotechnology suggested implies the following methods and approaches:
(i) site-directed in vivo interposon mutagenesis and construction of the (Brady-) Rhizobium glt Y::kan strains carrying kan-insertion in glt Y gene that is mutated in the "Super Fix" B. japonicum Asm Noes 108, 109 bacteria;
(ii) transference of the glt Y mutations from the Asm 108, 109 bacteria to wild type of (Brady-) Rhizobium by in vivo oligonucleotide-directed mutagenesis procedure, that will be developed for this purpose;
(iii) directed and coordinated modification of the activity of glt Y and/or gltBD, asm1, asm2 "super fix" genes, in wild type of rhizobia, or in the mutants mentioned above (see i. and ii.). The modification will be achieved by the methods of the site- and oligonucleotide-directed mutagenesis as well as using the multicopy and pL-overexpression vectors and cassets accommodated for rhizobia.
The effective strains of B. japonicum and other rhizobia, adapted for corresponding agricultural areas of Russia, the Ukraine, Uzbekistan, the USA, China and Thailand will be used as parental bacteria and more efficient rhizobia will be selected by the most resultative methods of the above developed biotechnology.
The suggested biotechnology and methods to be developed for the selection of (Brady-) Rhizobium strains have the following advantages over the well-known approaches to the selection of efficient rhizobia.
1.The commercial (Brady-) Rhizobium strains adapted for various agricultural areas in different countries will be used as parental bacteria for the selection of more efficient rhizobia.
2.The biotechnology has the goal-oriented, as well as wide host ranged character, because it implies the directed and coordinated modification of certain genes belonging to the GOG A T, GS, Ntr, Asm genetic systems existing in all rhizobia.
The project implementation will result in the following accomplishments:
1. The structural organization and functional role of the B. japonicum gltBD (GOGAT), gltX, glt Y as well as novel asm1, asm2 genes and their products in nitrogen metabolism and N2-fixation will be analysed on genetic and biochemical levels.
2. The glt X and glt Y genes mutated in the B. japonicum Asm 103, 104 (Fix-) and Asm 108, 109 ("Super Fix") strains, respectively, will be identified and molecular genetic analysis of these genes will be performed.
3. Certain "super fix" genes belonging to the GOG AT, GS, Ntr, Asm, Fix genetic systems and contributing to high efficiency in the Asm 108, 109 mutants will be revealed and the molecular genetic mechanisms of this contribution will be analyzed.
4. Based on these results (points 1-3) the biotechnology for the construction of the efficient B. japonicum GltM strains will be developed by directed and coordinated modification of the glt Y and/or gltBD, asml, asm2 "super fix" genes, mentioned above.
5. The collection of B. japonicum GltM strains capable to increase the soy-bean yield by at least 15-20% as compared to the parental industrial strains and adapted for various agricultural areas in Russia, the Ukraine, Uzbekistan, the USA, China and Thailand will be selected by the above developed technology.
6. The biotechnology will be tested for selection of similar efficient GltM strains of R. meliloti, R. leguminosarum, R. fredii and R. trifoli.
The results of our and others previous investigations showed the functional similarity of the glt loci in different (Brady-) Rhizobium strains and agricultural characteristics of B. japonicum Asm 108, 109 mutants enable us to estimate the possibility of the project implementation as positive.
The Potential Role of Foreign Collaborators
The structural organization and functional role of the genes belonging to the GOGAT (glutamate synthase), Asm (ammonium assimilation), Amt (ammonium transport), Ntr (nitrogen regulation) genetic systems in nitrogen metabolism and symbiotic N2 - fixation by B. japonicum will be studied in cooperation with laboratories from the USA, Italy, Switzerland and other countries.
Similarly, the functions of these genes in Fix and highly efficient B. japonicum Asm 108, 109 mutants will be analyzed.
Based on above results the molecular genetic biotechnology for directed selection of the B. japonicum and, probably, other rizobial strains will be developed.
The collection of the highly efficient B. japonicum adapted for different agricultural areas in Russia, the Ukraine, Uzbekistan, the USA, China will be selected by joint work together with foreign partners.
Coordination of the project activities, analysis and discussion of the data obtained will be conducted.
Coordination of the project will enable the integration of skilled Russian specialists into the international scientific community.