Creation of an inducible vector system based on the rhizobia nodA gene promoter

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Abstract


Background: The possibility of changing the properties of rhizobial bacteria by giving them the ability to regulate the expression of additionally introduced genes into them is an urgent task both for fundamental science and for applied agrobiology, since this will make it possible to obtain microsymbionts with desired properties. An expression construct using the rhizobia regulatory system was created in this work. The rhizobia nodD gene encodes a regulatory protein that, in the presence of plant inducers, flavonoids, activates the transcription of nod-genes involved in the early stages of the formation of legume-rhizobium symbiosis.

Materials and methods: A vector construct containing the nodD gene from Rhizobium leguminosarum bv. trifoli under the regulation of its own promoter and the gfp gene under the regulation of the nodA gene promoter from the same rhizobia was obtained. Neorhizobium galegae CIAM 0702 were transformed with the vector construct.

Results: It has been shown that in recombinant strains synthetic flavonoids are capable of inducing expression of gfp gene to varying degrees.

Conclusion: In the future, the results can be used to obtain rhizosphere microorganisms with a controlled synthesis of growth-stimulating and protective substances.


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About the authors

Olga V. Chubukova

Institute of Biochemistry and Genetics – Subdivision of the Ufa Federal Research Centre of the Russian Academy of Sciences

Author for correspondence.
Email: chubukova@bk.ru
ORCID iD: 0000-0001-7553-9527
SPIN-code: 3297-4830
ResearcherId: A-3397-2014

Russian Federation, 71 October Av., 450054 Ufa

PhD, Cand. Sci. (Biol.), Researcher

Zilya R. Vershinina

Institute of Biochemistry and Genetics – Subdivision of the Ufa Federal Research Centre of the Russian Academy of Sciences

Email: zilyaver@mail.ru
ORCID iD: 0000-0001-5480-5320
SPIN-code: 1866-7896

Russian Federation, 71 October Av., 450054 Ufa

PhD, Cand. Sci. (Biol.), Researcher

Rustam T. Matnyazov

Institute of Biochemistry and Genetics – Subdivision of the Ufa Federal Research Centre of the Russian Academy of Sciences

Email: rmat@mail.ru
SPIN-code: 6798-7913

Russian Federation, 71 October Av., 450054 Ufa

PhD, Cand. Sci. (Biol.)

Andrey K. Baymiev

Institute of Biochemistry and Genetics – Subdivision of the Ufa Federal Research Centre of the Russian Academy of Sciences

Email: baymiev@anrb.ru
ORCID iD: 0000-0001-6637-9365
SPIN-code: 1919-5236

Russian Federation, 71 October Av., 450054 Ufa

PhD, Cand. Sci. (Biol.)

Aleksey K. Baymiev

Institute of Biochemistry and Genetics – Subdivision of the Ufa Federal Research Centre of the Russian Academy of Sciences

Email: alex@anrb.ru
ORCID iD: 0000-0003-0606-6740
SPIN-code: 3771-4063

Russian Federation, 71 October Av., 450054 Ufa

PhD, Cand. Sci. (Biol.), Head of Laboratory

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Supplementary files

Supplementary Files Action
1.
Fig. 1. Scheme of organization of nod-genes of rhizobia R. leguminosarum bv. trifoli, which control nodule formation

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2.
Fig. 2. Scheme for creating constructs with the nodD gene. DNA fragment of R. leguminosarum bv. trifoli containing the sequence of the nodD gene, including the promoter (PnodD) and the promoter region of nodA (PnodA), were amplified with primers containing BglII restriction sites were introduced. Next, the amplificate was cut with the BglII restriction enzyme and cloned at the BamHI site in the pTurboGFP-B vector. From the obtained pTurboNodDGFP construct, the fragment containing nodD + PnodA + gfp was recloned at the XhoI and DraI restriction sites into the pJN105 plasmid of a wide range of hosts. The ultimate genetically engineered design pJNNodDGFP transformed the N. galegae strain of rhizobia CIAM 0702

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3.
Fig. 3. Western blot analysis of total protein from N. galegae, transformed with the pJNNodDGFP plasmid under the action of various inducers (flavonoids): 1 - protein markers; 2 - GFP control, E. coli sample with pTurbo GFP-B plasmid; 3 - apigenin; 4 - taxifollin; 5 - genistein; 6 - quercetin; 7 - hesperidin; 8 - N. galegae, with plasmid pJNNodDGFP, without inductor

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Copyright (c) 2021 Chubukova O.V., Vershinina Z.R., Matnyazov R.T., Baymiev A.K., Baymiev A.К.



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