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

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

Russian Federation, 71 October Av., 450054 Ufa

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

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

Russian Federation, 71 October Av., 450054 Ufa

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

PhD, Cand. Sci. (Biol.)

Russian Federation, 71 October Av., 450054 Ufa

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

PhD, Cand. Sci. (Biol.)

Russian Federation, 71 October Av., 450054 Ufa

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

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

Russian Federation, 71 October Av., 450054 Ufa

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Supplementary files

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2. Fig. 1. Scheme of organization of nod genes of R. leguminosarum bv. trifoli which control nodule formation

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3. Fig. 2. Scheme for constructs creation with nodD gene. The 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 in which 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 pTurboNodDGFP construct obtained, a 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 final genetically engineered construct pJNNodDGFP was used to transform the rhizobia strain N. galegae CIAM 0702

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

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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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