Cloning, Prokaryotic Expression and Functional Characterization of Gene from the Associative Nitrogen-Fixing Bacteria DX120E.

Background: Biological nitrogen fixation (BNF) is a unique mechanism in which microorganisms utilize the nitrogenase enzyme to catalyze the conversion of atmospheric nitrogen (N) to ammonia (NH). Fe protein, encoded by the gene, is an essential component of the nitrogenase in DX120E. However, the function of this gene in regulating nitrogen fixing activity is still unclear.

Objectives: The objective of this study was to reveal the function of gene in associative nitrogen-fixing bacteria DX120E and micro-sugarcane system by immunoassay and gene editing.

Materials And Methods: In the current investigation, the gene was cloned in a pET-30a (+) vector and expressed in . The NifH protein was purified and used to immunize rabbit, and then the serum was collected and purified to obtain rabbit anti-NifH polyclonal antibodies. The CRISPR-Cas9 system was applied to produce mutant strains, and the nitrogen-fixing enzyme activity, gene, and protein expression were analyzed.

Results: Both and NifH proteins were detected by Western blotting, which were 43 and 32 kDa respectively. The expression of and genes was decreased, and nitrogenase activity was reduced in the mutant strain.

Conclusion: The gene mutant weakened the nitrogenase activity by regulating the expression of Fe protein, which suggests a potential strategy to study the nitrogen fixation-related genes and the interactions between endophytic nitrogen-fixing bacteria and sugarcane.