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Iranian Journal of Biotechnology، جلد ۲۱، شماره ۳، صفحات ۸۹-۹۹
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عنوان انگلیسی Cloning, Prokaryotic Expression and Functional Characterization of NifH Gene from the Associative Nitrogen-Fixing Bacteria Klebsiella Variicola DX120E
چکیده انگلیسی مقاله Background: Biological nitrogen fixation (BNF) is a unique mechanism in which microorganisms utilize the nitrogenase
enzyme to catalyze the conversion of atmospheric nitrogen (N2) to ammonia (NH3). Fe protein, encoded by the nifH
gene, is an essential component of the nitrogenase in Klebsiella variicola 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 nifH gene in associative nitrogen-fixing bacteria
Klebsiella variicola DX120E and micro-sugarcane system by immunoassay and gene editing.
Materials and Methods: In the current investigation, the nifH gene was cloned in a pET-30a (+) vector and expressed
in Escherichia coli. 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 nifH mutant
strains, and the nitrogen-fixing enzyme activity, gene, and protein expression were analyzed.
Results: Both in vitro and in vivo NifH proteins were detected by Western blotting, which were 43 and 32 kDa respectively. The expression of nifD and nifK genes was decreased, and nitrogenase activity was reduced in the nifH mutant strain.
Conclusion: The nifH 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 nitrogenfixing bacteria and sugarcane.
کلیدواژه‌های انگلیسی مقاله Antibody, Fe protein,Klebsiella variicola DX120E, Knockout, Nitrogenase, Prokaryotic expression
نویسندگان مقاله Ying Qin |
College of Agriculture, Guangxi University, Nanning, China

Yu-Yan Huang |
College of Agriculture, Guangxi University, Nanning, China

Qaisar Khan |
College of Agriculture, Guangxi University, Nanning, China

Kun-Kun Zhang |
College of Agriculture, Guangxi University, Nanning, China

Dao-Jun Guo |
College of Life Sciences and Engineering, Hexi University, Zhangye, China

Li-Tao Yang |
College of Agriculture, Guangxi University, Nanning, China

Yang-Rui Li |
Guangxi Key Laboratory of Sugarcane Genetic Improvement, Key Laboratory of Sugarcane Biotechnology and Genetic Improvement (Guangxi), Ministry of Agriculture and Rural Affairs, Sugarcane Research Institute of Guangxi Academy of Agricultural Sciences, Sugarcane Research Center of Chinese Academy of Agricultural Sciences, Nanning, China

Yong-Xiu Xing |
College of Agriculture, Guangxi University, Nanning, China

نشانی اینترنتی https://www.ijbiotech.com/article_171695_3331379086c6194f5c6ad4de00ba2f96.pdf
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