Glutamine synthetase gene of acinetobacter as well as encoding protein and cloning method of glutamine synthetase gene
A glutamine, gene-encoded technology, applied in biochemical equipment and methods, enzymes, enzymes, etc., can solve problems such as low nitrogen fertilizer utilization rate and water environment pollution
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Embodiment 1
[0021] Embodiment one: Acineb1 Cloning and analysis of the full-length coding region of the gene:
[0022] Acinetobacter ( Acinetobacter spp.) are mainly distributed in water bodies and soils, and are easy to survive in humid environments. Collect a small amount of river bank soil samples, take 1 gram of soil, and extract its metagenomic DNA. By analyzing the glutamine synthetase gene sequence of Acinetobacter bacteria in the NCBI database, the degenerate primers were designed: Acneb1F: 5'-ATGAGCATGGCGAACAAGGT-3' and Acneb1R: 5'-TTACARGCTRTARTACATAT-3'. Using this pair of primers, a glutamine synthetase gene was amplified by PCR from the extracted soil microbial metagenomic DNA, named as Acineb1 . The gene was inserted into the cloning vector pMD18-T, the corresponding positive clone was selected, and the full-length DNA sequence of the gene was carried out to obtain the DNA sequence containing the complete ORF of the gene. The sequencing results showed that: Acineb1...
Embodiment 2
[0023] Embodiment two: Acineb1 Functional analysis in E. coli mutants
[0024] Through DNA sequencing analysis, select Acineb1 Gene ORF transcription direction and lactose promoter on vector pMD18-T lacZ The clones with the same transcription direction were extracted and transformed into Escherichia coli mutant ET6017 (Genotype: F-,[araD139]B / r,Δ(argF-lac)169,flhD5301,Δ(fruK-yeiR) 725 (fruA25), relA1, rpsL150 (strR), Δ (glnG-glnA) 229, ha-10, deoC1). See literature: Merida, A., Flores E., Florencio F.J., Regulation of Anabaena sp. strain PCC7120 glutamine synthetase activity in a Synechocystis sp. strain PCC6803 derivative strain bearing the Anabaena glnA gene and a mutated host glnA gene. J Bacteri 1992, 174(2): 650-654. Due to the deletion of the gene encoding glutamine synthetase, the mutant cannot grow normally in the medium without glutamine. This strain was purchased from E. coli Genetic Stock Center, Yale University. Depend on Figure 4 and Figure 5 It shows...
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