Bacillus megaterium nitrate reductase catalysis-electron transfer subunit coexpression carrier

Abstract

The invention relates to a bacillus megaterium nitrate reductase catalysis-electron transfer subunit coexpression carrier in the technical field of genetic engineering. The coexpression carrier is constructed by the following steps: firstly determining a bacillus megaterium whole genome sequence as a template, amplifying into a gene sequence containing enzyme cutting site nitrate reductase catalysis subunit (Bm-Nas B) and electron transfer subunit (Bm-Nas C) according to a designed PCR (Polymerase Chain Reaction) primer, and sequentially connecting the gene sequence onto the coexpression carrier pETDuet-1, and selecting positive clone of escherichia coli DH5alpha containing target gene fragment through a colony PCR technology. According to the bacillus megaterium nitrate reductase catalysis-electron transfer subunit coexpression carrier, the problem that the expression quantity of the nitrate reductase catalysis-electron transfer subunit gene sequence in the prior art is low in the natural bacillus megaterium and the usage effect is seriously restricted can be overcome, and the great increase on the expression quantity of the gene can be realized.

Description

technical field

[0001] The present invention relates to a method in the technical field of genetic engineering, specifically a nitrate reductase catalytic-electron transfer subunit gene sequence, a recombinant vector containing the gene, a recombinant bacterium, and the expression of the gene produced by the recombinant bacterium products and their applications. Background technique

[0002] With the development of facility agriculture, the cultivation area continues to expand, and the use of nitrogen fertilizers has increased significantly, greatly exceeding the demand of plants. Because the nitrate in the soil is not absorbed and transformed by the plants in time, a large amount of salt accumulates in the plants and the soil, resulting in secondary salinization of the protected cultivation soil.

[0003] Studies have shown that HCO removal in protected cultivation secondary salinized soil 3 - Outside, Na + 、K + , Ca 2+ , Mg 2+ , Cl - , SO 4 2- , NO 3 - have dif...

Examples

Embodiment 1

[0020] Cultivation of Bacillus megaterium NCT-2

[0021] Bacillus megaterium NCT-2 was isolated from a greenhouse cultivated in Chongming Island, Shanghai for 12-15 years. The preservation number is CGMCC NO.4698. No. 3, No. 1 Yard, Beichen West Road, District, Institute of Microbiology, China; date of preservation is March 21, 2011. The bacteria were inoculated in LB liquid medium and cultured at 32°C for OD 600 To about 1.5, the bacteria were collected by centrifugation and used to extract the genomic DNA of the bacteria.

[0022] The components of the LB liquid medium are: 10 g of peptone, 5 g of yeast extract, 10 g of NaCl, 1 L of deionized water, and pH 6.8-7.2. LB solid medium is 15-20g agar added on the basis of LB liquid medium.

Embodiment 2

[0024] Sequence verification of nitrate reductase catalytic subunit gene (Bm-Nas B)

[0025] According to the results of whole genome sequencing, design PCR primers:

[0026] Forward primer Nas B-F: 5′-ATGACGAAACAAAAGCTAATATTAA-3′

[0027] Reverse primer Nas B-R: 5′-TATAATAAATATGTGATTGCACGAG-3′

[0028] Using the genomic DNA of Bacillus megaterium NCT-2 as a template, the above two pairs of primers were used for amplification. The PCR conditions were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 45 s, annealing at 53°C for 30 s, extension at 72°C for 90 s; and final extension at 72°C for 10 min after 30 cycles.

[0029] The PCR amplification product was detected by electrophoresis, and the result showed that the obtained fragment was about 2.4kb; then the PCR product was gel-cut and recovered, connected to the pMD19-T vector and sent to the company for sequencing. The nucleotide sequence obtained after sequencing is consistent with the genome sequencing resu...

Embodiment 3

[0031] Sequence verification of nitrate reductase electron transfer subunit gene (Bm-Nas C)

[0032] According to the results of whole genome sequencing, design PCR primers:

[0033] Forward primer Nas C-F: 5′-ATGACTGAGATGCTATTGAAATATT-3′

[0034] Reverse primer Nas C-R: 5′-TAATAGCTGCTTCTTTTTACAGAAG-3′

[0035] Using the genomic DNA of Bacillus megaterium NCT-2 as a template, the above two pairs of primers were used for amplification. The PCR conditions were: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 53°C for 30 s, extension at 72°C for 75 s; and final extension at 72°C for 10 min after 30 cycles.

[0036] The PCR amplification product was detected by electrophoresis, and the result showed that the obtained fragment was about 2.1kb; then the PCR product was gel-cut and recovered, connected to the pMD19-T vector and sent to the company for sequencing. The nucleotide sequence obtained after sequencing is consistent with the genome sequenc...

technical field

[0001] The present invention relates to a method in the technical field of genetic engineering, specifically a nitrate reductase catalytic-electron transfer subunit gene sequence, a recombinant vector containing the gene, a recombinant bacterium, and the expression of the gene produced by the recombinant bacterium products and their applications. Background technique

[0002] With the development of facility agriculture, the cultivation area continues to expand, and the use of nitrogen fertilizers has increased significantly, greatly exceeding the demand of plants. Because the nitrate in the soil is not absorbed and transformed by the plants in time, a large amount of salt accumulates in the plants and the soil, resulting in secondary salinization of the protected cultivation soil.

[0003] Studies have shown that HCO removal in protected cultivation secondary salinized soil 3 - Outside, Na + 、K + , Ca 2+ , Mg 2+ , Cl - , SO 4 2- , NO 3 - have dif...

Examples

Embodiment 1

[0020] Cultivation of Bacillus megaterium NCT-2

[0021] Bacillus megaterium NCT-2 was isolated from a greenhouse cultivated in Chongming Island, Shanghai for 12-15 years. The preservation number is CGMCC NO.4698. No. 3, No. 1 Yard, Beichen West Road, District, Institute of Microbiology, China; date of preservation is March 21, 2011. The bacteria were inoculated in LB liquid medium and cultured at 32°C for OD 600 To about 1.5, the bacteria were collected by centrifugation and used to extract the genomic DNA of the bacteria.

[0022] The components of the LB liquid medium are: 10 g of peptone, 5 g of yeast extract, 10 g of NaCl, 1 L of deionized water, and pH 6.8-7.2. LB solid medium is 15-20g agar added on the basis of LB liquid medium.

Embodiment 2

[0024] Sequence verification of nitrate reductase catalytic subunit gene (Bm-Nas B)

[0025] According to the results of whole genome sequencing, design PCR primers:

[0026] Forward primer Nas B-F: 5′-ATGACGAAACAAAAGCTAATATTAA-3′

[0027] Reverse primer Nas B-R: 5′-TATAATAAATATGTGATTGCACGAG-3′

[0028] Using the genomic DNA of Bacillus megaterium NCT-2 as a template, the above two pairs of primers were used for amplification. The PCR conditions were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 45 s, annealing at 53°C for 30 s, extension at 72°C for 90 s; and final extension at 72°C for 10 min after 30 cycles.

[0029] The PCR amplification product was detected by electrophoresis, and the result showed that the obtained fragment was about 2.4kb; then the PCR product was gel-cut and recovered, connected to the pMD19-T vector and sent to the company for sequencing. The nucleotide sequence obtained after sequencing is consistent with the genome sequencing resu...

Embodiment 3

[0031] Sequence verification of nitrate reductase electron transfer subunit gene (Bm-Nas C)

[0032] According to the results of whole genome sequencing, design PCR primers:

[0033] Forward primer Nas C-F: 5′-ATGACTGAGATGCTATTGAAATATT-3′

[0034] Reverse primer Nas C-R: 5′-TAATAGCTGCTTCTTTTTACAGAAG-3′

[0035] Using the genomic DNA of Bacillus megaterium NCT-2 as a template, the above two pairs of primers were used for amplification. The PCR conditions were: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 53°C for 30 s, extension at 72°C for 75 s; and final extension at 72°C for 10 min after 30 cycles.

[0036] The PCR amplification product was detected by electrophoresis, and the result showed that the obtained fragment was about 2.1kb; then the PCR product was gel-cut and recovered, connected to the pMD19-T vector and sent to the company for sequencing. The nucleotide sequence obtained after sequencing is consistent with the genome sequenc...

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