Application of guaA gene, plasmid and strain in expression of azotobacter siderophore

A technology of siderophilic and nitrogen-fixing bacteria, applied in the fields of application, genetic engineering, plant gene improvement, etc., to achieve wide application prospects, increase the yield of bacterial siderophilic, and cost-effective effects

Pending Publication Date: 2022-04-15
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Overexpression of guaA gene is used to regulate the synthesis of bacterial siderophiles, so as to achieve the purpose of high-yield siderophiles, and the research on this aspect has not been reported yet.

Method used

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  • Application of guaA gene, plasmid and strain in expression of azotobacter siderophore
  • Application of guaA gene, plasmid and strain in expression of azotobacter siderophore
  • Application of guaA gene, plasmid and strain in expression of azotobacter siderophore

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Cloning and bioinformatics analysis of guaA gene

[0064] (1) Whole genome sequencing of nitrogen-fixing bacteria GXGL-4A

[0065] Whole-genome sequencing and gene annotation of the nitrogen-fixing bacteria GXGL-4A were carried out ( figure 1 ), the whole genome has a full length of 5660197bp, a GC content of 53.94%, and a total of 5261 genes have been annotated. Contains 22 rRNAs, 86 tRNAs and 131 other non-coding RNAs. Among them, the accession number of the whole genome sequence of GXGL-4A is CP015113.1; the preservation number of GXGL-4A in China General Microorganism Culture Collection Center (CGMCC) is CGMCC No.12588.

[0066] 1) According to the gene annotation results, design PCR amplification primers containing the upstream and downstream sequences of the guaA gene for amplification. The forward primer used was 5'-ATAACGCTGCCGACAAACTGGTG-3'; the reverse primer was 5'-GTCGCCCGGAAATAGTTAAGCAA-3'. Amplification conditions: 94°C for 5min; 30cycle (94°C for 30s,...

Embodiment 2

[0078] Construction of guaA Gene Knockout Strain of Azotobacter

[0079] (1) Construction of guaA gene knockout system

[0080] 1) Primer synthesis

[0081] Using the guaA gene of nitrogen-fixing bacteria as a template, Primer Premier 5.0 was used to design upstream and downstream primers (Table 1), and to synthesize knockout primers. Related expression vector identification primers and guaA gene transformant identification primers are listed in Table 1.

[0082] Table 1 guaA gene knockout primer sequence

[0083]

[0084] 2) Knockout plasmid construction

[0085] Using the DNA of nitrogen-fixing bacteria GXGL-4A as a template, use guaA-SmaI-UP-F / guaA-UP-R, guaA-SmaI-down-R / guaA-down-F respectively according to the following system (Table 2) and conditions Downstream sequences are amplified:

[0086] Table 2 Amplification system

[0087]

[0088] Amplification conditions: 94°C for 5min; 30cycles (94°C for 30s, 55°C for 30s, 72°C for 40s); store at 10°C.

[0089] A...

Embodiment 3

[0106] Construction of guaA Gene Overexpression Strain and Replenishment Strain of Azotobacter

[0107] (1) Construction of overexpression strain

[0108] According to the guaA gene sequence information, the whole gene synthesis was carried out, and the two ends were respectively introduced with BamHI and XhoI restriction sites, and then ligated into the double-cut pET28a(+) vector after BamHI / XhoI double cutting ( Figure 8 ), constructed into expression vector pET28a(+)-guaA. When the wild strain of nitrogen-fixing bacteria GXGL-4A was cultivated to OD 600 When it is 0.6-0.8, the pET28a(+)-guaA expression vector is transferred into the host bacteria by electric shock transformation method to obtain the guaA gene overexpression strain.

[0109] (2) Construction of guaA gene complementation strain

[0110] The plasmid pET28(+)-guaA was transferred into the nitrogen-fixing bacteria GXGL-4A knockout strain by electric shock transformation method, that is, the pET28a(+)-guaA p...

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Abstract

The invention relates to the technical field of bioengineering, in particular to application of a guaA gene, a plasmid and a strain in expression of azotobacter siderophore. A guaA gene expression vector of nitrogen-fixing bacteria GXGL-4A is constructed by using a prokaryotic expression vector pET28a (+), the guaA gene expression vector is transformed into a wild strain GXGL-4A to obtain a positive transformed strain, and the strain can highly express siderophore by adding trace IPTG (isopropyl-beta-d-thiogalactoside) for induction during culture. Compared with the prior art, the siderophore yield of the azotobacter can be remarkably improved by expressing the guaA gene in the genome of the azotobacter, the obtained transformant can be used as a plant growth-promoting bacterium, absorption of iron in soil is increased on the basis of original nitrogen fixation, a plant root system is helped to obtain more iron elements from the soil and other environments, and the yield of siderophore is improved. Meanwhile, iron nutrition of pathogenic bacteria in the soil is competed, and the disease resistance of the plants is improved.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to the application of guaA gene, plasmid and bacterial strain in expressing nitrogen-fixing bacteria siderophile. Background technique [0002] Iron is an essential nutrient element for the growth and development of organisms, and it participates in the synthesis of various enzymes and the metabolic functions of living organisms, such as cytochrome oxidase, ferredoxin, and nitrogenase. Iron is abundant in the earth's crust, but most forms cannot be directly utilized by organisms. Some plant rhizosphere growth-promoting bacteria can secrete small molecular substances that can efficiently bind iron ions, called siderophiles. In the case of iron deficiency, these bacteria can sequester the limited Fe in the soil by producing siderophilic 3+ , to supply the growth and development of plants and themselves. At this time, the iron concentration in the environment is reduced, and t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/74C12N1/21C12P1/04C12R1/065
Inventor 陈云鹏冯保云章梦婷
Owner SHANGHAI JIAO TONG UNIV
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