A method for studying the gene function of Bradyrhizobium

A technology of bradyrhizobium and gene, which is applied in the field of microorganisms, can solve the problems of researchers, the lack of suitable research methods, and the research of gene functions of bradyrhizobium, so as to speed up the progress of experimental research, save time and cost of consumables Effect

Active Publication Date: 2021-05-25
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

But it is very difficult for researchers
[0004] Due to the lack of suitable research methods, the research on the gene function of Bradyrhizobium is seriously restricted

Method used

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  • A method for studying the gene function of Bradyrhizobium
  • A method for studying the gene function of Bradyrhizobium
  • A method for studying the gene function of Bradyrhizobium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 Preparation of Pseudomonas aeruginosa PAO1 Electric Shock Competent Cells

[0064] The preparation of Pseudomonas aeruginosa PAO1 electric shock competent cells comprises the following steps:

[0065] (1) Pseudomonas aeruginosa PAO1 was shaken overnight for 8 hours, 6 mL was taken as a competent state, and divided into three 2 mL sterilized centrifuge tubes, room temperature was 10,000 rpm for 2 minutes, and the supernatant was removed;

[0066] (2) Resuspend with filter-sterilized 10% sucrose, wash once, room temperature 10000rpm, 2min, remove supernatant;

[0067] (3) Repeat step 2 twice;

[0068] (4) Concentrate into 100 μl with 10% glycerol and use for electroporation of the constructed plasmid.

Embodiment 2

[0069] Example 2 Construction of bradyrhizobium USDA110 bll5123 gene overexpression strain and colony phenotype observation

[0070] 1. Extraction of DNA

[0071] Genome kit (EE161-01) from Beijing Quanshijin Biotechnology Co., Ltd. was used to extract the whole genome of Bradyrhizobium USDA110.

[0072] 2. Amplification of the bll5123 gene

[0073] Utilize NEB high-fidelity Q5 polymerase, carry out following PCR amplification, obtain containing bradyrhizobium USDA110bll5123 (Gene ID: 1051644, namely NC_004463.1:c5683715-5682342 Bradyrhizobium japonicum USDA 110 chromosome, complete genome) gene product, the amplified product was connected to a cloning vector and transformed into Escherichia coli for sequencing after culture, the positive strains were retained, and the plasmid was extracted.

[0074] According to NCBI records, the nucleotide sequence of USDA110 bll5123 is shown in SEQ ID NO: 1, which is:

[0075] GTGTCCGCGCGTATCCTGGTTGTCGATGACGTTCCTGCCAACGTCAAACTCCTCGAGG...

Embodiment 3

[0092] Example 3 Determination of Growth Curve

[0093] 1. Shake the transformants PAO1-pBBR1MCS-5 and PAO1-pBBR1MCS-5-bll5123 constructed in Example 2 overnight for 8 hours (LB gentamicin 50 μg / mL), and then add LB gentamicin at a ratio of 1:100 Add 200 μl of damycin 50 μg / mL liquid to the plate for measuring the growth curve, and detect it with the instrument for measuring the growth curve. There are 8 replicates in each group, and the experimental results are repeated three times.

[0094] 2. Results

[0095] As determined by the growth curve, figure 2 It was shown that the strain PAO1-pBBR1MCS-5-bll5123 overexpressing Bradyrhizobium USDA110 bll5123 and the control strain PAO1-pBBR1MCS-5 had no effect on the growth rate, and the subsequent experiments excluded the effect of growth rate.

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Abstract

The invention discloses a bradyrhizobium ( Bradyrhizobium japonicum USDA110) gene function research method. The invention proposes a prokaryotic expression system using Pseudomonas aeruginosa as a host bacterium. Described Pseudomonas aeruginosa is Pseudomonas aeruginosa PAO1 ( Pseudomonas aeruginosa PAO1). The present invention directly conducts phenotype research on the synthesis and degradation gene functions of the intracellular messenger cyclic guanosine diphosphate (c-di-GMP) in Pseudomonas aeruginosa, whose background mechanism is relatively clear, and observes that Pseudomonas aeruginosa Changes in the traits of the single cell bacteria, the function of the target gene was preliminarily obtained, and a new prokaryotic expression system was proposed. The gene of Bradyrhizobium was expressed in this system, and the colony morphology, motility, exopolysaccharide production and biofilm formation were investigated, and the function of the Bradyrhizobium gene was initially obtained, which saved time and consumable costs, and accelerated the experiment The research progress is conducive to the study of the gene function of Bradyrhizobium.

Description

technical field [0001] The invention relates to the technical field of microorganisms, and more specifically relates to a method for researching the function of the intracellular messenger cyclic guanosine diphosphate synthesis and degradation gene of Bradyrhizobium bacteria. Background technique [0002] Bradyrhizobia can coexist with plants, form effective nodules, and synthesize nitrogen sources for plant growth and utilization (Zhang Yanhua, Wang Hui, Wang Yan, etc. Phylogenetic diversity of Bradyrhizobia in Liaoning Province[J]. Jiangsu Agricultural Science , 2016, Vol. 44(7):67-70.). However, due to the long growth cycle of Bradyrhizobium, it takes as long as 8-10 hours for Bradyrhizobium to reproduce for one generation compared with other bacteria (the production and application status and research direction of nitrogen-fixing bacteria). The experimental period is long, and the screening of functional genes is time-consuming. The relationship between the intracellul...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/78C12N15/31C12R1/385
CPCC07K14/195C12N15/78
Inventor 戴伟君程蒙蒙
Owner SOUTH CHINA AGRI UNIV
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