Promoter report gene for detecting superoxide radical ions in nodules and application thereof

A superoxide ion and reporter gene technology, applied in the direction of oxidoreductase, using vectors to introduce foreign genetic material, enzymes, etc., can solve the problem of unclear distribution pattern of ROS

Inactive Publication Date: 2019-01-04
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that ROS is one of the important regulatory factors for the development of nitrogen-fixing root nodules in legumes, but the distribution pattern of ROS in root nodules is still unclear. Therefore, it will be useful to develop a system to

Method used

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  • Promoter report gene for detecting superoxide radical ions in nodules and application thereof
  • Promoter report gene for detecting superoxide radical ions in nodules and application thereof
  • Promoter report gene for detecting superoxide radical ions in nodules and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment one: build sodB Promoter- uidA gene fusion plasmid

[0019] Using Sinorhizobium meliloti DNA as a template, each with a restriction enzyme site Pst I and Bam HI upstream and downstream primers , Cloned by polymerase chain reaction (PCR) sodB For the promoter sequence of the gene, refer to the DNA sequence shown in SEQ ID NO: 1 in the sequence listing. The PCR product was digested by restriction endonuclease and uidA Gene plasmid pRG960, and then use T4 ligase to connect the PCR product to the plasmid. Finally, the constructed plasmid was transformed into competent Escherichia coli DH5α by heat shock method.

Embodiment 2

[0020] Embodiment two: construction plasmid is transferred into S. meliloti

[0021] Transfer into mutant strains using the three-parent conjugation transfer experiment:

[0022] a. Inoculate Rhizobia (mutant strain), Escherichia coli (constructed fusion plasmid) and helper vector MT616, and culture at 28 ℃ and 37 ℃ until OD 600 is about 1;

[0023] b. Take 1000 µL of each of the above strains in a centrifuge tube, centrifuge at 8000 rpm for 1 min, remove the supernatant and retain the precipitate;

[0024] c. 1 ml of 0.85% sterilized normal saline to suspend and precipitate once to remove antibiotics;

[0025] d. Centrifuge at 8000 rpm for 1 min, remove the supernatant and retain the precipitate;

[0026] e. Use 300 µL of freshly prepared liquid LB medium to suspend the Rhizobia, E. coli and MT616 pellets;

[0027] f. Spot 5 µL of each of the three bacterial solutions on the same position on the non-resistant LB plate, and mix carefully to prevent the bacterial solution f...

Embodiment 3

[0045] Embodiment three: Inoculate the host plant Medicago sativa into the rhizobia with the fusion plasmid

[0046] (1) Germination of alfalfa seeds:

[0047] 1) Add alfalfa seeds to a sterile small triangular flask, add 75% ethanol and gently shake the triangular flask to make the seed

[0048] The seeds were fully exposed to ethanol for 5 min. Pour off all ethanol.

[0049] 2) Quickly pour a large amount of sterile water into the triangular flask and shake it quickly. Then pour out the water in the triangular flask,

[0050] Then repeat the water change three times to wash off the residual ethanol thoroughly.

[0051]3) Add 25% antiformin (sodium hypochlorite) solution to the Erlenmeyer flask. Shake gently for 15 min, then

[0052] After washing with water for 3-5 times, change the water every 10 minutes, repeat 5 times, in order to fully remove the seeds

[0053] Sodium hypochlorite residue on the surface.

[0054] 4) Add 20 mL of sterile water to the Erlenmeyer fl...

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Abstract

The invention relates to a promoter reporter gene for detecting superoxide radical ions in nodules and an application thereof. The promoter reporter gene is a base sequence shown by SEQ ID NO:1. A sodB promoter and a reporter gene uidA fusion plasmid are constructed for the first time, and can be directly transferred into the sinorhizobium meliloti strain to directly detect the distribution and the intensity condition of the superoxide ions in the nodule.

Description

technical field [0001] The present invention relates to a kind of Sinorhizobium meliloti ( Sinorhizobium meliloti ) Superoxide dismutase gene promoter fusion reporter gene detection system and its application in detecting superoxide ion accumulation in root nodules. Background technique [0002] Biological nitrogen fixation is one of the major issues in life sciences. Biological nitrogen fixation plays an important role in agricultural production, providing nitrogen for plants, especially food crops, and plays an important role in increasing crop yields, reducing fertilizer usage and production costs, reducing water and soil pollution, establishing ecological balance, and promoting sustainable agricultural development. significance. The legume-rhizobia symbiotic nitrogen fixation system is formed through long-term co-evolution of symbiotic partners, which is of great significance to the biological cycle of nitrogen in nature and human agricultural and forestry production. ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/82C12N15/65
CPCC12N9/0089C12N15/65C12N15/82C12Y115/01001
Inventor 于亮亮罗利李宁宁黄乐琦闫军辉
Owner SHANGHAI UNIV
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