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Gene capable of improving the nitrogen fixing ability of combined azotobacter and uses thereof

A nitrogen-fixing bacteria, horizontal technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problems of high energy consumption of biological nitrogen fixation, limited supply of carbon sources, low efficiency of combined nitrogen fixation in the field, etc. effect of function

Inactive Publication Date: 2004-06-02
INST OF AGRO FOOD SCI & TECH CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, energy limitation is the most important factor leading to the low efficiency of combined nitrogen fixation in the field. One of the reasons is that biological nitrogen fixation consumes a lot of energy. 2 Need to consume 16 ATP; Another reason is that the carbon source of the combined nitrogen-fixing bacteria on the root surface is very limited

Method used

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  • Gene capable of improving the nitrogen fixing ability of combined azotobacter and uses thereof
  • Gene capable of improving the nitrogen fixing ability of combined azotobacter and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The cloning of the DNA fragment that embodiment 1 contains dct transport system

[0042] 1. Collect soil samples rich in nitrogen-fixing bacteria that are non-symbiotic with grasses and isolate total DNA at the community level

[0043] Soil samples were collected from the natural environment of the swamp area where wild barnyardgrass and reeds were grown. Weigh 2 g of the collected soil sample, add 0.6 g of fine glass beads (d<0.11 mm), and shake twice at 4000 rpm. Add 300μl 2% SDS + 12% Tris-buffered phenol (pH8.0) solution on ice for 1 hour, add an equal amount of Tris-buffered phenol, pH8.0 (about 700ml), mix well, and centrifuge at 4°C, 13,000rpm 5 minutes. Add 0.1 volume of 3M NaAc pH 5.2 to the upper layer solution, mix well, add 0.6 volume of isopropanol and mix well. The DNA pellet was dissolved in 200 μl 1×TE (crude DNA). Weigh 100mg of cesium chloride and put it in a new 1.5ml Eppendorf centrifuge tube, add 100μl of crude DNA and mix gently, and let it stand...

Embodiment 2

[0048] Example 2 Identification of plant factor-induced promoters

[0049] The promoter sequence of the dctP gene was amplified by PCR (as shown in SEQ ID NO: 7), and Xho I digestion was inserted into the vector pVK100 (Tc resistance, Km resistance) and then inserted into the lacZ gene to screen for The only Tc-resistant transformant. Through the conjugation experiment, it was transferred into Pseudomonas stutzeri A15, and the recombinant bacteria were screened for Tc resistance. The recombinant bacteria expressed 1acZ in the medium added with fumaric acid, malic acid, succinic acid and other four-carbon dicarboxylic acids, and the colonies showed blue spots, indicating that the promoter was a plant factor-induced expression promoter.

Embodiment 3

[0050] Embodiment 3 Construction of combined nitrogen-fixing engineering bacteria

[0051] The XhoI fragment containing the dctPQM gene cluster was ligated into the shuttle vector pSZ21 to construct the recombinant plasmid pSZ24, transformed into E. Coli DH5α, and then transferred into Pseudomonasstutzeri A15 with the help of the helper plasmid pRK2013 through triparental conjugation. The recombinant bacteria that integrated the dct gene cluster into the chromosome of Pseudomonas stutzeri A15 were screened. Plasmid constructs such as figure 2 shown.

[0052] In the saline-alkali soil test field of Panjin City, Liaoning Province, the engineered bacteria were respectively carried out in the plot experiment of field release. The plot area was 10 square meters, and there were three repetitions. Wild bacteria and aseptic controls were set. The treatment method is to use the overnight culture solution to dilute 100 times to dip the rice roots. The test results show that the recom...

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Abstract

The present invention relates to one kind of gene capable of raising the nitrogen-fixing level of combined nitrogen-fixing bacteria and its encoding sequence, the sequence encoded polypeptide, and the application of the polypeptide in raising the nitrogen-fixing level of combined nitrogen-fixing bacteria. The present invention clones DNA segment containing dct transporting system and performs the sequence analysis; identifies plant factor inducing promote; and constitutes recombinant combined nitrogen-fixing engineering bacteria. The DNA segment encoded polypeptide the present invention provides may be used in preparing biological nitrogen-fixing fertilizer.

Description

Technical field: [0001] The invention relates to a gene capable of improving the nitrogen fixation level of combined nitrogen-fixing bacteria, its coding sequence, a polypeptide encoded by the sequence, and the application of the polypeptide in improving the nitrogen fixation level of combined nitrogen-fixing bacteria. Background technique: [0002] Biological nitrogen-fixing fertilizers, such as combined nitrogen-fixing bacteria, can improve soil and increase yields. However, the combined nitrogen-fixing wild 00110 strains directly isolated from the soil have very unstable nitrogen fixation efficiency and field inoculation effect under natural conditions, and it is difficult to exert the combined nitrogen fixation effect. It is of great significance to study the genetic control relationship of carbon-nitrogen metabolism of combined nitrogen fixation at the molecular level and gene level, and to solve or improve the energy limitation of combined nitrogen fixation. [0003] ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/04C07K14/195C12N1/21C12N15/31
Inventor 林敏阎春玲平淑珍徐玉泉
Owner INST OF AGRO FOOD SCI & TECH CHINESE ACADEMY OF AGRI SCI
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