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Method for improving transposition efficiency of transposons in super-slow-growing rhizobium arachis

A technology of ultra-slow growth and rhizobia, applied in the biological field, can solve problems such as hindering cell material exchange, cell toxicity, cell death, etc.

Active Publication Date: 2020-04-03
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Due to the high molecular weight of levotran, it will accumulate in the periplasmic space, and at the same time, the fructose residue will bind to various receptors on the cell, which hinders the normal material exchange of the cell and is potentially toxic to the cell effect, resulting in cell death

Method used

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  • Method for improving transposition efficiency of transposons in super-slow-growing rhizobium arachis
  • Method for improving transposition efficiency of transposons in super-slow-growing rhizobium arachis
  • Method for improving transposition efficiency of transposons in super-slow-growing rhizobium arachis

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Experimental program
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Effect test

Embodiment 1

[0056] Embodiment 1, the acquisition of recombinant plasmid pRL1063a-sacB

[0057] 1. Extraction of pRL1063a plasmid and pJQ200SK plasmid

[0058] 1. Inoculate a single colony of Escherichia coli containing the pRL1063a plasmid into 5 mL of LB liquid medium, shake overnight at 37°C at 180 rpm, and then centrifuge at 12,000 rpm for 1 min to collect the cells.

[0059] 2. After completing step 1, take the bacteria, use the plasmid mini-extraction kit and follow the instructions to extract the plasmid, and obtain A 260 / A 280 pRL1063a plasmid solution with a concentration of 1.8-2.0 and greater than 34ng / μL (the solvent is ddH 2 O).

[0060] 3. Inoculate the single colony of Escherichia coli containing the pJQ200SK plasmid into 5 mL of LB liquid medium, shake overnight at 37°C at 180 rpm, and then centrifuge at 12,000 rpm for 1 min to collect the cells.

[0061] 4. After completing step 3, take the bacteria, use the plasmid mini-extraction kit and follow the instructions to e...

Embodiment 2

[0086] Embodiment 2, establishment of bradyrhizobium peanut three-parent hybridization method

[0087] 1. Establishment of three-parent hybridization method of Bradyrhizobium peanut and analysis of Tn5 transposition efficiency of inserting Tn5 transposon into Bradyrhizobium peanut CCBAU 53363

[0088] The recipient bacterium was Bradyrhizobium peanut CCBAU 53363. For the colony morphology of Bradyrhizobium peanut CCBAU 53363 see image 3 .

[0089] The donor bacterium is the Escherichia coli containing the recombinant plasmid pRL1063a-sacB prepared in Example 1.

[0090] The auxiliary bacteria are Escherichia coli containing the auxiliary plasmid pRK2013.

[0091] The inventor of the present invention has established a three-parent hybridization method for Bradyrhizobium peanut through a large number of experiments. Specific steps are as follows:

[0092] 1. Inoculate the recipient bacteria on TY solid medium and culture at 28°C for 14 days to obtain activated recipient b...

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Abstract

The invention discloses a method for improving transposition efficiency of transposons in super-slow-growing rhizobium arachis. Experiments show that, Tn5 transposition efficiency after insertion of Tn5 transposons into rhizobium arachis CCBAU 53363 by adopting a conventional three-parent hybrid method for rhizobium is 1.25%; and Tn5 transposition efficiency after insertion of Tn5 transposons intorhizobium arachis CCBAU 53363 by adopting the method disclosed by the invention is up to 18.75-18.8%. Compared with the conventional three-parent hybrid method, the Tn5 transposition efficiency is increased by nearly 15 times after the Tn5 transposons are inserted into the rhizobium arachis CCBAU 53363 according to the method established by the invention. The method is very effective for geneticoperation on the transposons of the super-slow-growing rhizobium arachis, so that the method is of important application values.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for improving the transposition efficiency of a transposon in a super-brady peanut rhizobia. Background technique [0002] Rhizobium is a kind of Gram-negative bacteria widely distributed in soil. It can infect legumes and form root nodules. In the root nodules, it can differentiate into mature bacteroids that can fix nitrogen in the air and convert nitrogen into energy. Ammonia is absorbed and utilized by plants, while plants provide the carbon source needed for the life of rhizobia. Therefore, leguminous plants and rhizobia are in a mutually beneficial symbiotic relationship. According to statistics, the nitrogen fixed by this symbiotic relationship accounts for more than 65% of the total biological nitrogen fixation. The symbiosis between rhizobia and leguminous plants has great potential to improve soil fertility and reduce nitrogen fertilizer application. ...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N15/113C12N15/80C12R1/645
CPCC12N15/113C12N15/80
Inventor 隋新华吴月尚佼颖陈腊李可可霍斌贾玉文
Owner CHINA AGRI UNIV
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