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Construction method of genetic engineering strains for improving quantity of soybean nodules

The technology of a genetically engineered strain and a construction method is applied in the field of constructing genetically engineered strains, which can solve the problems of small increase in soybean yield of soybean plants, etc.

Inactive Publication Date: 2012-09-12
HEILONGJIANG UNIV
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  • Summary
  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0005] The present invention aims to solve the problem that the number of nodules formed by soybean plants and the increase in soybean yield are small compared with the original starting strain after inoculating soybean seedlings with genetic engineering strains of soybean rhizobia, and provides a method for increasing the number of soybean nodules. Construction method of genetic engineering strain

Method used

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  • Construction method of genetic engineering strains for improving quantity of soybean nodules
  • Construction method of genetic engineering strains for improving quantity of soybean nodules
  • Construction method of genetic engineering strains for improving quantity of soybean nodules

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specific Embodiment approach 1

[0012] Specific embodiment one: the construction method of the genetically engineered bacterial strain that this embodiment improves soybean nodulation quantity, carries out according to the following steps:

[0013] 1. Activate and expand the culture of S. fischeri 15067, and then extract the genomic DNA of S. The amplified product was obtained, and the amplified product was detected by 1% agarose gel electrophoresis, and then purified by a gel recovery kit to obtain the target gene dctA; 3. Sequencing and identification of the target gene dctA; 4. The target gene dctA and recombinant plasmid vector pET-P lac Carry out double enzyme digestion with NdeI and BamHI respectively, and digest the target gene dctA and the vector pET-P after digestion lac Ligated to obtain the recombinant vector pET-P lac -dctA; 5. Using the recombinant vector pET-P lac -dctA is used as a template for PCR amplification to construct P lac -dctA-T7 fragment, then P lac - The dctA-T7 fragment and t...

specific Embodiment approach 2

[0045] Specific embodiment two: the difference between this embodiment and specific embodiment one is: the reaction system of PCR amplification in step 2 is as follows:

[0046]

[0047] PCR amplification conditions were: denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 90 s, a total of 35 cycles, extension at 72°C for 7 min, and incubation at 4°C. Others are the same as in the first embodiment.

specific Embodiment approach 3

[0048] Specific embodiment three: the difference between this embodiment and specific embodiment one or two is: the double-enzyme digestion reaction system of the target gene dctA in step four is as follows:

[0049]

[0050] Enzyme digestion reaction conditions: keep warm in a water bath at 37°C for 3 hours. Others are the same as in the first or second embodiment.

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Abstract

The invention relates to a construction method of genetic engineering strains for improving quantity of soybean nodules. The construction method aims at solving the problems that when the conventional rhizobium japonicum genetic engineering strains are inoculated to soybean seedlings, the root nodule quantity of soybean plants and the yield increase of soybeans are small compared with those of the original starting strains. The construction method comprises the following steps of: performing activation and culture expansion on sinorhizobium fredii to extract genomic DNA (deoxyribonucleic acid); performing PCR (Polymerase Chain Reaction) amplification by virtue of a homologous sequence cloning method to obtain target gene dct A; performing sequencing identification on the target gene; constructing a prokaryotes expression vector pTR-Plac-dct A; and converting the prokaryotes expression vector into the sinorhizobium fredii to obtain the gene engineering strains. The quantity of the root nodules on the soybean plant with the transformed gene engineering strains is increased by 53.73%, and the yield of the soybeans is increased by 62.02%.

Description

technical field [0001] The invention relates to a method for constructing genetically engineered bacterial strains for increasing the number of soybean nodules. Background technique [0002] Nitrogen is an indispensable element of life in animals, plants, and microorganisms in nature. In living cells, nitrogen is the main component of all amino acids, nucleic acids, and many other important molecules. Nitrogen in the atmosphere can only be absorbed and utilized by plants through nitrogen fixation mainly through biological nitrogen fixation, and animals can absorb and utilize nitrogen directly or indirectly by eating plants as food. Biological nitrogen fixation refers to the process in which certain types of nitrogen-fixing microorganisms use nitrogenase in the body to reduce nitrogen in the atmosphere to ammonia at normal temperature and pressure. According to the nitrogen-fixing characteristics of nitrogen-fixing microorganisms and the relationship with plants, biological ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N1/21C12R1/41
Inventor 李海英于冰蒙明明陈思学马春泉李珊珊杨乐贾珊珊季琳吴川潘钰宫世龙王琳琳谷丹赵晨曦
Owner HEILONGJIANG UNIV
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