Constructing method of nifA gene-introduced rhizobium japonicum genetic engineering bacterial strain HD-SFH-01

A technology of genetically engineered strains, soybean rhizobia, applied in genetic engineering, microorganism-based methods, plant genetic improvement, etc., can solve problems such as small increase in soybean yield

Inactive Publication Date: 2010-11-10
HEILONGJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to solve the problem that the existing soybean rhizobia genetically engineered strains have little increase in the number of soybean nodules and a...

Method used

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  • Constructing method of nifA gene-introduced rhizobium japonicum genetic engineering bacterial strain HD-SFH-01
  • Constructing method of nifA gene-introduced rhizobium japonicum genetic engineering bacterial strain HD-SFH-01
  • Constructing method of nifA gene-introduced rhizobium japonicum genetic engineering bacterial strain HD-SFH-01

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specific Embodiment approach 1

[0010] Specific embodiment one: the construction method of soybean rhizobia genetic engineering strain HD-SFH-01 that imports nifA gene in this embodiment is carried out according to the following steps: 1, fast-growing soybean rhizobium 15067 is activated and expanded, and then the genome is extracted DNA; 2. Using the homologous sequence cloning method, using the fast-growing soybean rhizobium 15067 genomic DNA as a template for PCR amplification to obtain the target gene nifA, and then using TaKaRa Agarose GeL DNA Purification Kit to purify, and then purify the target gene The gene nifA was connected with the TA cloning vector pMD18-T to obtain the connection product; 3. The connection product was transformed into Escherichia coli DH5α and positive clones were screened and identified, and then the plasmid vector pUC19 was extracted by alkaline lysis, and the target gene nifA was combined with Plasmid vector pUC19 was digested by SacI and SalI respectively, and the desired ta...

specific Embodiment approach 2

[0037] Specific embodiment two: the difference between this embodiment and specific embodiment one is that the reaction system of PCR amplification in step two is a 25 μ L reaction system, which consists of the following components:

[0038] Ingredients Dosage

[0039] 400ng / μL fast-growing soybean rhizobia 15067 genomic DNA 1.0μL

[0040] 0.2 μM PnifA1 1.0 μL

[0041] (5'-CGC GTCGACT TATGACTGAATTATCC-3')

[0042] 0.2 μM PnifA2 1.0 μL

[0043] (5'-CGC GAGCTCA CCGAATTTAGATCTT-3')

[0044] 10×Ex Taq Buffer 2.5μL

[0045] 0.4mMdNTP Mixture 1.5μL

[0046] Taq DNA polymerase 0.5 μL

[0047] Sterile water 17.5μL

[0048] PCR amplification conditions were: denaturation at 94°C for 10 min, denaturation at 94°C for 1 min, annealing at 65°C for 30 s, extension at 72°C for 1 min, a total of 35 cycles, extension at 72°C for 7 min, and incubation at 4°C. Other steps and parameters are the same as those in Embodiment 1.

specific Embodiment approach 3

[0049] Embodiment 3: This embodiment differs from Embodiment 1 to Embodiment 2 in that the specific operation steps of TaKaRa Agarose GeL DNA Purification Kit in step 2 refer to the instruction manual. Other steps and parameters are the same as one of the specific implementation modes 1 to 2.

[0050] In this embodiment, TaKaRa Agarose GeL DNA Purification Kit is Takara Bio Agarose GeL DNA Purification Kit.

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Abstract

The invention relates to a constructing method of a nifA gene-introduced rhizobium japonicum genetic engineering bacterial strain HD-SFH-01, belonging to a constructing method of a rhizobium japonicum genetic engineering bacterial strain and solving the problems of less increase on soybean nodule quantity and little soybean yield increase after the traditional rhizobium japonicum genetic engineering bacterial strain is inoculated to a soybean seedling. The method comprises the steps of: extracting rhizobium japonicum genome DNA for carrying out PCR amplification to obtain a target gene nifA, purifying and then connecting with a TA clone carrier pMD18-T to obtain a connecting product and then converting into escherichia coli; carrying out multiple enzyme digestion, connection and verification to obtain a recombinant prokaryote expression carrier pTR-Plac-nifA; and converting the recombinant prokaryote expression carrier pTR-Plac-nifA into soybean indigenous rhizobium. The bacterial strain HD-SFH-01 is inoculated into the soybean seedling, the rhizobium quantity is increased, and the soybean yield is increased by 3.80 percent.

Description

technical field [0001] The invention relates to a method for constructing soybean rhizobia genetic engineering strains. Background technique [0002] Nitrogen is the main substance that constitutes protein and nucleic acid, and is an essential biological fertilizer in agricultural production. Biological nitrogen fixation refers to the process of molecular nitrogen forming ammonia through the catalysis of nitrogen-fixing microbial nitrogenase system. Organisms can directly absorb nitrogen in the air as nutrients. They first reduce molecular nitrogen to ammonia, and then convert it to amino acids and proteins, which is an important part of the nitrogen cycle on the earth. There are three forms of biological nitrogen fixation in nature. One is the authigenic nitrogen fixation of bacteria, which means that microorganisms live independently and fix nitrogen independently; Nitrogen fixation; the third is joint nitrogen fixation, which refers to a special symbiotic nitrogen fixat...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/74C12N1/21C12R1/41
Inventor 马春泉张喜波李海英陈思学蒙明明杨乐贾珊珊王海宁于海鹏于冰王宇光郭锡伟吴川季琳
Owner HEILONGJIANG UNIV
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