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Soybean symbiotic nitrogen-fixing lipopolysaccharide gene or its protein and application

A soybean and nitrogen fixation technology, applied in the field of molecular biology, can solve problems such as the impact of symbiotic nitrogen fixation

Active Publication Date: 2021-05-25
NORTHEAST INST OF GEOGRAPHY & AGRIECOLOGY C A S
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Lipopolysaccharide binding protein (LBP) is a glycoprotein. In animals, LBP acts as a carrier of lipopolysaccharide and has a high affinity with lipid A in lipopolysaccharide, thereby causing a series of inflammatory reactions. However, there have been no related reports on the effect of lipopolysaccharide-binding proteins on the symbiotic nitrogen fixation of legumes.

Method used

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  • Soybean symbiotic nitrogen-fixing lipopolysaccharide gene or its protein and application
  • Soybean symbiotic nitrogen-fixing lipopolysaccharide gene or its protein and application
  • Soybean symbiotic nitrogen-fixing lipopolysaccharide gene or its protein and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1. Isolation and structural analysis of the GmLBP6 gene in soybean

[0055] (1) Isolation of genes

[0056] The mRNA was isolated from young leaves of soybean variety—Williams82, and the first strand of cDNA was synthesized using the mRNA as a template and Oligo(T)17 as a primer. Then, using the first strand of the cDNA as a template, primers (5'-ATGGCACCCTCAATTGTTTTG-3') and primers (5'-TTATGAAACATAAGCTAACT-3') were used for PCR amplification, and a 1482bp cDNA fragment of the GmLPP6 gene was obtained , cloned into pGEM T Easy vector (TaKaRa Company) and named pGEM T Easy-GmLBP6.

[0057] The GmLBP6 CDS sequence contained in pGEM T Easy-GmLBP6 is shown in SEQ ID NO: 2, with a total of 1482 bp, which encodes a protein of 493 amino acids (SEQ ID NO: 1).

[0058] (2) Gene structure analysis

[0059] The young leaves of Williams82 were used as materials to extract DNA from them, and then the genomic DNA was used as a template to amplify the fragment of the GmLBP...

Embodiment 2

[0060] Embodiment 2. Soybean GmLBP6 gene does not affect plant growth

[0061] Using Williams82 as the background, we established a Tilling population containing about 5000 strains. Using this platform, we screened 1,200 strains and found a total of 15 mutant strains with nucleotide changes. After identification, 6 of the strains had missense mutations in the amino acids of the LBP6 protein. In the lbp6-5 mutant, LBP6 The 38th leucine of the protein is changed to proline. This position is a very conserved amino acid in the BPI domain, so we first selected lbp6-5 for subsequent experiments. By detecting the growth morphology of the plants before and after inoculation with rhizobia, it was found that the leaves of the Gmlbp6-5 mutant were slightly yellower than those of the Williams82 wild type, and there was no difference in other phenotypes such as plant height ( figure 2 ).

Embodiment 3

[0062] Example 3. Soybean GmLBP6 gene regulates the number of root nodules

[0063] (1) Soybean GmLP6 gene regulates the number of root nodules

[0064] Regardless of inoculation with rhizobia, the number of nodules in the Gmlbp6-5 mutant was more than that of the Williams82 wild type, and in the absence of inoculation with rhizobia, the number of nodules in the Gmlbp6-5 mutant was 13 times that of the Williams82 wild type, In the case of inoculation with rhizobia, the number of nodules of the Gmlbp6-5 mutant was more than twice that of the Williams82 wild type ( image 3 ).

[0065] (2) Soybean GmLBP6 gene regulation does not participate in the control of root nitrogenase activity

[0066] Although inoculated with rhizobia, the Gmlbp6-5 mutant formed more nodules than the wild type (the former was more than 2 times that of the latter). However, the nitrogenase activity of the mutant to form nodules was not significantly different from that of the wild type ( Figure 4 ). ...

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Abstract

The invention relates to the technical field of molecular biology, in particular to soybean symbiotic nitrogen-fixing lipopolysaccharide gene or its protein and application. The research of the present invention shows that after soybean symbiotic nitrogen-fixing lipopolysaccharide gene mutation causes GmLBP6 protein inactivation, the number of nodules of the mutant is significantly increased compared with that of the wild type, while the overexpression of the GmLBP6 gene on the mutant restores the number of nodules to consistent with wild type. Using GmLBP6 as a marker can realize early identification of nitrogen fixation ability of plant germplasm, especially soybean germplasm.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to soybean symbiotic nitrogen-fixing lipopolysaccharide gene or its protein and application. Background technique [0002] Compared with other commercial crops, leguminous crops such as soybean (Glycine max) have a unique agronomic trait - they can interact with matching soil rhizobia to form root nodules, and convert nitrogen in the air into ammonium that can be directly absorbed . The nitrogen fixed by the root nodules can not only provide nitrogen nutrition for the host plant, but also improve the soil and reduce the input of chemical fertilizers. Qin L et al pointed out that in Northeast China, inoculating high-efficiency rhizobia while reducing nitrogen fertilizer application by 50% increased soybean yield by more than 20% compared with conventional fertilization. It can be seen that applying rhizobia inoculation technology to agricultural production and giving full...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/54
CPCC07K14/415C12N15/8261
Inventor 冯献忠杨素欣高金珊李祯
Owner NORTHEAST INST OF GEOGRAPHY & AGRIECOLOGY C A S
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