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A method for improving plant nodulation nitrogen fixation efficiency

A nitrogen fixation efficiency and nodulation technology, applied in the field of genetic engineering, can solve problems affecting nitrogen fixation efficiency and achieve the effects of improving nitrogen fixation efficiency, increasing nodulation, and improving yield traits

Active Publication Date: 2022-04-22
HUAZHONG AGRI UNIV
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  • Claims
  • Application Information

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Problems solved by technology

Existing studies have found that miR156 affects the process from plant vegetative growth to reproductive growth by regulating the transcription factors of its target gene SPL family (Wang et al., 2009; Wu et al., 2009), overexpression of soybean miR156 can also inhibit The number of soybean nodules (Yan et al. 2013), but which target gene(s) miR156 regulates the development of root nodules, and whether it affects nitrogen fixation efficiency has not been reported

Method used

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  • A method for improving plant nodulation nitrogen fixation efficiency
  • A method for improving plant nodulation nitrogen fixation efficiency
  • A method for improving plant nodulation nitrogen fixation efficiency

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1, GmSPL9d Analysis of expression patterns of genes in response to rhizobia treatment.

[0025] 1) Material acquisition: The material used in the experiment was Williams 82 (W82 for short); the material was carried out according to the following process: soybean seeds were sterilized with 70% alcohol for 30 sec, and sowed in the vermiculite substrate soaked in low-nitrogen nutrient solution. They were cultured in a culture room with 16 h light / 8 h dark, light intensity 7000 LUX, temperature 26 °C, and relative humidity 70%. 4 days after sowing, the cotyledons were unearthed, inoculated with rhizobia, and a control (-R) not inoculated with rhizobia was set at the same time. Take samples of main root and lateral root 1 cm below the rhizome joint at 0 day, 1 day, 3 day, 5 day, and 7 days after inoculation, and freeze them in liquid nitrogen.

[0026] 2) Isolation of mRNA: Trizol method was used to extract soybean total RNA. ①Put the tissue into a grinder and g...

Embodiment 2

[0032] Example 2, the cultivation of transgenic soybeans with high ability of nodulation and nitrogen fixation.

[0033] 1) Extraction of total RNA from root tissue of Wilimas82 soybean

[0034]The mortar has been treated at 180 ℃ for 8 hours or burned to eliminate RNase contamination; reagents such as chloroform, isopropanol, and ethanol should be freshly opened and uncontaminated; other equipment such as pipette tips, centrifuge tubes and reagents such as ultrapure water , NaAc, were treated with 1 ‰ DEPC water overnight, and then sterilized at 121 ℃ for 30 minutes by high temperature and humidity.

[0035] (1) Take 100 mg of soybean root material and grind it with liquid nitrogen, add 1 mL of TRI pure reagent, after fully homogenizing, inhale the homogenate into a 1.5 mL centrifuge tube, and place it at room temperature for 5 min;

[0036] (2) Add 200 μl chloroform, vortex to mix, let stand for 5 minutes, 4 ℃, 12000 r / min, centrifuge for 10 minutes;

[0037] (3) Take the ...

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Abstract

The invention discloses a method for improving the efficiency of plant nodulation and nitrogen fixation. Firstly, construct a recombinant expression vector containing the gene fragment shown in SEQ ID NO: 1, then use the recombinant expression vector to construct a transformant, and then use the transformant to infect the target For plants, positive plants were screened to obtain transgenic plants with increased number of nodules and enhanced nodulation / nitrogen fixation ability compared with normal plants. The transgenic soybean constructed by the method of the invention has significantly increased number of nodules and higher nitrogen fixation efficiency.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to the transgenic technology and application of soybean nodulation and nitrogen fixation. Background technique [0002] Soybean is an important grain and oil economic crop and the main source of human plant protein intake. Therefore, soybean has a high demand for nitrogen nutrition. The nitrogen nutrition in the soil is often unable to meet the nitrogen demand of soybean. Excessive fertilization will cause Serious environmental pollution. During the long-term evolution process, soybean and rhizobia form mutualistic root nodules, in which nitrogen in the atmosphere is reduced to available nitrogen for plants, and symbiotic nitrogen fixation provides two-thirds of the nitrogen needed for soybean survival. This process directly affects the normal growth and yield of soybeans. Therefore, soybean nodulation and biological nitrogen fixation have always been a major research ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/84C12N15/29A01H5/00A01H6/54
Inventor 李霞孙政玺贠金霞姜琼
Owner HUAZHONG AGRI UNIV
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