Regulation gene GmRSD in middle and later periods of soybean nodulation and application method thereof
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A technology for regulating genes and application methods, which is applied in the field of genetic engineering to achieve the effects of increasing the symbiotic nitrogen fixation capacity of soybeans and improving the symbiotic nitrogen fixation capacity.
Pending Publication Date: 2019-12-20
JILIN ACAD OF AGRI SCI
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The functions and mechanisms of some genes have been clarified, and the pathways and some nodes involved in symbiotic nitrogen fixation have be...
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Embodiment 1
[0048] Example 1 Analysis of the expression pattern of GmRSD gene in response to rhizobia infection
[0049] Material acquisition: The material used in the experiment is Williams 82 (W82) for short; the material is carried out according to the following process: soybean seeds are sterilized with 70% alcohol for 30 sec, sown in the frog stone substrate soaked in nitrogen-free nutrient solution, and placed in the cultivation room Medium culture, 16h light / 8h dark, light intensity 7000LUX, temperature 26°C, relative humidity 70%. 15 days after sowing, inoculate rhizobia treatment, and each strain is inoculated with bradyrhizobia USDA110 bacterial liquid (OD 600 =0.08) 30 mL, and the roots of soybeans were taken 10, 18 and 28 days after inoculation respectively.
[0050] Extraction of mRNA: Mortar, small spoon, etc. are burned to eliminate RNase pollution, and reagents such as chloroform, isopropanol, and ethanol are used exclusively. Other equipment such as pipette tips and EP ...
Embodiment 2
[0057] A method for applying the regulating gene GmRSD in the middle and late stage of soybean nodulation, comprising the following steps:
[0058] Step 1, extracting total RNA in W82 soybean root nodule tissue:
[0059] (1) The mortar is treated at 180°C for 8 hours 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 and NaAc , were treated with 0.1% DEPC water overnight, then sterilized at 121°C for 30 minutes by high temperature and damp heat, and the pipette tip and centrifuge tube were dried at 65°C for later use; Trizol method was used to extract soybean total RNA:
[0060] (2) Take 100 mg of soybean root material and grind it with liquid nitrogen, add 1 mL of TRI pure reagent, after fully homogenizing, suck the homogenate into a 1.5 mL centrifuge tube, and place it at room temperature for 5 min;
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Abstract
The invention discloses an application method of a regulation gene GmRSD in the middle and later periods of soybean nodulation. The method comprises the following steps: firstly, constructing an overexpression vector containing a nucleotide sequence as shown in SEQ IDNO:1, then constructing a transformant by utilizing the overexpression vector, infecting a receptor soybean root system by utilizingthe obtained transformant, screening positive plants, and acquiring transgenic soybeans with high nodulation and nitrogen fixation capabilities compared with normal soybeans. The overexpression vector takes pEGAD as a skeleton vector, after EcorR I and HindIII enzyme digestion, a target gene segment is connected to the skeleton vector, and the construction of the overexpression vector is completed. Agrobacterium tumefaciens is transformed by the overexpression vector to obtain the transformant. Then, the transgenic plants are obtained by an agrobacterium tumefaciens K599-mediated hairy root transformation method, and the positive plants are screened so as to obtain the transgenic plants with high nodulation and nitrogen fixation capacities compared with normal plants. The overexpression vector constructed by the method transforms receptor soybean plants to obtain the transgenic plants. The method remarkably improves nodulation and nitrogen fixation capacity of soybeans, and has greatpractical significance in increasing soybean yield.
Description
technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a soybean nodulation regulating gene GmRSD and its application method. Background technique [0002] Soybean is a high-oil and high-protein crop, a very important source of vegetable oil and vegetable protein, and occupies a pivotal position in the world's agricultural production and agricultural economy. Soybean is considered as a land-growing crop because of its symbiotic nitrogen fixation function. Nitrogen has a great impact on the growth, yield and quality of soybean. Studies have shown that soybeans can fix 8 kilograms of nitrogen per mu, which is equivalent to applying 18 kilograms of urea per mu. It can be said that soybeans are one of the largest natural nitrogen fertilizer synthesis factories on the earth. Therefore, developing soybean production and making full use of the symbiotic nitrogen fixation function of soybean to synthesize nitrogen fertilizer is ...
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