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Culture method for high scaffolding nitrogen fixation plant

A breeding method and plant technology, applied in the field of genetic engineering, to achieve the effects of reducing nitrogen fertilizer application, increasing the number of root nodules, and increasing soybean yield

Inactive Publication Date: 2019-05-10
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are 6 calmodulins (CaMs) in the soybean genome, and the study of these calmodulin family proteins in the regulation of soybean root nodule number has not been reported

Method used

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  • Culture method for high scaffolding nitrogen fixation plant
  • Culture method for high scaffolding nitrogen fixation plant
  • Culture method for high scaffolding nitrogen fixation plant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1. Nomenclature and sequence of genes.

[0033] The overexpression vector constructed by the present invention can specifically overexpress the GmCam7Ls Family of 5 genes. The genes are located on soybean chromosomes 2, 3, 5, 14, and 19 respectively, and are named as follows, GmCam7La (gene number: Glyma.02G275600), having the coding sequence shown in SEQ ID NO: 1; GmCam7Lb (gene number: Glyma.03G004100), having the coding sequence shown in SEQ ID NO: 2; GmCam7Lc (gene number: Glyma.05G079700), having the coding sequence shown in SEQ ID NO: 3; GmCam7Ld (gene number: Glyma.14G040600), having the coding sequence shown in SEQ ID NO: 4; GmCam7Le (Gene number: Glyma.19G068300), having the coding sequence shown in SEQ ID NO:5.

Embodiment 2

[0034] Example 2. Expression analysis of GmCam7Ls family genes in response to rhizobia infection.

[0035] 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%. 15 days after sowing, inoculate with rhizobia, each plant was inoculated with 30 mL of bradyrhizobium USDA110 bacterial solution (OD600=0.08), and the soybean roots were collected on the 28th day after inoculation.

[0036] 2) Isolation of mRNA: Trizol method was used to extract soybean total RNA. ①Put the tissue into a grinder and grind it with liquid nitrogen for 3 times, then add 0.1-0.2 g of the ground tissue into a 1 mL centrifuge tube and t...

Embodiment 3

[0051] Example 3. Cultivation of transgenic soybeans with high ability of nodulation and nitrogen fixation.

[0052] 1) Extraction of total RNA from root tissue of Wilimas82 soybean.

[0053] 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.

[0054](1) Take 100 mg of soybean root material and grind it with liquid nitrogen, add 1 mL of TRI pure reagent, and homogenize thoroughly, then suck the homogenate into a 1.5 mL centrifuge tube, and place it at room temperature for 5 min.

[0055] (2) Add 200 μl chloroform, vortex to mix, let stand for 5 minutes, 4 ℃, 12000 r / min, centrifuge for 10 minutes.

[0056] (3) Take the...

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Abstract

The invention discloses a culture method for a high scaffolding nitrogen fixation plant. In a plant body, separated over-expression or simultaneous over-expression of nucleotide sequences shown in SEQID NO:1-SEQ ID NO:5 in a sequence list is performed, and a transgenic plant with the high scaffolding nitrogen fixation capability is obtained compared with a normal plant. By means of a constructedover-expression vector, a receptor soybean plant is converted to obtain the transgenic plant, the number of soybean root nodules can be remarkably increased, and the method has a great significance inreducing nitrogen fertilizer application and improving the soybean yield.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an application method for promoting the number of soybean root nodules by the soybean calmodulin protein family GmCaM7 coding gene. Background technique [0002] Soybean is an important grain and oil economic crop. Its unique symbiotic nitrogen fixation ability can convert nitrogen in the air into ammonia nitrogen that can be absorbed and utilized by plants, providing the necessary nitrogen nutrition for its own growth and development. Making full use of the symbiotic nitrogen fixation process mediated by soybean root nodules not only plays an important role in maintaining the normal growth and yield of soybean, but also helps reduce environmental pollution caused by excessive fertilization. Therefore, mining and improving the efficiency of symbiotic nitrogen fixation in soybean has become an agricultural possibility One of the important ways of sustainable developmen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/29A01H5/00A01H6/54
Inventor 纪洪涛李霞王延旭王倩
Owner HUAZHONG AGRI UNIV
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