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Application of soybean glyma.04g253500 disease resistance gene

A disease-resistant gene, soybean mosaic virus technology, applied in the field of genetic engineering, can solve the problems of affecting the commercial value of seeds, reducing production, reducing seed quality, etc., and achieve the effect of improving soybean disease resistance, simple synthesis method, and realizing industrial production

Active Publication Date: 2018-11-16
ZHEJIANG NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Diseased plants not only reduce yield but also reduce seed quality (such as germination rate, protein content and oil content), thus affecting the commercial value of seeds

Method used

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  • Application of soybean glyma.04g253500 disease resistance gene
  • Application of soybean glyma.04g253500 disease resistance gene
  • Application of soybean glyma.04g253500 disease resistance gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1. Synthesis of Glyma.04G253500 disease resistance gene sequence

[0042] 1. Extraction of total RNA, using the Trizol method to extract total RNA:

[0043] (1) Get the leaves of soybean seedling plants, cut the leaves of the plants into pieces, remove the petioles and thicker veins, put them into a mortar pre-cooled in liquid nitrogen, add liquid nitrogen to the mortar and grind to powder, and The powder was transferred to a 1.5 ml centrifuge tube cooled in liquid nitrogen.

[0044] (2) Add 1ml TRIZOL to a 1.5ml centrifuge tube, and vortex the centrifuge tube. After shaking for a while, uncap the centrifuge tube and deflate it to prevent the air pressure in the centrifuge tube from increasing and causing the liquid to splash. liquid.

[0045] (3) Put the prepared grinding solution into a new centrifuge tube, and put the centrifuge tube filled with the grinding solution on a centrifuge for centrifugation. The temperature of the centrifuge is 4° C., the rotatio...

Embodiment 2

[0080] Embodiment 2, construction of BPMV-Glyma.04G253500 vector:

[0081] 1. Enzyme digestion of vector BPMV-RNA1

[0082]

[0083] Place the centrifuge tube at 37°C for enzyme digestion for 3 hours, and then purify, that is, add 3 volumes of Buffer PCR-A to the PCR, enzyme digestion, enzyme labeling, or sequencing reaction solution (if Buffer PCR-A is less than 100 μl , to 100 μl); after mixing, transfer to a DNA preparation tube, place the DNA preparation tube in a 2ml centrifuge tube, centrifuge at 12,000×g for 1 min, and discard the filtrate. Put the preparation tube back into the 2ml centrifuge tube, add 0.7ml Buffer W2, centrifuge at 12,000×g for 1min, and discard the filtrate. Place the preparation tube in a clean 1.5ml centrifuge tube, add 20μl ddH to the center of the DNA preparation membrane 2 O, stand at room temperature for 1 min. Centrifuge at 12,000×g for 1 min to elute DNA.

[0084] 2. Enzyme digestion of gene fragment Glyma.04G253500

[0085]

[008...

Embodiment 3

[0101] Embodiment 3, gene bombardment creates soybean silent plant

[0102] 1. Preparation of microprojectiles

[0103] (1) Weigh 3 mg of gold powder (0.6 μm in diameter) and put it into a 1.5 mL centrifuge tube;

[0104] (2) Add 50 μL of absolute ethanol to the centrifuge tube, and vortex for 2-3 minutes;

[0105] (3) Place the vortexed centrifuge tube in an ice bath for 5 minutes and allow the gold powder to settle, centrifuge at 10,000 rpm for 30 seconds, and discard the supernatant;

[0106] (4) Repeat (2), (3) steps 2 times;

[0107] (5) Add 50 μL sterile water to the centrifuge tube, vortex for 2-3 minutes, and fully resuspend;

[0108] (6) Place the centrifuge tube in an ice bath for 5 minutes, centrifuge at 10,000 rpm for 30 seconds, and discard the supernatant;

[0109] (8) Repeat (5), (6) steps 2 times;

[0110] (9) Add 50 μL of sterile water to the centrifuge tube, vortex for 1 min, and store at -20°C.

[0111] 2. DNA embedding

[0112] (1) Put the microproje...

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Abstract

The invention relates to the field of gene engineering technologies, in particular to a soybean Glyma.04G253500 resistant gene and coded protein MEKK1 of the soybean Glyma.04G253500 resistant gene as well as a synthetic method. Specifically, the invention discloses application of the soybean Glyma.04G253500 resistant gene which is used for improving soybean disease resistance such as soybean virus resistance, soybean downy mildew resistance and the like; a sequence of cDNA nucleotide of the soybean Glyma.04G253500 resistant gene is shown in SEQ ID NO:1.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to soybean Glyma.04G253500 disease resistance gene and its encoded protein MEKK1 and its synthesis method. Background technique [0002] Soybean originates in China and has been cultivated in my country for more than 5,000 years. However, from 1996 to 2010, soybean imports soared from 1.1 million tons to 54.8 million tons, which has seriously affected my country's food security. Soybean is susceptible to infection by various pathogens such as viruses, fungi and nematodes in high-temperature and high-humidity environments. In my country, the main pathogens harmful to soybean production are soybean mosaic virus, Asian rust, downy mildew and soybean cyst nematode. These high-incidence soybean fungal diseases and virus diseases have caused the annual soybean production reduction in my country to be as high as 10-30%. Taking soybean mosaic virus disease as an example, it h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/54A01H5/00A01H6/54
CPCC12N9/12C12N15/8282C12N15/8283
Inventor 刘建中蒋续续倪萍萍
Owner ZHEJIANG NORMAL UNIVERSITY
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