Soybean mosaic virus SC18 line resistance gene GmNIK and application thereof

A soybean mosaic virus, SC18 technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve problems such as top withering, necrosis, leaf shrinkage, etc.

Inactive Publication Date: 2019-10-08
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After soybeans are infected with soybean mosaic virus, symptoms such as mosaic and leaf shrinkage, plant dwarfing, necrosis and even top ...

Method used

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  • Soybean mosaic virus SC18 line resistance gene GmNIK and application thereof
  • Soybean mosaic virus SC18 line resistance gene GmNIK and application thereof
  • Soybean mosaic virus SC18 line resistance gene GmNIK and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1: Obtaining of GmNIK gene

[0024] 1. RNA extraction:

[0025] (1) Take 50-100 mg of soybean leaf tissue and grind it in liquid nitrogen, add 1 ml Trizol to fully homogenize it, and let it stand at room temperature for 5 minutes.

[0026] (2) Add 0.2ml of chloroform, shake for 15s, and let stand for 2min.

[0027] (3) Centrifuge at 4°C, 12000g×15min, and take the supernatant.

[0028] (4) Add 0.5ml of isopropanol, mix the liquid in the tube gently, and let stand at room temperature for 10 minutes.

[0029] (5) Centrifuge at 4°C, 12000g×10min, discard the supernatant.

[0030] (6) Add 1ml of 75% ethanol to gently wash the precipitate. 4°C, 7500g×5min, discard the supernatant.

[0031] (7) Dry it and add an appropriate amount of DEPC H2O to dissolve (65°C for 10-15 minutes). Extracted RNA should be stored at -80°C.

[0032] 2. Synthesis of cDNA:

[0033] (1) Take the RNA (≤2 μg) stored at -80°C and dissolve it on ice. Configure the following reaction ...

Embodiment 2

[0052] Example 2: Analysis of the induced expression of GmNIK by SC18

[0053] The susceptible variety Nannong 1138-2 and the resistant variety Kefeng No.1 were planted, and SC18 was inoculated when the first true leaf unfolded, and phosphate buffered solution PBS was inoculated at the same time as the control. The leaves of inoculation 0h, 2h, 4h, 6h, 8h, 10h, 12h, 24h, 36h, 48h, 72h were collected, and qRT-PCR was performed. See Example 4 for quantitative primers. Induced expression analysis results such as figure 1As shown, the expression levels of GmNIK in Kefeng 1 and Nannong 1138 were up-regulated after inoculation with SC18. Up-regulated to the highest, but the peak expression of Kefeng 1 is much higher than that of Nannong 1138-2. It indicated that the high expression of GmNIK in the disease-resistant variety may be the reason for its resistance to SC18.

Embodiment 3

[0054] Embodiment 3: Construction of GmNIK silencing vector

[0055] 1. Use Primer5.0 software to design specific silent fragment primers: upstream primer F3: 5'-GAATCCTCTGCATGAGGATCCGATAAATGCAAAAACATTCAAT-3' (SEQ ID NO.5), downstream primer F4: 5'-CTCTCGAGGCCTGGAGTCGACGAAACCAAGTCCCAGAATTAG-3' (SEQ ID NO. 6), using the constructed vector pMD20-T-NIK (constructed in Example 1) as a template to amplify the silent fragment.

[0056] The reaction system is:

[0057]

[0058] The reaction conditions were 95°C for 2min; 95°C for 10s, 58°C for 10s, 72°C for 1min, 32 cycles; 72°C for 1min. The amplified product was purified and recovered and stored at -80°C.

[0059] The BPMV silencing vector pBPMV-IA-V2 was digested with restriction enzymes BamH I and SalI at 37°C for 3 hours, purified and recovered. The silent fragment was connected to pBPMV-IA-V2 by homologous recombination,

[0060] The reaction system is:

[0061]

[0062]

[0063] The reaction conditions were 37°C ...

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Abstract

The invention discloses a soybean mosaic virus SC18 line resistance gene GmNIK and an application thereof. A nucleotide sequence of the gene GmNIK is shown as SEQ ID NO. 1, and an amino acid sequenceof the encoded protein is shown as SEQ ID NO. 2. A bean pod mottle virus-induced gene silencing technology (VIGS) is used to silence the GmNIK gene in a diseased variety Kefeng No.1 and inoculate theSC18 line, the disease-resistant variety Kefeng No. 1 generates symptoms of necrosis and floral leaf disease sensitivity, after GmNIK is silenced, the resistance of the disease-resistant variety Kefeng No.1 to the soybean mosaic virus SC18 is changed, and the disease resistance is susceptible. The GmNIK gene of the Kefeng No.1 determines the resistance to the SC18 line. The introduction of the GmNIK gene by a transgenic technology or molecular marker-assisted selection is expected to increase soybean resistance to the soybean mosaic virus.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and relates to a soybean mosaic virus SC18 strain resistance gene GmNIK and its application. Background technique [0002] Soybean mosaic virus (SMV) disease is a common soybean disease in soybean producing areas all over the world, seriously affecting the yield and quality of soybean. After soybeans are infected with soybean mosaic virus, symptoms such as mosaic and leaf shrinkage, plant dwarfing, necrosis and even top wilting will appear, which reduces the photosynthetic efficiency of soybeans, resulting in a 35%-50% reduction in soybean yield, or even failure. The SC18 strain is a popular strain that exists in many soybean producing areas in my country. [0003] According to the conserved domains of the cloned R genes, plant R genes can be divided into five families: (1) with nucleotide binding sites and leucine repeats (NBS-LRR); (2) with leucine-containing Repeat transmembra...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82A01H5/00A01H6/54
CPCC07K14/415C12N15/8283
Inventor 智海剑方飞杨云华王丽群
Owner NANJING AGRICULTURAL UNIVERSITY
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