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BFR protein associated with resistance of wheat to powdery mildew, and coding gene and application thereof

A powdery mildew and protein technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of reduced wheat quality, threats to wheat production and food security, etc.

Inactive Publication Date: 2019-09-13
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Wheat powdery mildew can cause a 30%-40% yield loss in a pandemic year (Mehta, 2014), and also lead to a significant decrease in wheat quality, which seriously threatens my country's wheat production and food security

Method used

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  • BFR protein associated with resistance of wheat to powdery mildew, and coding gene and application thereof
  • BFR protein associated with resistance of wheat to powdery mildew, and coding gene and application thereof
  • BFR protein associated with resistance of wheat to powdery mildew, and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment one, the cloning of BFR gene cDNA and DNA sequence

[0034] 1. Acquisition of wheat cDNA and genome sequences

[0035] 1) Extraction of wheat genomic DNA and total RNA

[0036] Take 0.5 g of fresh wheat leaves, put them into a 2 mL centrifuge tube equipped with steel balls, freeze them with liquid nitrogen, and grind the materials into powder in a tissue grinder. Add 1.0mL lysate solution (1% SLS, 417mM Tris / HCl pH8.0, 417mM NaCl, 83mM EDTA), shake vigorously, vortex and mix thoroughly, and let stand on ice for 10min to fully lyse the cells. Add the extraction solution (Tris saturated phenol / chloroform / isoamyl alcohol = 25:24:1) equal to the volume of the lysate into the centrifuge tube, shake slowly until the emulsion is formed, let stand on ice (or room temperature) for 10 minutes, 12000rpm Centrifuge for 10 min, and transfer the supernatant to a new centrifuge tube. Repeat this operation once. Pipette the supernatant into another 1.5mL centrifuge tube,...

Embodiment 2

[0050] Example 2. Chromosomal mapping of the BFR gene using KASP markers

[0051] 1. Design and synthesis of KASP molecular marker primers

[0052] 1) The 3 BFR gene copies amplified from wheat genomic DNA in Example 1 were homologously compared, and 162 (A / G), 643 (G / A) and 1245 (C / T) were selected respectively Primers were designed for the SNP site at the bp position.

[0053]2) Design the selected SNP site and its upstream 19nt as two forward primers, and then add FAM: 5'-GAAGGTGACCAAGTTCATGCT-3' or VIC: 5'-GAAGGTCGGAGTCAACGGATT- at the 5' end of the two forward primers 3' fluorescent reporter tag sequence. Then select a suitable position downstream of the SNP site and select a nucleotide sequence of about 20 nt as a reverse primer, so that the length of the fragment obtained by the final PCR amplification is 50-80 bp. The reverse primer is not tagged. The KASP marker primers for the three SNP sites are:

[0054] SNP162F1: 5'-gaaggtgaccaagttcatgctCTGAGGGTCTTGTTGCTGCA-3...

Embodiment 3

[0071] Embodiment three, utilize BSMV-VIGS experiment to verify the anti-powdery mildew function of BFR gene

[0072] 1. Obtaining wheat with silenced BFR gene

[0073] 1) Induction of BFR gene down-regulation and construction of BSMV-VIGS vector system

[0074] (1) According to the BFR-3B gene sequence in Example 1, two silent fragments were selected in the non-conserved region outside the basic-Helix-Loop-Helix domain, and one fragment was located near the N-terminus of the coding region, the specific position It is +109bp~+314bp, 206bp long, named as the silent sequence BFR-1, using the primer pair V-BFR-F1: 5'-ATT GCTAGC TTTGCATACTTGACAAAAG-3' and V-BFR-R1:5'-TAA GCTAGC GGTTCACGGCAAGAGC-3' was obtained by PCR amplification. There is a SNP difference between this fragment and BFR-3A-xiaobaidongmai at the +161bp position, and there is a SNP difference between the BFR-3D-xiaobaidongmai at the +161bp and +203bp positions respectively. Another fragment is near the C-termi...

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Abstract

The invention relates to the field of wheat disease resistance, and specifically relates to a BFR protein associated with resistance of wheat to powdery mildew as well as a coding gene and applicationthereof. The BFR protein associated with resistance of wheat to powdery mildew has an amino acid sequence as shown in sequence 7, or a sequence constructed by replacing one or more amino acids in theamino acid sequence shown in sequence 7. Expression of the BFR protein in wheat leaves is reduced by adopting a virus-induced gene silencing technology and a CRISPR / Cas9 gene editing technology so asto have reaction type of infection of a powdery-mildew-resistant wheat variety, xiao-bai-dong (Triticum aestivum L.), on an avirulent strain E18 changed from grade 0-1 to grade 3-4. Over-expression of the BFR gene in wheat leaves is allowed by adopting biolistic transformation; and thus, pathogen spore germination of a virulent strain E18 on leaves of the wheat variety Kelong-199 is significantlyinhibited.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a BFR protein related to wheat powdery mildew resistance, its coding gene and its application. Background technique [0002] Wheat powdery mildew is a worldwide disease caused by wheat powdery mildew infection. Wheat powdery mildew (Blumeria graminisf.sp.tritici) belongs to the Ascomycetes class, the Brucella genus, and the gramineous powdery mildew wheat-specific type. It is a type of obligate living parasitic fungi (Wicker et al., 2013). [0003] The infection of wheat powdery mildew mainly affects the normal photosynthesis and metabolism of leaves, leading to adverse consequences such as decreased tillering, decreased seed setting rate, and decreased thousand-grain weight (Wiersma et al., 2017). Infection with powdery mildew at the seedling stage can even lead to plant death (Singh et al., 2016). In my country, the annual incidence area of ​​wheat powdery mildew is m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/83A01H5/00A01H6/46
CPCC07K14/415C12N15/8213C12N15/8218C12N15/8282
Inventor 张相岐刘亚培范仁春席海秀卫波
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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