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Wheat TaRPP13 gene and application thereof in wheat powdery mildew resistant breeding

A powdery mildew resistance gene and wheat technology, applied in the field of agricultural biological genetic engineering, can solve problems such as unseen sequence and function research

Inactive Publication Date: 2019-02-12
TIANJIN NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] So far, there is no information on the cultivation of wheat RPP13 Reports on related sequence and function studies of genes

Method used

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  • Wheat TaRPP13 gene and application thereof in wheat powdery mildew resistant breeding
  • Wheat TaRPP13 gene and application thereof in wheat powdery mildew resistant breeding
  • Wheat TaRPP13 gene and application thereof in wheat powdery mildew resistant breeding

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] TaRPP13 Gene sequence acquisition

[0046] 1 Experimental materials

[0047] 1.1 Plant material and strains

[0048] Jing 411, a wheat variety sensitive to powdery mildew, was donated by Professor Yang Zuomin of China Agricultural University. Powdery mildew resistant wheat variety Brock, a gift from Dr Ray Johnson. Near-Isogenic Lines (NILs) of powdery mildew-resistant hybrid varieties. In the previous experiments in our laboratory, the susceptible variety Jing 411 was used as the female parent to cross with the resistant variety Brock, and the powdery mildew in the F1 generation was selected. The resistant plant is the female parent, Jing 411 is used as the male parent for 7 consecutive generations of backcrossing, and the resulting offspring are bred after one generation of self-crossing (for the preparation method, please refer to the cultivation of near-isogenic lines of wheat Brock powdery mildew resistance gene by Zhang Rong et al. and Molecular Detection, Chi...

Embodiment 2

[0097] TaRPP13 Analysis of Differential Expression of Genes in Different Resistance Wheat

[0098] 1 Experimental materials and methods

[0099] 1.1 Plant material and strain (same as Example 1)

[0100] 1.2 TaRPP13 Analysis of gene expression patterns induced by powdery mildew

[0101] Wheat Jing 411, Brock and NILs were cultivated to the second-leaf stage. The powdery mildew was evenly inoculated on the back of the leaves, and the materials were weighed and quickly frozen in liquid nitrogen at 0hr, 2hr, 4hr, 8hr, 12hr, 24hr and 48hr after inoculation. RNA from each of the three varieties at 7 time points was extracted (same as in Example 1), and oligo(dT)18 was used as a reverse transcription primer to reverse transcribe into cDNA using the M-MLV system (same as in Example 1).

[0102] according to TaRPP13 Semi-quantitative RT-PCR primers semi-RPP13-F and semi-RPP13-R were designed for non-conserved segments in the gene sequence (see Table 1). Using Tabulin as an inte...

Embodiment 3

[0113] Evaluation using the VIGS method TaRPP13 Contribution of genes to powdery mildew resistance

[0114] 1 Materials and methods

[0115] 1.1 VIGS recombinant vector construction

[0116] The barley stripe mosaic virus (BMV) used for VIGS vector construction is a positive-strand RNA virus with 3 genomes: α, β and γ. Since the viral genome RNA is not suitable for direct manipulation, the viral RNA was reverse-transcribed into cDNA for further modification. Among them, add a place at the start codon of γb of ​​the γ chain Nhe I Restriction site for insertion of cloned DNA fragments.

[0117] 1. According to TaRPP13 Gene sequence, in the non-conserved region of the gene design added Nhe The primers RPP13-V-F and RPP13-V-R of the I recognition sequence (see Table 1 for the sequence). To contain TaRPP13 Plasmids with partial gene sequences were used as templates for PCR amplification. PCR reaction system: ExTaq 0.2μl, 10× ExTaq Buffer 2μl, dNTP Mixture 1.6μl, RPP13-V-...

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Abstract

The invention discloses a wheat TaRPP13 gene and application thereof in wheat powdery mildew resistant breeding. cDNA of a gene TaRPP13 of powdery mildew induced response in cultivating wheat Brock is3232 bp in total length, 5'UTR is 104 bp in length and 3'UTR is 194 bp in length, and an open reading frame is 2934 bp. A polypeptide chain of 977aa is coded, and has the homology of 93%, 79% and 66%with RPP13 gene-coded amino acid sequences in urartu wheat, brachypodium sylvaticum and corn respectively. The polypeptide chain has coiled-coil(CC), NB-ARC and LRR protein structural domains. Underthe stress of powdery mildew, a difference of expression of the gene in disease resistant wheat and disease infected wheat is apparent in time and expression quantity. Disease resistance of the disease resistant wheat Brock is reduced because of TaRPP13 gene silencing. The gene is capable of providing a new genetic resource for a wheat powdery mildew resistant mechanism and disease resistant breeding.

Description

[0001] National Natural Science Foundation of China (No. 31071671), Tianjin Municipal Science and Technology Commission Key Project (No. 17JCZDJC34100) Science and Technology Support Project (No. 18YFZCNC01100). technical field [0002] The invention belongs to the technical field of agricultural biological genetic engineering, in particular to a powdery mildew resistance gene in cultivated wheat Brock TaRPP13 and its application in wheat disease resistance breeding. Background technique [0003] Cultivated wheat Brock is highly resistant to powdery mildew and immune to dominant race 15 prevalent in North China. Analysis of powdery mildew resistance characteristics confirmed that Brock has a new powdery mildew resistance gene located on chromosome 3BL (Wang et al., Plant Production Science, 2004, 7(3): 319; Wang et al., Plant Production Science, 2005, 8 (5): 578). Due to the resistance degradation of powdery mildew resistance genes in wheat and the lack of resistance sourc...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415
CPCC07K14/415
Inventor 王振英刘晓颖范宝莉尚云涛兰桂霞张双力
Owner TIANJIN NORMAL UNIVERSITY
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