Wheat disease resistance protein, encoding gene and application of wheat disease resistance protein and encoding gene in regulation of plant disease resistance

A technology for encoding genes and transgenic plants, applied in the fields of applications, plant peptides, plant products, etc., can solve the problems of lack of easy utilization and slow progress of wheat resistant to sheath blight

Active Publication Date: 2016-07-13
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, conventional breeding methods have been slow in breeding sheath blight-resistant wheat varieties due to the lack of readily available germplasm resources for wheat sheath blight resistance

Method used

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  • Wheat disease resistance protein, encoding gene and application of wheat disease resistance protein and encoding gene in regulation of plant disease resistance
  • Wheat disease resistance protein, encoding gene and application of wheat disease resistance protein and encoding gene in regulation of plant disease resistance
  • Wheat disease resistance protein, encoding gene and application of wheat disease resistance protein and encoding gene in regulation of plant disease resistance

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Experimental program
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Effect test

Embodiment 1

[0093] Embodiment 1, the cloning of wheat disease resistance protein TaZnF2 and its coding gene

[0094] The inventors of the present application isolated and cloned a wheat disease resistance protein from sheath blight resistant wheat CI12633, and named it TaZnF2. The specific cloning method of TaZnF2 gene is as follows:

[0095]The total RNA of wheat CI12633 leaf sheath was extracted, and the extracted RNA sample was reverse-transcribed to synthesize the first-strand cDNA according to the procedure of Invitrogen’s first-strand cDNA synthesis kit, which was used as a template for gene cloning, using TaZnF2-3R1:5′-ATTCGGACGAGGAACCCC -3′, 3RACE-OuterPrimer: 5′-TACCGTCGTTCCACTAGTGATTT-3′ (Takara), for the first round of PCR amplification. The amplification program was as follows: pre-denaturation at 94°C for 3 minutes; then 35 cycles at 94°C for 45 seconds, 52°C for 45 seconds, and 72°C for 1 minute; extension at 72°C for 10 minutes. The first round of PCR amplification produc...

Embodiment 2

[0105] Example 2, the acquisition of sheath blight resistant transgenic wheat and the identification of disease resistance

[0106] 1. Construction of overexpressed transgene vector

[0107] The complete ORF sequence of the TaZnF2 gene was constructed on the monocotyledonous plant expression vector pAHC25-cMYC, and the construction process was as follows: figure 2 As shown, the specific steps are as follows:

[0108] 1. Preparation of linearized plasmid: Digest plant expression vector pAHC25-cMYC with SpeI and EcoICRI (is isolytic enzyme with SacI, but produce blunt ends after EcoICRI digestion), 1% agarose gel electrophoresis, agarose gel The DNA Purification and Recovery Kit recovers the linearized pAHC25-cMYC vector backbone.

[0109] 2. Acquisition of the target gene TaZnF2 containing restriction sites: Design a pair of primers TaZnF2-OT-F: 5′-TAA according to the ORF sequence of the TaZnF2 gene ACTAGT ATGTCGCCGGCGGAGATGGA-3' (the underlined sequence is the restrictio...

Embodiment 3

[0157] Example 3, Breeding Gene-Silenced Wheat with Reduced Sheath Blight Resistance

[0158] 1. The two ends of the DNA fragment shown in the 756th-955th nucleotides of sequence 1 in the sequence listing are respectively provided with the recognition sequence of the restriction endonuclease NheI. After NheI digestion, the DNA fragment (200bp) shown in the 756-955th nucleotide of sequence 1 in the sequence listing is inserted into the BSMV-γ (the γ vector of BSMV virus) after the NheI enzyme linearization by the reverse insertion method ), the DNA molecule (named antiTaZnF2) that is reverse complementary to the DNA fragment shown in the 756-955th nucleotide of sequence 1 in the sequence listing is driven by the T7 promoter of the γ vector to obtain the recombinant vector BSMV-γ : antiTaZnF2.

[0159] 2. At the two-leaf-one-heart stage, use the recombinant vector BSMV-γ: antiTaZnF2 to transfect the second leaf of the wheat sheath blight-resistant material—wheat CI12633. The sp...

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Abstract

The invention discloses a wheat disease resistance protein, an encoding gene and an application of the wheat disease resistance protein and the encoding gene in regulation of plant disease resistance. The wheat disease resistance protein TaZnF2 is a protein in A1), A2) or A3) as follows: A1) a protein with the amino acid sequence shown as sequence 2, A2) a protein related with the plant disease resistance, wherein the amino acid sequence shown in the sequence 2 in a sequence table is subjected to substitution and/or deletion and/or addition of one or more amino acid residues for acquisition of the protein, and A3) a fusion protein obtained by connecting labels to an N end or/and a C end of A1) or A2). Experiments prove that the resistance, to wheat sharp eyespot, of wheat genetically modified with TaZnF2 and having TaZnF2 gene overexpression is improved remarkably; while TaZnF2 gene expression in disease resistance wheat CI12633 is inhibited, resistance of plants to the wheat sharp eyespot is reduced, and the result that the TaZnF2 gene is a necessary disease resistance gene for the wheat sharp eyespot resistant reaction is proved.

Description

technical field [0001] The invention relates to wheat disease resistance protein and coding gene in the field of biotechnology and their application in regulating plant disease resistance. Background technique [0002] Wheat is one of the most important food crops in the world and plays a pivotal role in ensuring food security. With the improvement of fertilizer and water conditions, the increase of planting density and the change of farming system, wheat sheath blight has developed into a major disease of wheat production in my country, and has become an important factor limiting the high and stable yield of wheat in my country. Wheat sheath blight, also known as wheat sharp eye spot (wheatsharpeyespot), is caused by the fusion group of saprophytic trophic fungi Rhizoctonia acerealis CAG-1 and Rhizoctonia solani AG4, AG5 A worldwide soil-borne fungal disease of wheat. The main pathogen of wheat sheath blight in my country is Rhizoctonia acerealis. Sheath blight can gener...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C07K19/00C12N15/29C12N15/62C12N5/10C12N15/82A01H5/00A01N65/44A01P3/00
CPCA01N65/44C07K14/415C07K2319/20C07K2319/21C07K2319/22C07K2319/41C07K2319/43C12N15/8216C12N15/8282C12N2800/60
Inventor 张增艳刘鑫祝秀亮魏学宁杜丽璞徐慧君
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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