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Breeding method for genetically modified wheat capable of resisting both sheath blight and root rot and related biological material thereof

A technology of biological materials and transgenic plants, which is applied in the cultivation of transgenic wheat with resistance to sheath blight and root rot and the fields of related biological materials, can solve the problems of slow progress and lack of

Active Publication Date: 2019-01-01
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the lack of readily available wheat germplasm resources for resistance to sheath blight and root rot, conventional breeding methods have made slow progress in breeding wheat varieties resistant to sheath blight and root rot

Method used

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  • Breeding method for genetically modified wheat capable of resisting both sheath blight and root rot and related biological material thereof
  • Breeding method for genetically modified wheat capable of resisting both sheath blight and root rot and related biological material thereof
  • Breeding method for genetically modified wheat capable of resisting both sheath blight and root rot and related biological material thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Embodiment 1, the cloning of the wheat protein TaOCS and its encoding gene of concurrent resistance to sheath blight and root rot

[0088] 1. Cloning of TaOCS gene

[0089] The inventors of the present invention isolated and cloned a wheat disease resistance-related protein from sheath blight-resistant wheat germplasm CI 12633, whose amino acid sequence is shown in sequence 2 of the sequence listing, and named it TaOCS protein. The gene encoding the TaOCS protein is named TaOCS gene, as shown in sequence 1 of the sequence listing, and the specific cloning method is as follows:

[0090] Extract the total RNA from the stems of wheat CI12633 inoculated with Rhizoctonia solani, and reverse-transcribe the extracted RNA samples to synthesize the first-strand cDNA according to the procedure of the first-strand cDNA synthesis kit of Tiangen Biochemical Company, as a template for gene cloning, and use TaOCS -OF1: 5'-GCGCGGATGAAACCCTAGC-3' and TaOCS-OR1: 5'-GTATGGGTTACTGTTAGTCTT...

Embodiment 2

[0100] Example 2. Acquisition and identification of disease resistance of transgenic TaOCS wheat resistant to both sheath blight and root rot

[0101] 1. Construction of recombinant expression vector

[0102] Construct the complete ORF sequence of TaOCS gene into pWMB123, the specific operation is as follows:

[0103] 1. Using a primer pair composed of TaOCS-O-F and TaOCS-O-R, perform PCR amplification under the action of the high-fidelity amplification enzyme PRIMERSTAR (TAKARA Company), to obtain and recover the PCR amplification product.

[0104] TaOCS-O-F:5'-GAC GGATCC ATGGCCGCCGCCGCGACGAC-3' (the sequence indicated by the underline is the restriction site sequence);

[0105] TaOCS-O-R:5'-TCA GAGCTC TTAGGATCTATTCTTTGGAGGTTTGAC-3' (the sequence indicated by the underline is the restriction site sequence).

[0106] PCR reaction program: pre-denaturation at 98°C for 1min; 35 cycles of 98°C for 10s, 56°C for 15s, and 72°C for 2min; 72°C for 10min.

[0107] 2. Recover an...

Embodiment 3

[0157] Example 3, TaOCS reverse functional analysis of silencing the TaOCS gene in wheat CI12633

[0158] 1. Silencing the TaOCS gene in wheat CI12633 using virus-mediated gene silencing technology

[0159] 1. Add the NheI recognition sequence to the two ends of the DNA molecule shown in the 2055-2246th nucleotide of sequence 1 in the sequence listing, respectively, to obtain a DNA fragment with the NheI recognition sequence at both ends, and pass the DNA fragment through NheI After digestion, insert it into the BSMV-γ (the γ vector of BMSV virus) linearized by NheI enzyme by the reverse insertion method to make the DNA molecule (antiTaOCS) reverse complementary to No. 2055-2246 of sequence 1 in the sequence listing Driven by the T7 promoter of the gamma vector, the recombinant vector BSMV-gamma: antiTaOCS for silencing TaOCS was obtained.

[0160] 2. At the three-leaf one-heart stage, use the recombinant vector BSMV-γ: antiTaOCS to transfect the second and third leaves of wh...

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Abstract

The invention discloses a breeding method for a genetically modified wheat capable of resisting both sheath blight and root rot and a related biological material thereof. The method for breeding the disease-resistant genetically modified wheat comprises the following step: the coding gene of a protein with an amino acid sequence shown as SEQ ID No. 2 is introduced into a receptor wheat, so that the genetically modified wheat, the disease resistance of which is higher than the disease resistance of the receptor wheat, is obtained; and the disease resistance is resistance to sheath blight and / orresistance to root rot. A transgenic experiment in which TaOCS is introduced into wheat proves that compared with the resistance of the receptor wheat, the resistance of the genetically modified wheat overexpressed by TaOCS to sheath blight and root rot is remarkably enhanced, indicating that TaOCS is a protein related to the sheath blight and root rot of plants, and TaOCS and the coding gene thereof can be used for enhancing the resistance of plants to the sheath blight and / or root rot.

Description

technical field [0001] The invention relates to a breeding method of transgenic wheat resistant to sheath blight and root rot and related biological materials in the fields of molecular biology and genetic engineering. Background technique [0002] More than 50% of the world's population uses wheat as a staple food crop, so wheat plays a pivotal role in ensuring food security. With the increase of planting density, changes in fertilizer and water conditions and farming systems, wheat sheath blight and root rot often occur. Wheat sheath blight, also known as wheat sharp eyespot, is mainly caused by saprotrophic trophic fungus Rhizoctonia cerealis in China. Sheath blight can generally reduce wheat production by 10% to 30%, and severe plots can reduce wheat production by more than 50%. Wheat root rot (common root rot) is caused by Bipolaris sorokiniana (Bipolaris sorokiniana) [0003] Cochliobolus sativus) and other wheat root diseases have occurred in wheat producing areas ...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/46
CPCC07K14/415C12N15/8282
Inventor 祝秀亮张增艳
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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