Cultivation method of genetically modified WMYB-R wheat resistant to root rot and sheath blight and related biological material thereof

A technology of biomaterials and transgenic plants, applied to the cultivation of WMYB-R genetically modified wheat resistant to root rot and sheath blight and related biomaterials, which can solve the problems of slow research progress, lack of germplasm resources of wheat disease resistance, etc. question

Active Publication Date: 2015-12-02
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 fact that the resistance to root rot and sheath blight of wheat is controlled by multiple genes, there is a lack of ideal germplasm resources for whea

Method used

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  • Cultivation method of genetically modified WMYB-R wheat resistant to root rot and sheath blight and related biological material thereof
  • Cultivation method of genetically modified WMYB-R wheat resistant to root rot and sheath blight and related biological material thereof
  • Cultivation method of genetically modified WMYB-R wheat resistant to root rot and sheath blight and related biological material thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Embodiment 1, the cloning of wheat resistance-related protein WMYB-R coding gene WMYB-R gene

[0096] Using the wheat gene chip to analyze the gene differential expression data of resistant wheat CI12633 and susceptible wheat Wenmai 6 in response to the sheath blight pathogen, combined with the correlation analysis of resistance expression, the highly expressed wheat resistant to sheath blight was identified in wheat CI12633 The important gene of blight and root rot is named WMYB-R gene.

[0097]The leaves of wheat CI12633 seedlings inoculated with the pathogenic bacteria of wheat sheath blight-Rhizoctoniacerealis R0301 were treated with liquid nitrogen, and the total RNA of the leaves was extracted according to the instructions of the Invitrogen TRIZOL Reagent total RNA extraction reagent. According to the procedures of Invitrogen's first-strand cDNA synthesis kit, the extracted RNA samples were reverse-transcribed to synthesize first-strand cDNA, which was used as a t...

Embodiment 2

[0098] Embodiment 2, WMYB-R gene is subjected to the induced expression analysis of wheat root rot pathogen

[0099] The leaves of wheat Yangmai 16 seedlings were rubbed and inoculated with the mycelium block of Bipolarissorokiniana, the pathogenic fungus of wheat root rot, and the mycelium block of Bipolarissorokiniana was placed on The leaves of the first and second basal leaf sheaths were taken 12h, 24h, 48h, 72h, and 96h after inoculation respectively, and the leaves of Yangmai 16 that were not inoculated with Bipolarissorokiniana were used as a control (0h). After quick-freezing in liquid nitrogen, store in a -80°C ultra-low temperature freezer for later use.

[0100] Total RNA (about 5 μg total RNA per sample) was extracted from leaves of Yangmai 16 at each treatment time point, and reverse-transcribed into cDNA according to the procedure of the Invitrogen First-Strand cDNA Synthesis Kit. Using the actin gene constitutively expressed in wheat as an internal reference, t...

Embodiment 3

[0108] Embodiment 3, the acquisition and identification of resistance to root rot and sheath blight transgenic wheat

[0109] 1. Construction of recombinant expression vector

[0110] 1. Inoculate wheat CI12633 blades with wheat root rot pathogenic bacteria-Bipolarissorokiniana, extract the RNA of wheat CI12633 blades after 48 hours, reverse transcribe into cDNA; take cDNA as a template, use the following WMYB-R A primer pair composed of -TF and WMYB-R-TR was used for PCR amplification to obtain a PCR amplification product (WMYB-R gene carrying a SpeI site).

[0111] WMYB-R-TF:5’-AT ACTAGT ATGGGACGTCCGTCGTCC-3' (underlined as SpeI enzyme recognition site);

[0112] WMYB-R-TR: 5'-TCAGAAGTATGGTTCCAATT-3'.

[0113] PCR reaction program: pre-denaturation at 94°C for 3 minutes; then denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 1 min, 15 cycles; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 1 min, 20 cycles; ...

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Abstract

The invention discloses a cultivation method of genetically modified WMYB-R wheat resistant to root rot and sheath blight and a related biological material thereof. The cultivation method includes the step of guiding coding gene of WMYB-R protein into a receptor plant to obtain a genetically modified plant with disease resistance higher than that of the receptor plant. If the WMYB-R gene is modified into plants, resistance, of the plants, to root rot and sheath blight can be improved, so that pesticide consumption is lowered to reduce environmental pollution; the cultivation method has important theoretical and practical significance and can play an important role in genetic improvement of the plants.

Description

technical field [0001] The invention relates to a breeding method of WMYB-R gene transgenic wheat resistant to root rot and sheath blight and related biological materials in the fields of molecular biology and genetic engineering. Background technique [0002] Wheat (Triticum aestivum) is one of the four major crops that humans rely on for survival. More than one-third of the world's population uses wheat as a staple food. The yield and quality of wheat directly affect human survival and quality of life. With the changes of farming systems, fertilizer and water conditions, and climate conditions, soil-borne diseases such as root rot and sheath blight are increasing in my country's wheat production areas, and have become one of the important factors restricting high and stable yields of wheat. [0003] Wheat root rot caused by Bipolarissorokiniana (Cochliobolus sativus in the sexual form), etc., occurs in all countries in the world, especially in the northern wheat areas of m...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82C12N5/10C12N1/21A01H5/00A01N47/44A01P3/00
CPCA01N47/44C07K14/415C12N15/8282
Inventor 张增艳单天雷洪彦涛魏学宁杜丽璞徐惠君
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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