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Application of ZmHIR3 protein or coding gene thereof in regulation and control of maize rough dwarf virus resistance

A technology that encodes genes and rough dwarf disease, applied in the field of genetic engineering, can solve problems such as environmental pollution, labor and time-consuming, poor control effect, etc., and achieve the effect of improving the ability to resist rough dwarf disease

Active Publication Date: 2022-05-31
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above-mentioned control measures are labor-intensive, time-consuming, easy to cause environmental pollution, and have poor control effects. Breeding and planting disease-resistant varieties is an effective way to prevent and control maize rough dwarf disease.

Method used

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  • Application of ZmHIR3 protein or coding gene thereof in regulation and control of maize rough dwarf virus resistance
  • Application of ZmHIR3 protein or coding gene thereof in regulation and control of maize rough dwarf virus resistance
  • Application of ZmHIR3 protein or coding gene thereof in regulation and control of maize rough dwarf virus resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 ZmHIR3 Gene Overexpression Vector and Editing Vector Construction and Transformation

[0035] The vector used for overexpression in the present invention is pCUB-eGFP-3×FLAG, which is based on the pCUB vector (Song Pan, 2015, construction and genetic transformation of corn ZmPOT gene TALEN knockout and overexpression vector) using BamHI restriction enzyme point, the GFP protein and 3×FLAG tag were inserted between the promoter and terminator on the pCUB vector to construct.

[0036] Insert the ZmHIR3 gene (the nucleotide sequence shown in SEQ ID NO.2, which can encode the amino acid sequence shown in SEQ ID NO.1) into the promoter and GFP tag protein on the pCUB-eGFP-3×FLAG vector between. Carry out transformation, pick a single spot, sequence, and use the heat shock method to transform the overexpression vector plasmid pCUB-ZmHIR3-eGFP-3×FLAG into Agrobacterium EHA105 competent cells, pick a single colony of recombinant Agrobacterium, and extract the plasmid...

Embodiment 2

[0040] Example 2 Molecular detection of ZmHIR3 gene overexpression T2 generation strain

[0041]From the transgenic lines of the T1 generation, the plants that were positive in both PCR and test strip detection were selected for strict selfing, and the grains were harvested, and the T2 generation lines were obtained by planting in ear rows in the second year. Using the total leaf DNA of the T2 generation transgenic plants, design specific primers (SEQ ID NO.6-7) according to the sequence of the promoter plus the target gene plus the terminator, using the recipient control maize leaf DNA as a negative control, and the plasmid DNA as a positive control, Two rounds of PCR testing were performed. The result is as figure 2 Shown: There are 21 positive transgenic plants and the plasmids can amplify the 2104bp gene fragment and the 429bp Bar gene fragment, and no amplified bands are seen in the negative control and water control.

[0042] The leaves of different T2 generation tran...

Embodiment 3

[0043] Example 3 Molecular detection of ZmHIR3 gene edited T2 generation strains

[0044] The total DNA of the two-leaf stage leaves of the gene-edited transgenic plants of the T2 generation obtained by the CTAB method was extracted. Design specific primers (SEQ ID NO.8-9) according to the edited target site, strictly self-cross the gene-edited plants of the T1 generation to harvest the ears, and plant all the ear rows in the second year to obtain the T2 generation plants, design according to the sequence of the target site Specific primers, with wild-type maize leaf DNA as a negative control and plasmid DNA as a positive control, two rounds of PCR detection were performed. Test results such as Figure 4 Shown: Both the positive transgenic plants and the plasmids can amplify a total of 618bp fragments including 3 targets, and carry out sequencing identification. There are 6 types of stable genetic homogeneity and variation.

[0045] The leaves of the T2 transgenic line seedl...

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Abstract

The invention relates to the technical field of gene engineering, in particular to application of ZmHIR3 protein or a coding gene thereof in regulation and control of maize rough dwarf virus resistance. According to the invention, pathogen and host interaction relationship, resistance identification and spatio-temporal expression pattern analysis are carried out on RBSDV infected corn, and a protein, namely the ZmHIR3 protein, related to resistance to rough dwarf virus of corn is found. A further research finds that the coding gene of the over-expressed ZmHIR3 protein in a plant material can effectively improve the rough dwarf disease resistance of a corn material, so that reliable data support is provided for research on a molecular biological mechanism for enhancing the rough dwarf disease resistance of plants such as corn, and the ZmHIR3 protein has important significance in the field of rough dwarf disease resistant corn variety cultivation.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to the application of ZmHIR3 protein or its coding gene in regulating the ability of maize to resist rough dwarf disease. Background technique [0002] Maize rough dwarf disease (MRDD) is one of the most serious viral diseases that harm maize widely, and one of its causes is Rice Black-Streaked Dwarf Virus (RBSDV). At present, there is a lack of disease resistance resources and related resistance genes for maize rough dwarf disease, and the pathogenic and resistance mechanisms are unclear. The breeding of disease-resistant varieties provides theoretical support. [0003] The virus that causes maize rough dwarf disease is mainly transmitted by the gray planthopper (Laodelphax striatellus Fallen) in a persistent manner, and cannot be transmitted through soil, seed transmission, sap friction, and grafting. After the SBPH acquires the poison, it can carry and transmit the ...

Claims

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Application Information

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IPC IPC(8): C12N15/84C07K14/415C12N15/29A01H5/00A01H6/46
CPCC12N15/8283C07K14/415Y02A40/146
Inventor 周羽王振华卢晴邸宏张林李益晨董玲祖洪月曾兴刘显君
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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