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Application of ZmPGK gene to prevention and treatment of maize dwarf mosaic disease

A technology of sugarcane mosaic virus and corn, applied in application, genetic engineering, angiosperms/flowering plants, etc., can solve problems such as loss of resistance to viruses, production hazards, etc., and achieve the goal of improving research levels, promoting and ensuring production safety Effect

Inactive Publication Date: 2017-08-18
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the emergence of highly pathogenic strains of SCMV, some original virus-resistant varieties lost resistance, causing greater harm in production

Method used

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  • Application of ZmPGK gene to prevention and treatment of maize dwarf mosaic disease
  • Application of ZmPGK gene to prevention and treatment of maize dwarf mosaic disease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Discovery of ZmPGK protein and its coding gene

[0054] Using isobaric tagging for relative and absolute quantification (iTRAQ) proteomics technology and method, the proteins that were significantly differentially expressed in the upper first and second systematically infected leaves after SCMV infection were screened, and identified A protein ZmPGK that is significantly differentially expressed in the two leaves is found, the sequence of which is shown in SEQ ID NO:1. The gene encoding the ZmPGK protein is named ZmPGK gene, and its sequence is shown in SEQ ID NO:2.

Embodiment 2

[0055] Example 2 Construction of recombinant plasmid pC13 / F3-13m-PGK

[0056] 1. Extract the total RNA of maize inbred line Va35 and reverse transcribe it into cDNA.

[0057] 2. Using the cDNA obtained in step 1 as a template, perform PCR amplification with a primer pair composed of ZmPGK-silencing-F and ZmPGK-silencing-R to obtain a PCR amplification product.

[0058] ZmPGK-silencing-F: 5'-GA CCTAGG TCTCTTATTGAGAAGGC-3'

[0059] ZmPGK-silencing-R: 5'-TC CCATGG GGCCGTTCCAAAT-3'

[0060] 3. Digest the PCR amplified product in step 2 with restriction endonucleases Nco I and Avr II, and recover the digested product.

[0061] 4. Digest the vector pC13 / F3-13m with restriction endonucleases Nco I and Avr II, and recover about 5000 bp of the vector backbone.

[0062] 5. Ligate the digested product of step 3 with the vector backbone of step 4 to obtain the recombinant vector pC13 / F3-13m-PGK. According to the sequencing results, the structure of the recombinant vector pC13 / F3-13...

Embodiment 3

[0064] Example 3 Transient silencing experiment of ZmPGK gene

[0065] 1. Expansion of BMV virus particles in Nicotiana benthamiana

[0066] 1. Infiltrating Nicotiana benthamiana with Agrobacterium carrying BMV vector

[0067] (1) Transform the correctly sequenced pC13 / F3-13m-PGK vector into Agrobacterium C58C1 competent cells; at the same time, the Agrobacterium carrying pC13 / F1+2 and pC13 / F3-13m-GFP stored at -80°C Streak on LB plates (containing 50 μg / ml Kan, 100 μg / ml Rif), and culture at 28°C for about 48 hours. Pick a single colony on the LB plate and inoculate it in 3-4 ml of LB liquid medium (containing 50 μg / ml Kan, 100 μg / ml Rif), culture at 28°C, 180 rpm for 14-18 hours;

[0068] (2) Transfer 200-300 μl of the bacterial solution to 20-30 ml LB liquid medium (containing 50 μg / ml Kan, 100 μg / ml Rif) at a ratio of 1:100, and incubate at 28°C and 180 rpm for 10-12 hours;

[0069] (3) Room temperature, 4500rpm centrifuge 8min to collect the bacteria, use 20ml infiltra...

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Abstract

The invention relates to application of a ZmPGK gene to prevention and treatment of a maize dwarf mosaic disease. The application of the ZmPGK gene is characterized in that a genetic engineering method is utilized, and through inhibiting the expression of the ZmPGK gene in maize, the disease resistance of crops to the maize dwarf mosaic disease is improved. The invention discovers for the first time that the silencing ZmPGK gene can lead to a significant reduction in replication and proliferation levels of SCMV, which indicates that a maize protein ZmPGK and an encoding gene thereof play an important role in the replication and the proliferation of the SCMV; the ZmPGK gene is a maize-encoded recessive anti-virus gene against the SCMV and can be used for molecular breeding of the maize against the SCMV and other works. The invention provides a basis for research on the role of a plant protein in virus infection and the fields of maize antiviral breeding and the like, improves a maize antiviral research level in China, and promotes and ensures the safety of maize production in China.

Description

technical field [0001] The invention relates to the technical fields of biotechnology and crop disease prevention and control, in particular to the application of ZmPGK gene in the prevention and treatment of maize dwarf mosaic disease. Background technique [0002] Maize is the main food crop in my country, but maize dwarf mosaic disease (MDMD) caused by sugar cane mosaic virus (SCMV) infection poses a serious threat to the high and stable yield of maize in my country. SCMV is the main pathogen of maize dwarf mosaic disease in the maize producing areas of northern my country, often causing more than 30% yield loss of maize. Due to the emergence of highly pathogenic strains of SCMV, some original virus-resistant varieties lost their resistance, causing greater harm in production. There is an urgent need to identify new disease-resistant genes or resistant materials for sustainable and more effective control of SCMV and its hazards, and to ensure the safety of maize producti...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01N57/16A01P1/00A01H5/00
CPCA01N57/16C12N15/8218C12N15/8283
Inventor 周涛陈晖谢吉鹏袁雯范在丰
Owner CHINA AGRI UNIV
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