Application of ZmPAO gene in maize dwarf mosaic virus resistance

A gene and maize technology, which is applied in the application field of ZmPAO gene in maize resistance to dwarf mosaic disease, can solve problems such as production hazards, loss of resistance to viruses, and achieve the effects of improving research level, promoting and ensuring production safety.

Inactive Publication Date: 2017-09-19
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 ZmPAO gene in maize dwarf mosaic virus resistance
  • Application of ZmPAO gene in maize dwarf mosaic virus resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] The discovery of embodiment 1ZmPAO protein and its coding gene

[0048] 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 ZmPAO that is significantly differentially expressed in the two leaves is found, and its sequence is shown in SEQ ID NO:1. The gene encoding the ZmPAO protein is named ZmPAO gene, and its sequence is shown in SEQ ID NO:2.

Embodiment 2

[0049] Example 2 Construction of recombinant plasmid pC13 / F3-13m-PAO

[0050] 1. Extract the total RNA of maize inbred line Va35 and reverse transcribe it into cDNA.

[0051] 2. Using the cDNA obtained in step 1 as a template, perform PCR amplification with a primer pair composed of ZmPAO-silencing-F and ZmPAO-silencing-R to obtain a PCR amplification product.

[0052] ZmPAO-silencing-F: 5'-TA CCTAGG CTACGGAGTGTGGCAG-3'

[0053] ZmPAO-silencing-R: 5'-TA CCATGG GAGTTGTCCTCTGTCTTG-3'

[0054] 3. Digest the PCR amplified product in step 2 with restriction endonucleases Nco I and Avr II, and recover the digested product.

[0055] 4. Digest the vector pC13 / F3-13m with restriction endonucleases Nco I and Avr II, and recover about 5000 bp of the vector backbone.

[0056] 5. Ligate the digested product of step 3 with the vector backbone of step 4 to obtain the recombinant vector pC13 / F3-13m-PAO. According to the sequencing results, the structure of the recombinant vector pC13 / F...

Embodiment 3

[0058] Example 3 Transient silencing experiment of ZmPAO gene

[0059] 1. Expansion of BMV virus particles in Nicotiana benthamiana

[0060] 1. Infiltrating Nicotiana benthamiana with Agrobacterium carrying BMV vector

[0061] (1) Transform the correctly sequenced pC13 / F3-13m-PAO 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;

[0062] (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;

[0063] (3) Room temperature, 4500rpm centrifuge 8min to collect the bacteria, use 20ml infiltra...

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Abstract

The invention relates to application of ZmPAO gene in maize dwarf mosaic virus resistance. The ZmPAO gene is overexpressed in maize by means of gene engineering, so that maize dwarf mosaic virus resistance in crops is improved. It is discovered for the first time that SCMV (sugarcane mosaic virus) replication and proliferation levels can be significantly increased by silencing the ZmPAO gene, it is indicated that the maize protein ZmPAO and its encoding gene play an important role in resisting SCMV infections in maize and in proliferation, and the encoding gene is a maize-encoded anti-SCMV antiviral gene applicable to molecular breeding of anti-SCMV maize and other works. Basis is provided for the study on the role of vegetable proteins in viral infections, antiviral breeding of maize and other fields, viral resistance research level for maize is increased for China, and the production safety of Chinese maize is promoted and guaranteed.

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 ZmPAO gene in maize resistance to 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 production. [0003] As a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/29A01H5/00
CPCC07K14/415C12N15/8283
Inventor 周涛陈晖谢吉鹏袁雯范在丰
Owner CHINA AGRI UNIV
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