Application of ZmPDIL gene in controlling maize dwarf mosaic

A sugarcane mosaic virus, corn technology, applied in application, genetic engineering, recombinant DNA technology and other directions, can solve problems such as production hazards, loss of resistance to viruses, and achieve the effect of improving research level, promoting and ensuring production safety

Inactive Publication Date: 2017-08-22
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 vi

Method used

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  • Application of ZmPDIL gene in controlling maize dwarf mosaic
  • Application of ZmPDIL gene in controlling maize dwarf mosaic

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Discovery of ZmPDIL 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 To a protein ZmPDIL significantly differentially expressed in the two leaves, the sequence is shown in SEQ ID NO:1. The gene encoding ZmPDIL protein is named ZmPDIL gene, and its sequence is shown in SEQ ID NO:2.

Embodiment 2

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

[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 ZmPDIL-silencing-F and ZmPDIL-silencing-R to obtain a PCR amplification product.

[0058] ZmPDIL-silencing-F: 5'-AT CCTAGG GGATCTGCGACGTGTT-3'

[0059] ZmPDIL-silencing-R:5'-TA CCATGG TGCTGGTGGACACGTCAC-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-PDIL. According to the sequencing results, the structure of the recombinant vector p...

Embodiment 3

[0064] Example 3 Transient silencing experiment of ZmPDIL 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-PDIL 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 of 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 inf...

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Abstract

The invention relates to an application of ZmPDIL gene in controlling maize dwarf mosaic. A gene engineering method is utilized, and through inhibiting the expression of the ZmPDIL gene in maize, the resistance of crops to maize dwarf mosaic is improved. The invention initially discovers that silencing the ZmPDIL gene can result in obvious reduction of the reproduction and proliferation levels of SCMV, which shows that the zein ZmPDIL and encoded genes thereof can play an important role in SCMV infestation and proliferation, are recessive antivirus genes for maize encoded anti-SCMV, and can be used for molecular breeding works for maize anti-SCMV, and the like. The invention provides a basis for studying the action of vegetable protein in virus infestation, maize antivirus breeding and other fields, thereby improving the level of research in maize antivirus in China and promoting and ensuring the production safety of maize 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 ZmPDIL 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 product...

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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