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Plant nicotinic acid methyltransferase gene NANMT and application thereof in plant broad-spectrum disease resistance

A technology of nicotinic acid methyl and Arabidopsis nicotinic acid methyl, applied in the direction of plant genetic improvement, angiosperms/flowering plants, transferase, etc.

Pending Publication Date: 2022-07-15
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, whether NANMT can play a role in plant disease resistance has not been reported yet.

Method used

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  • Plant nicotinic acid methyltransferase gene NANMT and application thereof in plant broad-spectrum disease resistance
  • Plant nicotinic acid methyltransferase gene NANMT and application thereof in plant broad-spectrum disease resistance
  • Plant nicotinic acid methyltransferase gene NANMT and application thereof in plant broad-spectrum disease resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1. Cloning of ZmNANMT

[0053] 1.1 Extraction of total RNA from maize inbred line B73

[0054] (1) Put the corn B73 leaf material into a high-temperature sterilized mortar, pour it into liquid nitrogen and grind it into powder; continuously add liquid nitrogen during the grinding process to prevent melting;

[0055] (2) Transfer the ground leaf powder into a 1.5ml centrifuge tube, quickly add 1ml RNAiso Plus extract, vortex, and let stand for 10min at room temperature;

[0056] (3) Add 200 μl of chloroform, gently shake and mix until the liquid is stratified, and leave it at room temperature for 5 minutes;

[0057] (4) 4°C, 12,000rpm, centrifuge for 15min, transfer 500μl of supernatant to a new 1.5ml centrifuge tube, add 500μl of isopropanol, invert up and down to mix, and place at room temperature for 10mim;

[0058] (5) 4°C, 12,000rpm, centrifuge for 15min, discard the supernatant, wash the precipitate with 1ml 75% ethanol, flick the bottom of the tube to ma...

Embodiment 2

[0091] Example 2. Construction of ZmNANMT overexpression vector and verification of disease resistance function of transgenic lines

[0092] 2.1 Construction of maize ZmNANMT overexpression vector

[0093] (1) Double enzyme digestion of maize overexpression vector pCAMBIA3301 with BamH1 and Xma1;

[0094] (2) BamH1 and Xma1 restriction sites were added at both ends of the gene ZmNANMT;

[0095] (3) The target band is recovered by gel after double enzyme digestion of the gene, and connected to the pCAMBIA3301 carrier through T4 DNA ligase;

[0096] (4) Transform E. coli competent DH5α, identify the positive clones by colony PCR, and sequence the plasmid in advance. The plasmid vector after correct sequencing is named pCAMBIA3301::ZmNANMT, and its map is shown in Figure 4 .

[0097] 2.2 Plasmid transformation Agrobacterium

[0098] (1) Add 1 μg of plasmid vector to 50 μl of Agrobacterium competent EHA105, mix well, and take an ice bath for 30 minutes;

[0099] (2) Quick-fr...

Embodiment 3

[0152] Example 3. Construction of ZmNANMT Gene Editing Vector and Verification of Disease Resistance of Edited Lines

[0153] 3.1 Construction of maize ZmNANMT gene editing vector

[0154] The CRISPR / CAS9 base vector was single digested with HindIII. Based on the principle of CRISPR / CAS9 technology, and according to the ZmnANMT genome sequence, two target sites, gRNA1 and gRNA2, were designed. The nucleotide sequence of gRNA1 is shown in SEQ ID No.3, and the nucleotide sequence of gRNA2 is shown in SEQ ID No.4. Show. The U-S (U6-gRNA-sgRNA cassette) fragment was used as the template to design specific amplification primers with vector nicked homology arms and overlapping PCR overlapping part (20bp gRNA part), and U-S was used as the template to amplify the target fragment U6-2 promoter and sgRNA scanfold fragment, and finally obtained CPB::U6-2::gRNA::sgRNA::CPB overlapping fragment by overlapping PCR, after sequencing the overlapping PCR product, use T4 DNA ligase (TaKaRa) ...

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Abstract

The invention discloses a plant nicotinic acid methyltransferase gene NANMT, which comprises a corn nicotinic acid methyltransferase gene ZmNANMT, or an arabidopsis nicotinic acid methyltransferase gene AtNANMT, or a plant nicotinic acid methyltransferase gene NANMT. Wherein the cDNA (complementary deoxyribonucleic acid) nucleotide sequence of the ZmNANMT is as shown in SEQ ID No. 1, and the cDNA nucleotide sequence of the AtNANMT is as shown in SEQ ID No. 7. The invention further discloses application of the plant nicotinic acid methyltransferase gene NANMT to improvement of the broad-spectrum disease resistance of the plant by reducing the expression of the plant nicotinic acid methyltransferase gene NANMT. Experiments prove that through inoculation identification of pathogenic fungi of a ZmNANMT transgenic overexpression positive strain, it is found that the ZmNANMT overexpression positive strain shows a susceptible phenotype to various corn diseases, and it is proved that ZmNANMT is a new susceptible gene. Two target spots are designed for ZmNANMT by further utilizing a CRISPR / CAS9 technology, pathogenic bacteria inoculation identification is carried out on a ZmNANMT edited strain, and the ZmNANMT edited strain is found to have broad-spectrum disease resistance to various corn fungal diseases. The invention provides a new gene, a new material and a new method for disease-resistant breeding of corn, and the application prospect is wide.

Description

technical field [0001] The invention belongs to the technical field of biological genetic engineering, in particular to plant (maize or Arabidopsis thaliana) nicotinic acid methyltransferase gene NANMT and its application to improve broad-spectrum disease resistance of plants by reducing the expression of NANMT. Background technique [0002] Corn (Zea mays L.) is an important food crop in my country, and it occupies an important position in the national economy and agricultural production as a feed crop and an industrial raw material. Maize faces many problems in the production process, such as drought, high temperature, salinity and other adversity stress and biotic stress such as pests and diseases. Among them, disease is one of the main factors affecting the yield of maize in my country, and the annual yield loss accounts for about 10% of the total maize yield, which seriously threatens the food security of our country. Affected by global climate change, maize diseases a...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/10C12N15/84A01H5/00A01H6/46A01H6/20
CPCC12N9/1007C12N15/8218C12N15/8282C12N15/8281C12Y201/01007
Inventor 王官锋李雅洁
Owner SHANDONG UNIV
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