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Method of breeding tobacco plant having broad-spectrum resistance to potyviruses

A tobacco plant, potato technology, applied in the field of biology, tobacco biotechnology breeding

Active Publication Date: 2019-02-22
YUNNAN ACAD OF TOBACCO AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no report of resistance to potato virus Y (PVY), capsicum vein mottle virus (ChiVMV) and tobacco vein band mosaic virus (TVBMV) in tobacco

Method used

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  • Method of breeding tobacco plant having broad-spectrum resistance to potyviruses
  • Method of breeding tobacco plant having broad-spectrum resistance to potyviruses
  • Method of breeding tobacco plant having broad-spectrum resistance to potyviruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Construction of simultaneously editing tobacco eIFiso4E-S gene and eIFiso4E-T gene knockout vector

[0023] The plasmid pRGEB31 used for the construction of the gene knockout expression vector of the present invention is described in the document "Xie, K.and Y.Yang (2013). "RNA-guided genome editing in plants using a CRISPR-Cas system." MolPlant 6(6): 1975-1983." published in;

[0024] 1. According to the principle of designing target sites by CRISPR / Cas9 technology, the target sites of the present invention are designed on the first exon of eIFiso4E-S gene and eIFiso4E-T gene. When looking for the target site, first find the PAM (NGG or CCN, in the form of 5'-NNNNNNNNNNNNNNNNNNNNNNNGG-3' or 5'-CCNNNNNNNNNNNNNNNNNNNNNNNN-3') site from the first exon sequence,

[0025] 2. Oligo synthesis and annealing of gRNA

[0026] 2.1 Using the gRNA target site as a template, design the primer oligo according to the following format. In order to ensure the efficiency of ...

Embodiment 2

[0042] Example 2: Plant Transformation of Gene Knockout Vectors and Detection of Gene Knockout Plants

[0043] The correct plasmid will be ligated and transformed into Agrobacterium GV3101 by electroporation. Agrobacterium-mediated transformation of tobacco callus to obtain transgenic plants. Tobacco 2-1398 containing homozygous va gene locus was used as material to induce callus, and Agrobacterium-mediated tobacco transformation experiments were carried out. Agrobacterium GV3101 was used for infection and transformation, and after hygromycin resistance selection, the resistant callus was differentiated and regenerated to obtain transgenic positive lines.

[0044] A. Detection of eIFiso4E-S gene mutants in transgenic tobacco

[0045] Design target gene detection primers. Design primers according to the target gene upstream and downstream of the target site sequence, and the primer sequences are:

[0046] eIFiso4E-SEditestF 5'-gattaccggcccagtctgtcatcat-3'

[0047] eIFiso4E...

Embodiment 3

[0053] Example 3: Polymerization of eifiso4e-t KO , eifiso4e-t KO and va's three-burst material obtained

[0054] The CRISPR-Cas9 gene editing vector constructed in Example 1 was used to transform va tobacco 2-1398 (genotype eIFiso4E-S / eIFiso4E-T / va) (see Example 2 for the method). The sequence of the eIFiso4E-S biallelic mutation of the single plant 2-1398g1-2B and 2-1298g1-2B of the eIFiso4E-S heterozygous mutation was screened by PCR amplification and sequencing, as shown in SEQ ID No.1 , the 11th to 18th nucleotides are deleted at site 1, and the 11th to 17th nucleotides are deleted at site 2. Causes a translational frameshift of the eIFiso4E-S polypeptide, resulting in a functionally inactive polypeptide.

[0055] Table 2 Mutation sites of eIFiso4E-S gene exon1 obtained by gene editing

[0056]

[0057] Screening out the single plant 2-1398g1-2B of the homozygous mutation of eIFiso4E-T, the sequence of the homozygous mutation of eIFiso4E-T of 2-1298g1-2B is ...

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Abstract

The invention relates to a method of breeding a tobacco plant having broad-spectrum resistance to potyviruses. The method comprises the following steps: polymerizing an eIFiso4E-S gene mutant, an eIFiso4E-T gene mutant and an eIF14E1 gene mutant in the tobacco plant to achieve the resistance of the tobacco to four potyviruses comprising a potato virus Y (PVY), a chilli veinal mottle virus (ChiVMV)and a tobacco vein banding mosaic virus (TVBMV). The method has great application prospect for cultivating the anti-Potyviruses tobacco.

Description

technical field [0001] The invention belongs to the field of biotechnology, and further belongs to the field of tobacco biotechnology breeding, in particular to a kind of polymerizing eIFiso4E-S gene mutant, eIFiso4E-T gene mutant and eIF4E1 gene mutant to obtain tobacco with broad-spectrum resistance to potyvirus Y virus Plant approach. Background technique [0002] The genus Potyvirus (Potyvirus) includes more than 10 members, and mainly harms crops of the family Solanaceae (Solanaceae). Potyviruses mainly harm Solanaceae crops such as tobacco, tomato and pepper. In recent years, potato virus Y (PVY), pepper vein mottle virus (ChiVMV) and tobacco vein band mosaic virus (TVBMV) have become common diseases on tobacco. In the farmland ecology where crops of the same family are planted next to each other, controlling the occurrence of Potyviruses on one crop is not only beneficial to the disease control of this crop, but also beneficial to the disease control of other crops. ...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00A01H6/82
CPCC12N15/8243C12N15/8283C07K14/00
Inventor 刘勇黄昌军于海芹曾建敏
Owner YUNNAN ACAD OF TOBACCO AGRI SCI
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