Tobacco NtMLO1 and NtMLO2 fixed-point co-knockout system and application thereof

A tobacco and co-cultivation technology, applied in the field of plant genetic engineering, can solve the problems of high identification difficulty and long breeding cycle, and achieve the effects of easy screening, simple steps, and shortening of the breeding cycle

Pending Publication Date: 2021-05-28
TOBACCO RES INST CHIN AGRI SCI ACAD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the breeding of tobacco powdery mildew-resistant varieties is to use hybridization to transfer two disease-resistant allelic variations into susceptible varieties at the same time, which has problems such as high difficulty in progeny identification and long breeding cycle.

Method used

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  • Tobacco NtMLO1 and NtMLO2 fixed-point co-knockout system and application thereof
  • Tobacco NtMLO1 and NtMLO2 fixed-point co-knockout system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Construction of Tobacco NtMLO1 and NtMLO2 Site-directed Co-knockout System

[0029] According to the gene sequence of tobacco NtMLO1 (Gene ID: LC089758) and NtMLO2 (Gene ID: LC089759), a homologous sequence was found on the first exon of tobacco NtMLO1 gene and NtMLO2 gene, and its nucleotide sequence is as shown in SEQ Shown in ID NO.1; Based on the homologous sequence, use biological software to design target sites, and simultaneously target the gRNA target site sequences of genes NtMLO1 and NtMLO2, and its nucleotide sequence is shown in SEQ ID NO.2. Synthesize MT-F and MT-R, the sequence is shown in SEQ ID NO.3, 4, make it anneal to form oligo double-stranded DNA with cohesive ends, insert the oligo double-stranded DNA into the two BsaI of Cas9 / gRNA expression vector Between the enzyme cutting sites, the tobacco NtMLO1 and NtMLO2 site-specific co-knockout system containing the target sites was obtained, named pORE-MT, and the positive plasmids verified by ...

Embodiment 2

[0030] The acquisition of embodiment 2 recombinant Agrobacterium tumefaciens

[0031]The pORE-MT vector system obtained in Example 1 was transformed into Agrobacterium strain EHA105 by heat shock to obtain a recombinant Agrobacterium containing the pORE-MT plasmid, which was named EHA / pORE-MT.

Embodiment 3

[0032] Embodiment 3 creates the method for resisting powdery mildew tobacco variety

[0033] The method comprises the steps of:

[0034] 1) Soak the seeds of China Tobacco 300 in sterile water for 30 minutes, soak in 75% alcohol for 30 seconds, rinse with sterile water for 3 times, soak in 15% hydrogen peroxide for 8 minutes, and wash with sterile water for three times; use sterile filter paper to dry the water on the seeds , planted in MS medium with a sterilized toothpick, placed in an artificial climate chamber with a temperature of 24°C, an illumination of 1000Lx, and an illumination of 16h / d, and selected tissue-cultured seedlings with a leaf age of 5-6 weeks for use.

[0035] 2) Inoculate the recombinant Agrobacterium in Example 2 in LB liquid medium (containing 50 mg / L kanamycin and 50 mg / L rifampicin), culture at 28° C. with shaking at 200 r / min to OD 600 The value is 0.5-0.6, centrifuge at 12000rpm for 1min, resuspend the bacteria with sterile water containing 20μg / m...

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Abstract

The invention belongs to the field of plant genetic engineering, and particularly relates to a tobacco NtMLO1 and NtMLO2 fixed-point co-knockout system and application thereof. The invention discloses a homologous sequence of tobacco genes NtMLO1 and NtMLO2, a target sequence is designed based on the homologous sequence, a tobacco NtMLO1 and NtMLO2 fixed-point co-knockout system is constructed, the tobacco NtMLO1 and NtMLO2 genes can be knocked out at the same time, and the simultaneous mutation efficiency of the two genes is 50.0%. The invention provides an efficient breeding mode for creating powdery mildew resistant germplasm resources of tobacco and cultivating powdery mildew resistant tobacco varieties.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and in particular relates to a tobacco NtMLO1 and NtMLO2 co-knockout system and its application. Background technique [0002] Powdery mildew is one of the leaf diseases that commonly occur on tobacco. It generally occurs in the middle and late stages of tobacco growth. The main diseased part is the leaves, usually starting from the lower leaves and spreading from bottom to top. In severe cases, all leaves fall off, leaving only Leaf veins, stems, petioles, pedicels, capsules and other parts can also be affected. MLO is a kind of plant-specific protein, which is located on the plasma membrane and has a complete set of seven transmembrane domains. Specific homologous genes in the MLO family are susceptibility genes of powdery mildew, and the resistance of plants to powdery mildew can be increased by loss-of-function mutation or silencing. Bai et al. (2008) cloned a powdery mildew resista...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/84C12N15/55A01H4/00A01H5/00A01H6/82C12Q1/6895
CPCC07K14/415C12N15/8218C12N15/8282C12N15/8205C12N9/22A01H4/001A01H4/008C12Q1/6895C12Q2600/156C12Q2600/13
Inventor 刘旦杨爱国李丹丹王学波申展程立锐蒋彩虹冯全福
Owner TOBACCO RES INST CHIN AGRI SCI ACAD
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