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Agrobacterium rhizogenes-mediated transformation method for pumpkin root system and gene editing method

A technique of Agrobacterium rhizogenes and gene editing, applied in the field of plants, can solve the problems of no pumpkin root transformation reports, unfavorable pumpkin molecular biology and gene function, etc., to promote molecular biology and gene function research, facilitate molecular biology and the effects of gene function studies

Inactive Publication Date: 2020-06-02
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, there is currently no report on the transformation of pumpkin roots, which is not conducive to promoting the study of pumpkin molecular biology and gene function

Method used

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  • Agrobacterium rhizogenes-mediated transformation method for pumpkin root system and gene editing method
  • Agrobacterium rhizogenes-mediated transformation method for pumpkin root system and gene editing method
  • Agrobacterium rhizogenes-mediated transformation method for pumpkin root system and gene editing method

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Experimental program
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Effect test

Embodiment 1

[0044] This example provides an Ar-qual strain of Agrobacterium rhizogenes containing pCAMBIA1305.4, its construction method refers to the Ar-qual instruction manual, the transformation method is liquid nitrogen freeze-thaw method, and the successfully transformed Ar-qual strain of Agrobacterium rhizogenes Store at -80°C.

Embodiment 2

[0046] This embodiment provides a method for constructing an Agrobacterium rhizogenes Ar-qual strain containing a gene editing vector, comprising the following steps:

[0047] S1, for the gene IDs CmoCh10G011690.1, CmoCh14G010850.1 and CmoCh01G014300.1, respectively, download the genome sequence of each target gene from the Cucurbitaceae genome database (http: / / cucurbitgenomics.org / ), and then submit the sequence to the Geneious software, Mark the exons and introns, select the genome file, run the program, get the sgRNA sequence set, and select the sgRNA sequence located on the exon and with the highest score.

[0048] The sgRNA primer pair shown in Table 1 was obtained according to the sequence design:

[0049] Table 1 Sequences of sgRNA primer pairs

[0050]

[0051] Send each sgRNA primer pair to a primer synthesis company (Beijing, Qingke) for synthesis.

[0052] Anneal the synthesized primer pair, and configure the primer annealing reaction system according to the fo...

Embodiment 3

[0065] Such as image 3 As shown, this embodiment provides a method for Agrobacterium rhizogenes mediated transformation of pumpkin roots, comprising the following steps:

[0066] (1) Sterilize plump pumpkin seeds:

[0067] Select plump and healthy pumpkin seeds, soak them in hot water at 55°C for 2-4 hours, then rinse them with clean water, remove the seed shells, and then transfer them to an ultra-clean workbench for operation. In the triangular flask, add 75% alcohol to sterilize for 1min, then pour off the alcohol, and use sterilized ddH 2 O rinse the seeds once, pour off the sterilized water, then sterilize the seeds with 0.3% sodium hypochlorite (ready to use) solution, rinse with sterile water to completely separate the seed coat from the seed kernel, pour off the water, and obtain the seed separated from the seed coat Benevolence.

[0068] (2) Obtain the explant containing the cotyledon base and the hypocotyl connecting segment

[0069] Such as image 3 As shown i...

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Abstract

The invention relates to an agrobacterium rhizogenes-mediated transformation method for a pumpkin root system and a gene editing method. According to the invention, agrobacterium rhizogenes-mediated transformation of the pumpkin root system is realized for the first time through specific process steps, and gene editing of the transformed root system is realized, so promotion of molecular biology and gene function research on pumpkins is facilitated.

Description

technical field [0001] The invention relates to the field of plant technology, in particular to a pumpkin root transformation method mediated by Agrobacterium rhizogenes and a gene editing method. Background technique [0002] Cucurbitaceae is one of the most important edible plant families in the world, including cucumbers, pumpkins, loofahs, watermelons and other common vegetables and fruits. Among them, the fruit, young stems and leaves, and flowers of pumpkin are all edible, with high nutritional value and certain health care functions. At the same time, pumpkins have the advantages of being resistant to barrenness and stress, and can also be used as rootstocks for other cucurbit crops such as watermelons, melons, and cucumbers to solve problems such as continuous cropping obstacles. [0003] However, there is no report on the transformation of pumpkin roots, which is not conducive to the promotion of pumpkin molecular biology and gene function research. Contents of t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/113C12N15/11C12Q1/6895A01H4/00A01H5/06A01H6/34
CPCC12N15/8205C12N15/8218C12N15/113C12Q1/6895A01H4/00C12N2310/20
Inventor 别之龙杨丽曹海顺周利剑孙静宇
Owner HUAZHONG AGRI UNIV
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