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Method for screening homogenized rice chloroplast transgenic plants

A screening method and homogenization technology, applied in botany equipment and methods, biochemical equipment and methods, genetic engineering, etc., can solve problems such as failure

Inactive Publication Date: 2018-07-06
JILIN ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, the above-mentioned plants that achieve chloroplast transformation are all dicotyledonous plants, and the chloroplast transformation of monocotyledonous plants has not been successful, and many important crops, such as rice, corn, wheat and sorghum, are all monocotyledonous plants.

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  • Method for screening homogenized rice chloroplast transgenic plants
  • Method for screening homogenized rice chloroplast transgenic plants
  • Method for screening homogenized rice chloroplast transgenic plants

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Embodiment 1

[0038] 1 Materials and methods

[0039] 1.1 Test material

[0040] 1.1.1 Plant material

[0041] The test materials for testing are 20 early-maturing rice lines (code-named: 12, 14-17, 15, 17, 19, 35, 37-14, 47, 58, 111, 117, 198, 201, 239, 450, 501 , 808, 848, 1471, Z291), selected and bred by the Rice Breeding Laboratory of Jilin Agricultural University.

[0042] 1.1.2 Plasmids and strains

[0043] Design of the Prrn-T7g10-MSI99-smGFP-HPT-Trps16 expression cassette required for rice chloroplast expression. Prrn promoter and Trps16 terminator are 16S rRNA promoter and 3' end mature sequence respectively (GenBank accession number: BA000010.8); the three genes MSI99, smGFP and HPT included are all based on rice chloroplast Codon optimization for codon bias. The attP site and attB site recognized by homologous recombinase were inserted in the upstream of the smGFP gene and downstream of the HPT gene to excise the selection marker gene. The fragment was synthesized by Jinst...

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Abstract

The invention discloses a method for screening homogenized rice chloroplast transgenic plants. The method comprises the following steps: step (1), rice chloroplast transformation and homogenized screening; step (2), molecular detection. According to the method disclosed by the invention, homogenized rice chloroplast transgenic callus and plant materials are obtained for the first time, and relevant molecular evidence is obtained. A method is provided for judging whether the transgenic plants pass through long-term screening and achieve a homogenization level. Moreover, rice materials suitablefor chloroplast transformation are obtained by virtue of screening, and the improvement has the following characteristic: high-frequency regeneration capacity; the induced II callus also has excellentdifferentiation capacity after long-term (more than six months) subculture. In addition, comparison proves that compared with common spectinomycin and other screening reagents (such as antibiotics orherbicides), the hygromycin is more suitable to use as a screening reagent of rice chloroplast transformation.

Description

technical field [0001] The invention relates to a screening method for homogeneous rice chloroplast transgenic plants. Background technique [0002] Plant chloroplasts are semi-autonomous organelles with their own genomes and are therefore transformable. Chloroplast transformation is usually achieved by direct transformation by biolistic method or PEG method. Although there have been reports of transformation by Agrobacterium-mediated transformation, they have not been repeated. Chloroplast transformation is the direct delivery of exogenous gene DNA to chloroplasts, and then site-specific integration of exogenous genes into the chloroplast genome by means of homologous recombination, which was gradually established with the invention of the gene gun method. [0003] The research on chloroplast transformation started later than that of nucleus transformation. Boynton et al. transformed atpB gene mutant cells with wild-type Chlamydomonas chloroplast DNA, which completely rest...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00A01H6/46C12Q1/6895
CPCC12N15/8261C12Q1/6895
Inventor 邢少辰王云鹏韦正乙
Owner JILIN ACAD OF AGRI SCI
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