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Rice chloroplast genetic transformation method

A genetic transformation method, chloroplast technology, applied in biochemical equipment and methods, using vectors to introduce foreign genetic material, recombinant DNA technology, etc., can solve problems such as failure

Pending Publication Date: 2019-07-23
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. 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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  • Rice chloroplast genetic transformation method
  • Rice chloroplast genetic transformation method
  • Rice chloroplast genetic transformation method

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Experimental program
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Effect test

Embodiment 1

[0042] 1 Materials and methods

[0043] 1.1 Test material

[0044] 1.1.1 Plant material

[0045] 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.

[0046] 1.1.2 Plasmids and strains

[0047] Design of the Prrn-T7g10-MSI99-smGFP-HPT-Trps16 expression cassette required for rice chloroplast expression. The Prrn promoter and Trps16 terminator are the 16S rRNA promoter and the 3' end mature sequence respectively (GenBank accession number: BA000010.8); the three genes MSI99, smGFP, and HPT included are based on the 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 synthes...

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Abstract

The invention discloses a rice chloroplast genetic transformation method. The rice chloroplast genetic transformation method comprises the following steps of step (1) constructing a rice chloroplast expression vector; step (2) performing rice chloroplast transformation and homogenization screening; and step (3) performing molecular detection. According to the rice chloroplast genetic transformation method disclosed by the invention, homogenized rice chloroplast transgenic callus tissue and plant materials are obtained for the first time, and relevant molecule evidences are obtained. Through screening, rice materials suitable for chloroplast transformation are obtained. The improvement has the following characteristics of having high-frequency regeneration capacity, and II property callus tissue produced through inducing still has favorable differentiation capacity after long-term (more than 6 months) subculture. Through comparison, the situation that hygromycin is more suitable for being used as a screening agent of rice chloroplast transformation than frequently-used spectinomycin and other screening agents.

Description

technical field [0001] The invention relates to a rice chloroplast genetic transformation method. Background technique [0002] Plant chloroplasts are semi-autonomous organelles with their own genomes and are therefore transformable. Chloroplast transformation is usually achieved through direct transformation by biolistic method or PEG method. Although there have been reports of transformation through 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 nuclear transformation. Boynton et al. transformed atpB gene mutant cells with wild-type Chlamydomonas chloroplast DNA, which completely restored the ph...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82
CPCC12N15/8261
Inventor 邢少辰王云鹏韦正乙
Owner JILIN ACAD OF AGRI SCI
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