Method for increasing chloroplast genetic transformation efficiency through genome editing technique

A technology of genetic transformation efficiency and genome editing, applied in the direction of introducing foreign genetic material using vectors, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problem of low homologous recombination rate of exogenous genes, unobtained homogeneous plants, etc. question

Active Publication Date: 2019-03-12
HUNAN HYBRID RICE RES CENT
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  • Claims
  • Application Information

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Problems solved by technology

However, although there are reports about chloroplast transformation in the main monocotyledonous plants rice and wheat, homogeneous plants that can be stably inherited have not been obtained. The main reason is that the homologous recombination rate of exogenous genes is low. Homologous recombination rate of source genes is an important way to improve chloroplast transformation efficiency

Method used

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  • Method for increasing chloroplast genetic transformation efficiency through genome editing technique
  • Method for increasing chloroplast genetic transformation efficiency through genome editing technique
  • Method for increasing chloroplast genetic transformation efficiency through genome editing technique

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1: Construction of Rice Chloroplast Genome CRISPR / Cas9 Gene Editing Vector

[0032] Ligation constructs containing the prrn promoter, T7Leader, Cas9 Gene, hpt Gene, eGFP Expression cassette for gene and TpsbA terminator and added at both ends of the expression cassette trnI and trnA Rice chloroplast homologous recombination sequence, at the same time in trnA An expression cassette containing prrn promoter, sgRNA and TpsbA terminator was added downstream (attached figure 1 ). Specific steps are as follows:

[0033] 1.1 Construction of intermediate binary vector pC1304

[0034] Digest the pCTE04 vector (containing chloroplast homologous fragments) with restriction endonucleases NdeI and XbaI trnA , trnI , hpt Gene, eGFP gene), recover the target fragment of about 1580bp; use XbaI and HindIII to double-digest the pCE4 vector, and recover the target fragment of about 3160bp; use HindIII and XbaI to double-enzyme pCAMBIA1300 vector, and recover ...

Embodiment 2

[0071] Example 2: Application of rice chloroplast genome CRISPR / Cas9 gene editing vector in rice chloroplast transformation

[0072] 2.1 Biolistic transformation

[0073] The pC1304-Cas9-sgRNA vector was transformed into rice TP309 immature embryo-induced calli (2000) by the method of gene gun, and the transformed calli were grown on the selection medium with a hygromycin concentration of 50mg / L, hygromycin On the differentiation medium with a concentration of 20mg / L, multiple subcultures were screened and differentiated, and the grown shoots were transferred to a rooting medium containing hygromycin with a concentration of 20mg / L for rooting, until they grew to a suitable size. After the height, move the seedlings to the hardening room to harden the seedlings, and transfer them to the greenhouse for cultivation after 3-5 days.

[0074] 2.2 Molecular detection of transgenic plants

[0075] After several times of screening and differentiation, a total of 9 transformed plants ...

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Abstract

The invention discloses a method for increasing chloroplast genetic transformation efficiency through the genome editing technique. According to the method, chloroplast genome is subjected to fixed shear through the genome editing technique, thereby increasing recombination efficiency and integration efficiency of exogenous gene in the chloroplast genome, and further increasing the chloroplast genetic transformation efficiency.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology and discloses a method for improving the efficiency of chloroplast genetic transformation by using genome editing technology. Background technique [0002] Plant transgenic technology mostly involves introducing foreign genes into the genome of the nucleus for expression. However, with the deepening of research, a series of disadvantages of this conventional method have emerged one after another. For example, the expression efficiency of exogenous genes is low, gene silencing, inactivation and position effects are prone to occur, it is difficult to operate the transformation of multiple genes at the same time, and the expression of exogenous genes in offspring is unstable. More importantly, exogenous genes can be inherited with pollen, and the safety of genetically modified environments brought about by the spread of pollen has aroused people's concerns. In plant cells, DNA genetic mate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00A01H6/46
CPCC12N15/8213C12N15/8214C12N2810/10
Inventor 曹孟良唐宁李丁夏玉梅石跃兵卜小兰余木兰曹晨影米微微朱虹瑾
Owner HUNAN HYBRID RICE RES CENT
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